ALLOGRAFT REJECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/246,665, filed September 21, 2021, the entirety of which is hereby incorporated by reference.

FIELD OF INVENTION

[0002] This invention relates to gene expressions in kidney allografts after transplantation and their use in identifying transplant rejections.

BACKGROUND

[0003] Analysis of gene expression in allograft biopsies is now accepted as a valid diagnostic methodology for antibody rejection (AMR). However, questions remain regarding the sensitivity and specificity of detected genes associated with ARM.

[0004] The Banff 2017 classification of renal allograft pathology adopted “thoroughly validated gene transcripts” in allograft biopsies as one of the criteria for diagnosis of antibody- mediated rejection (AMR or ABMR). However, no specific recommendation was given regarding the “molecular classifiers/transcripts sets” best suited for diagnosis, or the sample types and technical platforms to be utilized for assessment. Moreover, patient populations may differ, especially at centers performing desensitization with patients at high risk for antibody- mediated rejection (AMR or ABMR), thereby enriching desensitized patients, coupled with another factor - preemptive therapies aimed at antibody reduction and B-cell depletion - which may alter gene expression profiles. Therefore, it remains challenging to clearly define biopsy- derived gene expression signatures to enhance and refine conventional pathologic diagnosis.

[0005] Therefore, it is an objective of the present invention to provide markers and their gene expression signatures that are associated with ABMR or cell-mediated rejection (CMR) pathology of kidney transplants, so as to provide diagnosis or risk assessment of transplant rejections.

[0006] It is another objective of the present invention to provide treatment methods for subjects determined to have ABMR or CMR of kidney transplants, and/or assessment methods for subjects receiving therapies against kidney transplant rejection, based on expression profiles of signature genes. [0007] It is another objective of the present invention to provide devices or assay platforms supporting the measurement of signature genes.

[0008] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

BRIEF DESCRIPTION OF THE FIGURES

[0009] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0010] FIG. 1 depicts a study design and workflow of the entire study.

[0011] FIG. 2 depicts quantity and quality of total RNA extracted from 50 biopsies.

Fifty (50) biopsies were randomly selected from all 133 biopsies included in this study. RNA quantity and quality are expressed as nanograms (ng) per biopsy (several 5 pm sections of biopsy) and RNA integrity number (RIN), respectively. Horizontal long and short lines indicate mean and standard deviation of the results from 50 biopsies, respectively.

[0012] FIG. 3A-3K depict differential levels of 11 genes’ mRNA transcripts in a discovery cohort of 69 biopsies with definitive pathology diagnosis divided into 4 different groups. 3A-3E: 5 AMR genes, i.e., KLRF1 (FIG. 3A), CCL3 (FIG. 3B), CCL4 (FIG. 3C), SH2D1B (FIG. 3D), and CD 160 (FIG. 3E). 3F-3K: 6 CMR genes, i.e., IL10 (FIG. 3F), SIRPG (FIG. 3G), IL21R (FIG. 3H), ETV7 (FIG. 31), STAT1 (FIG. 3J), and CCL8 (FIG. 3K). Horizontal long and short lines indicate mean and standard deviation of results in each group of 30 AMR, 14 AMR+CMR,14 CMR, or 11 no rejection control biopsies, respectively. Statistical analysis results by multiple comparisons for each gene are indicated on each graph. ** p<0.01, * 0.01<p<0.05; # 0.05<p<0.10.

[0013] FIG. 4A-4D depict AMR and CMR gene scores in a discovery cohort of 69 biopsies and ROC curves of gene scores. 4A: Differential AMR gene scores based on the mRNA transcript levels of 5 selected genes in a discovery cohort of 69 biopsies with definitive pathology diagnosis in 4 different groups, and the dotted line indicates the cutoff level of AMR gene score at 0.49 for AMR diagnosis. 4B: ROC curve of AMR gene score evaluated in the discovery cohort of 69 biopsies. 4C: Differential CMR gene scores based on the mRNA transcript levels of 6 selected genes in 69 biopsies with definitive pathology diagnosis in 4 different groups, and the dotted line indicates the cutoff level of CMR gene score at 0.92 for CMR diagnosis. 4D: ROC curve of CMR gene score evaluated in the discovery cohort of 69 biopsies. Horizontal long and short lines indicate the mean and standard deviation of results in each biopsy group, respectively. Statistical analysis results by multiple comparisons: ** p<0.01, * 0.01<p<0.05, # 0.05<p<0.10.

[0014] FIG. 5A and 5B depict gene scores in a test cohort of 56 biopsies with ambivalent pathology findings for either AMR or CMR. 5A: AMR gene scores in these 56 biopsies divided into 4 different groups according to AMR-related pathology findings (20 with active/ chronic active AMR, 12 with chronic [inactive] AMR, 16 with suspicious active/chronic active AMR, and 8 non- AMR biopsies), and the dotted line indicates the cutoff level of AMR gene score at 0.49 for AMR diagnosis. 5B: CMR gene scores in these 56 biopsies divided into 4 different groups according to CMR-related pathology findings (9 with acute CMR, 34 with borderline acute CMR, 3 with chronic active CMR, and 10 without any type of CMR), and the dotted line indicates the cutoff level of CMR gene score at 0.92 for CMR diagnosis. Horizontal long and short lines indicate the mean and standard deviation of results in each group, respectively. Statistical analysis results by multiple comparisons: ** p<0.01, * 0.01<p<0.05.

[0015] FIG. 6A-6D depicts CMR activity in biopsies with AMR. 6A: CMR gene scores in 88 biopsies with acute CMR or complete absence of CMR activity divided into 3 groups (37 with acute, 11 with no rejection; and 40 with various levels of AMR activity but no CMR), and the dotted line indicates the cutoff level of CMR gene score at 0.92 for CMR diagnosis. 6B: Tubulitis (t) Banff lesion scores in 40 biopsies with various levels of AMR activity but no CMR from Fig. 6A divided into 2 groups by their CMR gene scores: above 0.92 (n=5) or below 0.92 (n=35). 6C: AMR gene scores in the same 2 groups of biopsies shown in Fig. 6B, and the dot line indicates the cutoff level of AMR gene score at 0.49 for AMR diagnosis. 6D: AMR gene score vs. CMR gene score in 40 biopsies with various types of AMR, and the vertical dotted line indicates the cutoff level of AMR gene score at 0.49 for AMR diagnosis while the horizontal dotted line indicates the cutoff level of CMR gene score at 0.92 for CMR diagnosis. Horizontal long and short lines indicate the mean and standard deviation of results in each group, respectively. Statistical analysis results by either multiple comparison in Fig. 6A or MW test in Fig. 6B and 6C: ** p<0.01, * 0.01<p<0.05, or p as indicated on the graph. The correlation coefficient (p) by Spearman’s test is indicated in Fig. 6D.

[0016] FIG. 7A-7C depict ABMR-related gene score in the discovery cohort (Fig. 7A), in the validation cohort (Fig. 7B), and in both cohorts combined (Fig. 7C). [0017] FIG. 8A-8C depict TCMR-related gene score in the discovery cohort (Fig. 8A), in the validation cohort (Fig. 8B), and in both cohorts combined (Fig. 8C).

[0018] FIG. 9A depicts MVI Banff lesion scores in 12 biopsies with pathology diagnosis of chronic (inactive) ABMR from Fig. 5A. Twelve (12) biopsies were divided into 2 groups: 6 had ABMR gene scores above 0.49, and the other 6 had ABMR gene scores below 0.49. Horizontal long and short lines indicate mean and standard deviation of results of each group, respectively. Statistical analysis result by MW test: p as indicated on the graph.

[0019] FIG. 9B depicts ABMR gene scores in 64 biopsies with active/chronic active ABMR by pathology diagnosis. Sixty-four (64) biopsies were divided into 4 groups: 30 TCMR-free, 14 with acute TCMR, 18 with borderline acute TCMR, and 2 with non-acute chronic active TCMR. Horizontal long and short lines indicate mean and standard deviation of results in each group, respectively. Statistical analysis results by multiple comparisons: * 0.01<p<0.05.

[0020] FIG. 10A depicts the expression levels of 8 genes in 31 graft kidney biopsies examined to exhibit ABMR pathology (“ABMR biopsies”), as well as in 32 graft kidney biopsies examined to show no-rejection in histology (“no-rejection biopsies”). FIG. 10B depicts the fold change (or ratio) of the expression level in ABMR biopsies relative to the expression level in no-rejection biopsies, for each of the 8 genes in figure 10A as well as VWF. [0021] FIG. HA depicts the gene scores (calculated according to procedures described in Example 1) over different Banff score ‘g+ptc’, for each of the 8 genes in ABMR biopsies. FIG. 11B is a line plot with linear regression analysis of the gene scores over different Banff score ‘g+ptc’ for CCL4 in ABMR biopsies, showing an inverse relationship. FIG. 11C is a line plot of the gene scores over different Banff score ‘g+ptc’ for CCL3 in ABMR biopsies.

SUMMARY OF THE INVENTION

[0022] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0023] Various methods are provided for detecting expression levels of a set of genes in a subject, wherein the subject has received a kidney transplant (or transplantation of a graft kidney) and/or the subject desires a determination regarding rejection of the graft kidney or exhibits a symptom/sign of rejection of the graft kidney, and the methods include: assaying a biological sample obtained from the subject, and measuring expression levels of a set of genes in the biological sample, wherein the set of genes is: set (i) comprising, or consisting of, killer cell lectin-like receptor subfamily F member 1 (KLRF1), chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), SH2 domain containing IB (SH2D1B), and cluster of differentiation 160 (CD 160): or set (ii) comprising, or consisting of, KLRF1, fibroblast growth factor binding protein 2 (FGFBP2), granulysin (GNLY), and SH2D1B: or set (iii) comprising, or consisting of, KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, optionally further comprising interferon gamma (IFNG) or set (iv) comprising, or consisting of, interleukin 10 (I I ), interleukin 21 receptor (IL21R), signal-regulatory protein gamma (SIRPG), Ets variant 7 (ETV7), signal transducer and activator of transcription 1 (STAT1), and chemokine (C-C motif) ligand 8 (CCLS); or set (v) comprising, or consisting of, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), SIRPG, ADAM like decysin 1 (ADAMDEC1), IL21R, cluster of differentiation 8 a (CD8A), and TNFAIP3 -interacting protein 3 (TNIP3); or set (vi) comprising, or consisting of, IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3,- or a combined set of any two or more of the sets (i)-(vi).

[0024] In some embodiments, the sets (i)-(vi) each comprises the respective genes. In other embodiments, the sets (i)-(vi) each consists of the respective genes. In additional embodiments, one or more housekeeping genes (e.g., GAPDH, or PWF) is detected along with the genes in the set(s).

[0025] In various implementations, a biological sample is a biopsy of the graft kidney. In various embodiments, the rejection may be active antibody -mediated rejection or chronic active antibody-mediated rejection; and in other embodiments, the rejection may be acute T cell-mediated rejection.

[0026] In various implementations, measuring expression levels of the set of genes includes measuring transcription levels of the genes. For example, this can be performed via one or more techniques such as real-time quantitative polymerase chain reaction (RT-qPCR), microarray analysis, RNA sequencing, and/or high throughput RNA sequencing. In further implementations, measuring transcription levels of the genes includes measuring levels of mRNA of each gene in the set. mRNA levels can be measured via qPCR on complementary DNA (cDNA) converted from total RNA extracted from a biological sample. In some implementations, between 5 ng and 20 ng total RNA, between 20 ng and 50 ng total RNA, or between 50 ng and 100 ng total RNA, or at least 5 ng, 20 ng, 50 ng, or at least 100 ng of total RNA, is extracted from the biological sample. In some aspects, the measured mRNA level is a relative expression level to reference RNA. In other aspects, the measured mRNA level is the number of mRNA copies per biological sample (e.g., per biopsy of the graft kidney).

[0027] In other implementations, measuring expression levels of the set of genes includes measuring expression levels of proteins encoded by the set of genes. This can be performed via one or more techniques such as western blotting, ELISA or another immunohistochemical assays.

[0028] In some embodiments, the subject exhibits a symptom or sign of, or desires determination of, presence of antibody-mediated rejection (ABMR) of the graft kidney, and the set of gene is the set (i), the set (ii), the set (iii).

[0029] In some embodiments, the subject exhibits a symptom or sign of, or desires determination of, presence of cell-mediated rejection (CMR) of the graft kidney, and the set of gene is the set (iv), the set (v), the set (vi).

[0030] In various implementations, all the genes in a set is detected to be higher than respective expression levels in a reference sample, wherein the reference sample may be a biological sample obtained from a subject having no rejection of a kidney transplant, or from a subject with a normal/healthy kidney. In one example, the genes in any of sets (i)-(iii) from subject with ABMR of a kidney transplant are detected to be higher than respective gene expression levels from a subject with CMR of a kidney transplant, or from a subject with no rejection of the kidney transplant. In another example, the genes in any of sets (iv)-(vi) from subject with CMR of a kidney transplant are detected to be higher than respective gene expression levels from a subject with CMR of a kidney transplant, or from a subject with no rejection of the kidney transplant. In additional implementations, the detected expression levels of genes in any of sets (i)-(vi) are computed into a gene score, and the gene score from a subject having a rejection is higher than that from a subject having no rejection of a kidney transplant, or from a subject with a normal/healthy kidney. In one example, the gene score based on expression levels of genes in any of sets (i)-(iii) from a subject having ABMR of a kidney transplant is higher than that from a subject with CMR of a kidney transplant, or from a subject with no rejection of the kidney transplant. In another example, the gene score based on expression levels of genes in any of sets (iv)-(vi) from a subject having CMR of a kidney transplant is higher than that from a subject with ABMR of a kidney transplant, or from a subject with no rejection of the kidney transplant. [0031] Various methods for treating a kidney transplant recipient are provided, wherein the recipient may experience a pathology of, exhibit symptoms of, or be suspected of developing ABMR of the kidney transplant, and the methods include:

(a) performing plasma exchange and administering intravenous immune globulins (IVIG), so as to remove circulating donor-specific antibodies (DSA) and/or reduce DSA production,

(b) administering an effective amount of one or more complement inhibitors selected from Eculizumab and a Cl esterase inhibitor, so as to reduce damage to the kidney transplant from the DSA,

(c) administering an effective amount of rituximab or another anti-CD20 antibody, so as to reduce or deplete B cells,

(d) administering an effective amount of Imlifidase, so as to reduce anti-HLA DSA,

(e) administering an effective amount of antithymocyte globulin, so as to reduce or deplete T cells,

(f) administering an effective amount of an IL-6/IL-6R inhibitor, optionally the IL- 6/IL-6R inhibitor comprising Tocilizumab or clazakizumab, or

(g) performing surgical splenectomy, splenic embolization, and splenic radiation,

(h) administering an effective amount of daratumumab or another anti-CD38 antibody, so as to reduce or deplete plasma cells, or performing a combination of any two or more of (a)-(h), to a kidney transplant recipient who has been detected with a higher expression level of each gene in one or more of gene sets (i)-(iii) in a biological sample obtained from the subject relative to a respective reference value, and wherein the gene set (i) comprises: KLR 1, CCL3, CCL4, SH2D1B, and CD160, the gene set (ii) comprises: KLRF1, FGFBP2, GNLY, and SH2D1B,' and the gene set (iii) comprises: KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, optionally the gene set (iii) further comprising IFNG.

[0032] Various methods for treating a kidney transplant recipient are further provided, wherein the recipient may experience a pathology of, exhibit symptoms of, or be suspected of developing CMR of the kidney transplant, and the methods include

(a) administering an effective amount of steroid or corticosteroid,

(b) administering an effective amount of a lymphocyte-depleting antibody or Murom onab-CD3, or

(c) performing a combination of (a) and (b), to the kidney transplant recipient who has been detected with a higher expression level of each gene in one or more gene sets of (iv)-(vi) in a biological sample obtained of the subject relative to a respective reference value, wherein the gene set (iv) comprises: ELIO, EL21R, SIRPG, ETV7, STAT1, and CCZS; the gene set (v) comprises: CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,' and the gene set (vi) comprises: IL 10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0033] System for detection or measurement of expression levels of a set of genes are also provided. In some embodiments, a system is provided for detection of expression levels of a set of genes indicative of ABMR of kidney allograft, wherein the system includes a plurality of reagents, each reagent specifically binding to one of the genes in the set or binding to a protein encoded by one of the genes in the set, wherein the set of genes is the set (i), the set (ii), or the set (iii) above. In other embodiments, a system is provided for detection of expression levels of a set of genes indicative of CMR of kidney allograft, wherein the system includes a plurality of reagents, each reagent specifically binding to one of the genes in the set or binding to a protein encoded by one of the genes in the set, wherein the set of genes is the set (iv), the set (v), or the set (vi) above. In additional embodiments, a system is provided for detection of expression levels of a set of genes, wherein the system includes a plurality of reagents, each reagent specifically binding to one of the genes in the set or binding to a protein encoded by one of the genes in the set, wherein the set of genes comprises any two or more of the sets (i)-(vi). Preferably, the system includes a plurality of reagents, each reagent specifically binding to a different gene in the set or binding to a protein encoded by a different gene in the set. Also preferably, the system further includes a reagent that specifically binds to a housekeeping gene or a protein encoded by the housekeeping gene. In some aspects, the plurality of reagents in the system specifically bind to only the genes (or proteins encoded thereby) in the set, wherein the set consists of the genes listed above.

[0034] Additional embodiments provide that a system further includes a chip, an array, a surface or a device, wherein the plurality of reagents is immobilized thereto. For example, in detection of transcription levels of genes, the plurality of reagents comprises a plurality of polynucleotides, each fully complementary to at least a portion of a different gene in the set, and the polynucleotides are immobilized in the chip, array, or device or on the surface. As another example, in detection of expression levels of proteins encoded by the genes, the plurality of reagents comprises a plurality of antibodies (or antigen-binding fragments thereof), each specifically binding to an epitope of a different protein encoded by the genes in the set, and the antibodies (or antigen-binding fragments thereof) are immobilized in the chip, array, or device or on the surface. In further embodiments, a system also includes one or more pharmaceutically acceptable excipients, diluents, and/or vessels containing them.

[0035] Various embodiments provide methods of providing prognosis or diagnosis for a human subject having received a kidney transplant. The diagnosis or prognosis methods may include measuring expression levels of each gene in a set in a biological sample of the human subject, wherein the set is the set (i)-set (vi), and wherein a diagnosis or prognosis of AB MR of the kidney transplant is indicated when the expression levels of each gene in any one of the sets (i)-(iii) are higher than a respective reference value, and a diagnosis or prognosis of CMR of the kidney transplant is indicated when the expression levels of each gene in any one of sets (iv)-(vi) are above a respective reference value. In some additional embodiments, the subject indicated with a diagnosis/prognosis of the ABMR has expression levels of a set of endothelial- specific genes in the biopsy sample about the same to or below corresponding reference values in a kidney biopsy sample of a control subject, and the set of endothelial-specific genes are DARC, ECSCR, PECAM1, NOS3, and VWF.

[0036] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

DESCRIPTION OF THE INVENTION

[0037] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 ^rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 ^th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 ^th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2 ^nd ed. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al. U. S. Patent No. 5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep;23(9): 1126- 36).

[0038] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0039] A subject, patient, or individual (used interchangeably) can be one who has been previously diagnosed with or identified as suffering from or having a disease-state (e.g., rejection of transplant allograft, transplant failure, loss of function of transplant) in need of monitoring or one or more complications related to such a disease-state, and optionally, have already undergone treatment for the disease-state or the one or more complications related to the disease/condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease-state or one or more complications related to the disease/condition. For example, a subject can be one who exhibits one or more risk factors for a disease-state or one or more complications related to a disease-state or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular disease-state (e.g., antibody-mediated rejection of transplant allograft, active ABMR, chronic active ABMR, or cell-mediated rejection of transplant allograft) can be a subject having that disease/condition, diagnosed as having that condition, or at risk of developing that disease. The terms, “patient”, “individual” and “subject” are used interchangeably herein. A “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. In various embodiments, the subject is a human. In some embodiments, the subject is a human recipient of a kidney allograft.

[0040] The term “expression level(s)” or “expression level(s) of one or more genes” refers to a quantity reflected in or derivable from the gene or protein expression data, whether the data is directed to gene transcript accumulation or protein accumulation or protein synthesis rates, etc. The term “expression level” refers to the amount of either gene transcript accumulation or protein transcript accumulation, unless the context suggests gene transcription level or protein/peptide expression level, or unless specifically noted. For example, “gene expression/transcription level” refers to the amount of gene transcript accumulation; “protein expression level” refers to the amount of protein accumulation.

[0041] Gene expression is regulated at different stages, including: transcription (copy genetic information from genomic DNA into RNA), post-transcriptional modification (convert primary transcript RNA into messenger RNA [mRNA]), translation (produce polypeptide chains based on mRNA), and post-translation modification (chemical changes of protein after translation). Some embodiments provide that gene expression level(s) to be measured/ detected/ determined in one or more methods disclosed herein refer to the level at any of the above-mentioned stages. In some embodiments, the gene expression level(s) in one or more methods disclosed herein is mRNA level(s). In some embodiments, the gene expression level(s) in one or more methods disclosed herein is protein expression level(s).

[0042] In various embodiments, measuring the expression level of a marker set (also called a gene set) include measuring the expression level of each marker (or each gene) in the set, and a decreased or increased level of the expression level of a marker set (or a gene set) include a decreased, or increased respectively, level of each marker (or gene) in the set.

[0043] As of 2019, the Banff schema recognizes four diagnostic categories of AB MR: 1. Active ABMR, 2. Chronic active ABMR, 3. Chronic (inactive) ABMR, and 4. C4d staining without evidence of rejection. The first category, “active ABMR,” requires 3 diagnostic criteria: histologic evidence of acute tissue injury, evidence of current or recent antibody interaction with the endothelium (usually C4d), and serologic evidence of DS A (although C4d staining or validated transcripts may substitute for DSA). Histologically, microvascular inflammation (MVI), also known as capillaritis, qualifying for this “active ABMR” category is a glomerulitis (g) score + peritubular capillaritis (ptc) score of 2 or greater. Other acute tissue injury patterns are acute tubular injury, thrombotic microangiopathy, and less commonly arterial lesions of endothelialitis, fibrinoid necrosis, or transmural inflammation. The second category, chronic active ABMR, has a similar three criteria, but with histologic evidence of chronic tissue injury, such as transplant glomerulopathy (TG) attributable to ABMR. The third category, chronic (inactive) ABMR, shows histologic evidence of chronic tissue injury, but without capillaritis and without C4d deposition in peritubular capillaries. The final category is peritubular capillary C4d staining without evidence of rejection, previously referred to as the category of “accommodation.” This category primarily applies to ABO blood group incompatible transplants, which show positive C4d staining in even 80% of protocol biopsies and the staining does not correlate with peritubular capillaritis. Despite positive C4d staining, ABO blood group incompatible kidney transplants show the same rate of capillaritis as conventional kidney transplants. Although some protocol biopsies in patients with positive crossmatch (anti-HLA DSA) transplants show positive C4d staining and no histologic evidence of tissue injury, and thus qualify for the category of accommodation, the state of accommodation in patients with anti-HLA DSA is likely temporary and unstable. [0044] As of 2019, the Banff schema recognizes two diagnostic categories of TCMR: 1. Acute TCMR, and 2. Chronic active TCMR, as well as a category of Borderline (Suspicious) for acute TCMR. In “acute TCMR”: Grade IA requires interstitial inflammation involving >25% of non-sclerotic cortical parenchyma (i2 or i3) with moderate tubulitis (t2) involving 1 or more tubules, not including tubules that are severely atrophic; Grade IB requires interstitial inflammation involving >25% of non-sclerotic cortical parenchyma (i2 or i3) with severe tubulitis (t3) involving 1 or more tubules, not including tubules that are severely atrophic; Grade IIA requires mild to moderate intimal arteritis (vl), with or without interstitial inflammation and/or tubulitis; and Grade IIB requires severe intimal arteritis (v2), with or without interstitial inflammation and/or tubulitis; and Grade III requires transmural arteritis and/or arterial fibrinoid necrosis involving medial smooth muscle with accompanying mononuclear cell intimal arteritis (v3), with or without interstitial inflammation and/or tubulitis. In “chronic active TCMR”: Grade IA requires interstitial inflammation involving >25% of sclerotic cortical parenchyma (i-IFTA2 or i-IFTA3) AND > 25% of total cortical parenchyma (ti2 or ti3) with moderate tubulitis (t2 or t-IFTA2) involving 1 or more tubules, not including severely atrophic tubules; other known causes of i-IFTA should be ruled out; Grade IB requires interstitial inflammation involving >25% of sclerotic cortical parenchyma (i-IFTA2 or i-IFTA3) AND > 25% of total cortical parenchyma (ti2 or ti3) with severe tubulitis (t3 or t-IFTA3) involving 1 or more tubules, not including severely atrophic tubules; other known causes of i-IFTA should be ruled out; and Grade II requires chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell inflammation in fibrosis and formation of neointima). This may also be a manifestation of chronic active or chronic AB MR or mixed ABMR/TCMR. Yet, “borderline (suspicious) for acute TCMR” requires foci of tubulitis (tl, t2, or t3) with mild interstitial inflammation (il), or mild (tl) tubulitis with moderate-severe interstitial inflammation (i2 or i3), and no intimal or transmural arteritis (v = 0).

[0045] The pathological requirements listed above can (histological) symptoms or signs of ABMR or CMR of graft kidneys. Further detail, updates, and clarification of criteria for T cell- and antibody-mediated rejection from the Banff 2019 Kidney Meeting are published in Am J Transplant, 2020;20:2318-2331, which is incorporated herein in its entirety, to demonstrate a standardized reporting for TCMR and ABMR diagnoses.

[0046] The terms “treating” or “treatment” or “to treat” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic disease or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder if the subject shows, e.g., total, partial, permanent, or transient, alleviation or elimination of any symptom associated with the disease or disorder.

[0047] The term “about” or “approximately” when used in connection with a referenced numeric indication (in percentage) means the referenced numeric indication (in percentage) plus or minus up to 5% of that referenced numeric indication (in percentage), unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” or “approximately” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0048] Previous reports on gene expression signatures or mRNA transcripts used samples including freshly collected renal allograft biopsy tissues with immediate RNALATER® storage or formalin-fixed paraffin-embedded (FFPE) biopsy tissues, or peripheral blood mononuclear cells (PBMC). The associations were compared to specific histologic lesions and presence of donor-specific-antibodies (DSA), but these studies did not reveal clearly defined biopsy-derived gene expression signatures. Moreover, patient populations vary, and preemptive treatment such as desensitization or therapies aimed at antibody reduction and B-cell depletion may alter gene expression profiles.

[0049] To address these issues, we have herein conducted a gene expression study using frozen tissue sections derived from renal allograft biopsies to analyze gene expression signatures from patients diagnosed with AMR, cell-mediated rejection (CMR), and mixed AMR/CMR compared to biopsies with no pathologic findings.

Detection/Sample Processing Methods

[0050] Various embodiments provide for methods for detecting expression of each gene in a set of genes or detecting level of each protein encoded by a different gene in the set - collectively referred to detecting expression levels of a set of genes, or detecting gene expression levels, unless otherwise noted - in a subject or in a biological sample, wherein the subject desires a determination regarding rejection (ABMR, CMR, or in general rejection) of a kidney transplant in the subject, or wherein the subject exhibits a symptom or sign of rejection (ABMR, CMR, or in general rejection) of a kidney transplant, or wherein the detection is in a biological sample of the graft kidney before and/or after transplantation in a subject. In some implementations, the methods for detecting expression levels of genes are detecting mRNA levels of the genes, optionally via reverse transcription to obtain cDNA as template for qPCR assays. In other implementations, the methods for detecting expression levels of genes are detecting expressed protein levels, wherein each protein is encoded by a different gene in the set. Generally, methods for detecting expression levels of a set of genes in a subject comprises assaying a biological sample obtained from a subject in need of or having undergone a kidney transplant, and detecting the expression levels of the set of genes in the biological sample. Preferably, the biological sample includes or is a biopsy (or a specimen) of the graft kidney.

[0051] Some embodiments provide methods for detecting gene expression levels in a biological sample (e.g., a biopsy of a renal allograft) obtained from a subject, and the methods include measuring expression levels of a set of genes in the biological sample, wherein the set of genes is:

Set (i) comprising, or consisting of, killer cell lectin-like receptor subfamily F member 1 (KLRF1), chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), SH2 domain containing IB (SH2D1B), and cluster of differentiation 160 (CD 160): or

Set (ii) comprising, or consisting of, KLRF1, fibroblast growth factor binding protein 2 (FGFBP2), granulysin GNLY , and SH2D1B: or

Set (iii) comprising, or consisting of, KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, or

Set (iv) comprising, or consisting of, interleukin 10 (LL10, also known as IL-10), interleukin 21 receptor (IL21R, also denoted as IL-21R), signal -regulatory protein gamma (SIRPG), Ets variant 7 (ETV7), signal transducer and activator of transcription 1 (STA Tl), and chemokine (C-C motif) ligand 8 (CCLS); or

Set (v) comprising, or consisting of, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), SIRPG, ADAM like decysin 1 (ADAMDEC1), IL21R, cluster of differentiation 8 a (CD8A), and TNFAIP3 -interacting protein 3 (TNIP3); or

Set (vi) comprising, or consisting of, IL 10, IL21R, SIRPG, ETV7, STATl, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3,- or

(vii) a combination of any two or more of subsets (i)-(vi).

[0052] In some implementations of the methods, the subject exhibits a symptom of, or is suspected of developing, antibody-mediated rejection (ABMR) of the renal allograft after the transplantation, or the subject’s biopsy sample of the renal allograft indicates a pathology of the ABMR of the renal allograft, and the set of genes comprises, or the set of genes is selected from the group consisting of:

(i) KLRF1, CCL3, CCL4, SH2D1B, and CD160,' or

(ii) KLRF1, FGFBP2, GNLY, and SH2D1B: or (iii) KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY.

[0053] In other implementations of the methods, the subject exhibits a symptom of, or is suspected of developing, cell mediated rejection (CMR) of the renal allograft after the transplantation, or the subject’s biopsy sample of the renal allograft indicates a pathology of the CMR of the renal allograft, and the set of genes comprises, or the set of genes is selected from the group consisting of:

(iv) /Z70, 11.21 R, SIRPG, ETV7, STAT1, and CCL8- or

(v) CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,- or

(vi) IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0054] In some implementations, because endothelial-specific genes, Duffy antigen/chemokine receptor (DARC, also known as ACKR1 encoding atypical chemokine receptor 1), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR), platelet and endothelial cell adhesion molecule 1 (PECAM1), nitric oxide synthase 3 (NOS3), and Von Willebrand factor (VWF), do not significantly associate with ABMR, the method does not include measuring an expression level of any one of these five genes in a biopsy kidney sample. In additional implementations, methods may further include detecting expression levels of additional genes in any one of Tables 4, 7, 8, or 12.

[0055] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising all seven of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY. In some aspects, the detected set of genes further comprises a housekeeping gene, interferon gamma (IFNG), or both.

[0056] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising any six of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY. In some aspects, the detected set of genes further comprises a housekeeping gene, IFNG, or both.

[0057] In some embodiments, methods of detecting expression levels of a set of genes in a subject are provided, which includes: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising any five of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some embodiments, the detected set of genes comprises KLRF1, CCL3, CCL4, SH2D1B and CD160, but does not comprise FGFBP2 and GNLY. In some aspects, the detected set of genes further comprises a housekeeping gene, IFNG, or both. [0058] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising any four of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY. In some embodiments, the detected set of genes comprises KLRF1, FGFBP2, GNLY, and SH2D1B, but does not comprise CCL3, CCL4, and CD160. In some aspects, the detected set of genes further comprises a housekeeping gene, IFNG, or both.

[0059] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising any three of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some aspects, the detected set of genes further comprises a housekeeping gene, IFNG, or both. [0060] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising any two of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY. In some aspects, the detected set of genes further comprises a housekeeping gene, IFNG, or both.

[0061] In other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes selected from the group consisting of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, GNLY, and a combination ofKLRFl, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY. In some aspects, the methods include detecting expression levels of a set of genes consisting of a combination of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG. [0062] In yet other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some aspects, the methods include detecting the expression levels of genes being or consisting of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG.

[0063] In yet other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of any six of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY. In some aspects, the methods include detecting the expression levels of genes being or consisting of any six of KLBF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG.

[0064] In yet other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of any five of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of KLRF1, CCL3, CCL4, SH2D1B and CD160. In some aspects, the methods include detecting the expression levels of genes being or consisting of any five of KLBF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG.

[0065] In yet other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of any four of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some aspects, the methods include detecting the expression levels of genes being or consisting of any four of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG.

[0066] In yet other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of any three of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some aspects, the methods include detecting the expression levels of genes being or consisting of any three of KLBF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG.

[0067] In yet other embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes being or consisting of any two of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY. In some aspects, the methods include detecting the expression levels of genes being or consisting of any two of KLBF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY, and one or both of a housekeeping gene and IFNG.

[0068] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes comprising all ten oilLIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene. [0069] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any nine of IL 10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene.

[0070] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any eight of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene.

[0071] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any seven of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene.

[0072] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any six of ELIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the methods include detecting the expression levels of a set of genes comprising CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene. [0073] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any five of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene.

[0074] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any four of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the methods include detecting expression levels of a set of genes comprising KLRF1, FGFBP2, GNLY, and SH2D1B. In some aspects, the detected set of genes further comprises a housekeeping gene.

[0075] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any three of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene.

[0076] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes comprising any two of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further comprises a housekeeping gene. [0077] In some embodiments, methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of a set of genes selected from or consisting of IL 10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0078] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any nine of ELIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further is selected from, or consists of, a housekeeping gene and any nine of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0079] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any eight of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes further is selected from, or consists of, a housekeeping gene and any eight of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0080] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any seven of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and any seven of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0081] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any six of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the methods include detecting the expression levels of a set of genes selected from or consisting of CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and any six of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3.

[0082] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any five of ELIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and any five of ELIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0083] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any four of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the methods include detecting expression levels of a set of genes selected from or consisting of KLRF1, FGFBP2, GNLY, and SH2D1B. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and any four of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and KERF 1, FGFBP2, GNLY, and SH2D1B.

[0084] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any three of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and any three of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0085] In some embodiments, methods of detecting expression levels of a set of genes in a subject include detecting in a biological sample obtained from the subject the expression levels of a set of genes selected from or consisting of any two of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the detected set of genes is selected from, or consists of, a housekeeping gene and any two of IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0086] Some embodiments provide that methods of detecting expression levels of a set of genes in a subject include: assaying a biological sample obtained from the subject, optionally wherein the subject desires a determination regarding kidney transplant rejection or the subject exhibits a symptom of kidney transplant rejection, and detecting the expression levels of genes comprising all 17, or two or more (e.g., 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2), of KLRF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, GNLY, IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In some aspects, the methods include detecting expression levels of genes comprising one or both of a housekeeping gene and /FAG, and all 17, or two or more (e.g., 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2), oiKLRFl, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, GNLY, IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In other embodiments, the methods include detecting expression levels of genes selected from, or consists of, all 17, or two or more (e.g., 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2), o KLRFl, CCL3, CCL4, SH2D1B, CD160, FGFBP2, GNLY, IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. In yet other aspects, the methods include detecting expression levels of genes selected from, or consists of, one or both of a housekeeping gene and IFNG, and all 17, or two or more (e.g., 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2), of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, GNLY, IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0087] Additional embodiments provide methods for detecting expression levels of a set of genes further include computing a gene score for each gene in the set or computing a gene score for the set of genes collectively detected from a same biological sample (also referred to as a collective gene score for the biological sample). A gene score is computed based on the detected expression level of the gene. For example, the amount of a gene of interest is first normalized to the amount of a house-keeping gene (e.g., GAPDFL, or FWF for kidney transplant biopsy samples) from the same assay sample that the amount of the gene of interest is measured from, so as to obtain a relative quantity (RQ) of the gene of interest; and optionally followed by normalizing the RQ of the gene of interest from that one assay sample to an average RQ of that gene of interest in a pool of reference samples.

[0088] Reference samples can be a plurality of samples obtained from a plurality of subjects (“control”). In some aspects, the plurality of subjects can be a plurality of healthy subjects. In some aspects, the plurality of subjects can be a plurality of subjects with kidney transplants but showing no symptoms or signs of transplant rejection. In other aspects, the plurality of subjects can be a plurality of subjects with kidney transplants, where some exhibiting signs of transplant rejection and some exhibiting no signs of transplant rejection.

[0089] Additional embodiments provide methods for detecting expression levels of a set of genes further include computing (e.g., operating a computer to calculate) gene scores for each gene in the set or computing a collective gene score for the set, wherein the gene scores for each gene in the set are higher than a respective reference level for each gene, or wherein the collective gene score for the set is higher than a reference level for the gene set.

[0090] Some embodiments provide that the methods for detecting gene expression levels include detecting a higher expression level of a set of genes in a biological sample relative to a respective control or to a respective reference level. A higher expression level of a set of genes generally refers to a higher expression level of each gene in the set, relative to the expression level of that specific gene (called “respective gene”) in a control, or relative to a reference level of that specific gene (or the respective gene). Typically, a higher expression level is at least two-fold, three-fold, four-fold, or five-fold higher than a respective control or a respective reference level.

[0091] Further embodiments provide that one or more methods disclosed herein include detecting one or more of: an expression level of SH2D1B in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, or 15-25 times, or at least 22 times the respective level in a reference sample; an expression level of KLRF1 in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or between 3 times and 10 times the respective level in a reference sample; an expression level of GNLY in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, or 15-20 times, or at least 16 times the respective level in a reference sample; an expression level of FGFBP2 in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, 15-20 times, 20-25 times, or at least 19 times the respective level in a reference sample; an expression level of CCL3 in a biological sample obtained from a subject which is at least 8 times the respective level in a reference sample, (i.e., at least 7 times higher than the respective level in a reference sample), or which is between 5-10 times, 11-15 times, or 15-20 times the respective level in a reference sample; an expression level of CCL4 in a biological sample obtained from a subject which is at least 5 times the respective level in a reference sample, (i.e., at least 4 times higher than the respective level in a reference sample), or which is between 3-7 times, 7-10 times, or 15-20 times the respective level in a reference sample; an expression level of CD 160 in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is between 1.5-3 times, 3-5 times, or 5-10 times the respective level in a reference sample; and/or an expression level of IFNG in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is between 1.5-3 times, 3-5 times, or 5-10 times the respective level in a reference sample; and optionally wherein the reference sample comprises biological samples obtained from a subject having a kidney transplant but showing no signs of rejection of the transplant.

Alternatively, the reference sample may be biological samples obtained from a subject having a kidney transplant showing signs of CMR of the transplant, or the reference sample may be biological samples obtained from a subject showing no signs of transplant rejection and a subject having a kidney transplant showing signs of CMR of the transplant.

Treatment Methods

[0092] Treatment for ABMR after kidney transplantation has been recommended in the 2019 expert consensus from the Transplantation Society Working Group, which is described in Transplantation, May 2020, volume 104, number 5. Exemplary treatments for ABMR of kidney allograft include: performing plasma exchange and administering intravenous immune globulins (IVIG) to remove circulating donor-specific antibodies (DSA) and/or reduce DSA production in the subject; administering an effective amount of one or more complement inhibitors of Eculizumab or Cl esterase inhibitors (e.g., BERINERT, CINRYZE, RUCONEST, RHUCIN; plasma-derived, or recombinantly produced Cl esterase inhibitors) to reduce damage to the kidney transplant from the DSA; administering an effective amount of a CD20 inhibitor such as an anti-CD20 antibody (e.g., rituximab) to deplete B cells; administering an effective amount of Imlifidase to reduce anti-HLA DSA; administering an effective amount of antithymocyte globulin to deplete T cells; administering an effective amount of an IL-6/IL-6R inhibitor (e.g., Tocilizumab or clazakizumab); administering an effective amount of an anti- CD38 antibody (e.g., daratumumab, or isatuximab) or an anti-plasma cell agent; administering an effective amount of Bortezomib to reduce antibody-producing plasma cells; and/or performing surgical splenectomy, splenic embolization, and splenic radiation.

[0093] Treatments of acute rejections, including acute cellular rejection and antibody- mediated acute rejection, are described in Chapter 6, American Journal of Transplantation 2009; 9 (Suppl 3): S21-S22. Exemplary treatments of acute cellular rejection of kidney transplant includes administering pulse steroid; administering a corticosteroid therapy, e.g., intravenous solumedrol 250-500 mg daily for 3 days; and/or administering an anti-T-cell antibody (muromonab [OKT3], ATG or ALG), to restore kidney function and prevent graft loss.

[0094] If a subject has a pathology of both ABMR and TCMR of the kidney transplant, a combination of treatments for ABMR and CMR can be administered or performed to the subject.

[0095] Various methods provide for a method for treating a kidney transplant recipient experiencing a pathology of, exhibiting symptoms of, or suspected of developing antibody- mediated rejection (ABMR) of the kidney transplant, comprising administering or performing a therapy directed to treatment or management of ABMR to a subject detected with expression levels of each of a set of genes above a reference value in a biopsy sample of the kidney transplant of the subject, wherein the set of genes comprises, or is selected from a group consisting of, or consists of:

(i) KLRF1, CCL3, CCL4, SH2D1B, and CD160,- or

(ii) KLRF1, FGFBP2, GNLY, and SH2D1B,- or

(iii) KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, and optionally IFNG.

[0096] In some implementations, a therapy directed to treatment or management of ABMR includes: performing plasma exchange and administering intravenous immune globulins (IVIG) to remove circulating donor-specific antibodies (DSA) and/or reduce DSA production in the subject; administering an effective amount of one or more complement inhibitors of Eculizumab or a Cl esterase inhibitor (e.g.,Berinert, Cinryze) to reduce damage to the kidney transplant from the DSA; administering an effective amount of an anti-CD20 antibody (e.g., rituximab) to deplete or reduce B cells; administering an effective amount of Imlifidase to reduce anti-HLA DSA; administering an effective amount of antithymocyte globulin to deplete or reduced T cells; administering an effective amount of an IL-6/IL-6R inhibitor (e.g., Tocilizumab or clazakizumab); administering an effective amount of an anti- CD38 antibody (e.g., daratumumab, isatuximab) to deplete or reduce plasma cells, especially malignant plasma cells where CD38 is overly expressed; administering an effective amount of Bortezomib to reduce antibody-producing plasma cells; and/or performing surgical splenectomy, splenic embolization, and splenic radiation.

[0097] Further embodiments provide for a method for treating a kidney transplant recipient experiencing a pathology of, exhibiting symptoms of, or suspected of developing cell- mediated rejection (CMR) of the kidney transplant, comprising administering or performing a therapy directed to treatment or management of CMR to a subject detected with expression levels of each of a set of genes in a biopsy sample of the kidney transplant of the subject above a reference value, wherein the set of genes comprises, or is selected from a group consisting of, or consists of:

(iv) /Z70, IL2IR, SIRPG, ETV7, STAT1, and CCL8- or

(v) CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,- or

(vi) ILIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0098] In some implementations, a therapy directed to treatment or management of CMR includes: administering an effective amount of steroid or corticosteroid; administering an effective amount of a lymphocyte-depleting antibody or Muromonab-CD3; or both.

[0099] Some embodiments provide that methods for treating ABMR of kidney transplant in a subject include: diagnosing the ABMR by detecting the expression levels of a set of genes as being above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (i), (ii), or (iii) above; and administering a therapy directed to treatment or management of ABMR to the subject.

[0100] In further implementations, the methods for treating ABMR of kidney transplant in a subject include: diagnosing the ABMR by detecting the expression levels of a set of genes as being above respective reference level, and administering a therapy directed to treatment or management of ABMR to the subject, wherein the detection of the expression levels as being above respective reference level comprises one or more of: detecting an expression level of SH2D1B in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, or 15-25 times, or at least 22 times the respective level in a reference sample; detecting an expression level of KLRF1 in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or between 3 times and 10 times the respective level in a reference sample; detecting an expression level of GNLY in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, or 15-20 times, or at least 16 times the respective level in a reference sample; detecting an expression level oiFGFBP2 in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, 15-20 times, 20-25 times, or at least 19 times the respective level in a reference sample; detecting an expression level of CCL3 in a biological sample obtained from a subject which is at least 8 times the respective level in a reference sample, (i.e., at least 7 times higher than the respective level in a reference sample), or which is between 5-10 times, 11-15 times, or 15-20 times the respective level in a reference sample; detecting an expression level of CCL4 in a biological sample obtained from a subject which is at least 5 times the respective level in a reference sample, (i.e., at least 4 times higher than the respective level in a reference sample), or which is between 3-7 times, 7-10 times, or 15-20 times the respective level in a reference sample; detecting an expression level of CD 160 in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is between 1.5-3 times, 3-5 times, or 5-10 times the respective level in a reference sample; and/or detecting an expression level of IFNG in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is between 1.5-3 times, 3-5 times, or 5-10 times the respective level in a reference sample; and optionally wherein the reference sample comprises biological samples obtained from a subject having a kidney transplant but showing no signs of rejection of the transplant, or wherein the reference sample comprises biological samples obtained from a subject having a kidney transplant showing signs of CMR of the transplant, or wherein the reference sample comprises biological samples obtained from a subject showing no signs of transplant rejection and a subject having a kidney transplant showing signs of CMR of the transplant. [0101] Some embodiments provide that methods for treating ABMR of kidney transplant in a subject include: obtaining the results of an analysis of expression levels of a set of genes in a biological sample obtained from the subject; and administering a therapy directed to treatment or management of ABMR to the subject when the expression levels of the set of genes are above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (i), (ii), or (iii).

[0102] Some embodiments provide methods for treating ABMR of kidney transplant in a subject include: requesting the results of an analysis of expression levels of a set of genes in a biological sample obtained from the subject; and administering a therapy directed to treatment or management of ABMR to the subject when the expression levels of the set of genes are above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (i), (ii), or (iii).

[0103] Some embodiments provide that methods for treating ABMR of kidney transplant in a subject include: administering a therapy directed to treatment or management of ABMR to a subject who has been determined to have expression levels of the set of genes above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (i), (ii), or (iii).

[0104] In further implementations of the methods, expression levels of the set of genes above respective reference levels comprise one or more of: an expression level of SH2D1B in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, or 15-25 times, or at least 22 times the respective level in a reference sample; an expression level of KLRF1 in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or between 3 times and 10 times the respective level in a reference sample; an expression level of GNLY in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, or 15-20 times, or at least 16 times the respective level in a reference sample; an expression level of FGFBP2 in a biological sample obtained from a subject which is at least 4 times the respective level in a reference sample, (i.e., at least 3 times higher than the respective level in a reference sample), or which is between 4-10 times, 11-15 times, 15-20 times, 20-25 times, or at least 19 times the respective level in a reference sample; an expression level of CCL3 in a biological sample obtained from a subject which is at least 8 times the respective level in a reference sample, (i.e., at least 7 times higher than the respective level in a reference sample), or which is between 5-10 times, 11-15 times, or 15-20 times the respective level in a reference sample; an expression level of CCL4 in a biological sample obtained from a subject which is at least 5 times the respective level in a reference sample, (i.e., at least 4 times higher than the respective level in a reference sample), or which is between 3-7 times, 7-10 times, or 15-20 times the respective level in a reference sample; an expression level of CD 160 in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is between 1.5-3 times, 3-5 times, or 5-10 times the respective level in a reference sample; and/or an expression level of IFNG in a biological sample obtained from a subject which is at least 3 times the respective level in a reference sample, (i.e., at least 2 times higher than the respective level in a reference sample), or which is between 1.5-3 times, 3-5 times, or 5-10 times the respective level in a reference sample; and optionally wherein the reference sample comprises biological samples obtained from a subject having a kidney transplant but showing no signs of rejection of the transplant, or wherein the reference sample comprises biological samples obtained from a subject having a kidney transplant showing signs of CMR of the transplant, or wherein the reference sample comprises biological samples obtained from a subject showing no signs of transplant rejection and a subject having a kidney transplant showing signs of CMR of the transplant.

[0105] Some embodiments provide that methods for treating CMR of kidney transplant in a subject include: diagnosing the CMR by detecting the expression levels of a set of genes as being above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (iv), (v), or (vi); and administering a therapy directed to treatment or management of CMR to the subject.

[0106] Some embodiments provide that methods for treating CMR of kidney transplant in a subject include: obtaining the results of an analysis of expression levels of a set of genes in a biological sample obtained from the subject; and administering a therapy directed to treatment or management of CMR to the subject when the expression levels of the set of genes are above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (iv), (v), or (vi).

[0107] Some embodiments provide that methods for treating CMR of kidney transplant in a subject include: requesting the results of an analysis of expression levels of a set of genes in a biological sample obtained from the subject; and administering a therapy directed to treatment or management of CMR to the subject when the expression levels of the set of genes are above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (iv), (v), or (vi).

[0108] Some embodiments provide that methods for treating CMR of kidney transplant in a subject include: administering a therapy directed to treatment or management of cmr to a subject who has been determined to have expression levels of the set of genes above respective reference levels, wherein the set of genes comprises, is selected from a group consisting of, or consists of, those listed in set (iv), (v), or (vi).

[0109] In various implementations of one or more treatment methods disclosed herein, the detection step, and/or the result acquisition step, may be performed more than once, e.g., periodically, before and/or after a therapy is initiated, continued, or discontinued. For example, one or more treatment methods disclosed herein may further include a repeated detection step and/or a repeated result acquisition step. The repeated detection step and/or result acquisition step may be 2 weeks, 1 month, 2 months, 3 months, or 6 months from the last or most recent detection or result acquisition, and/or may be carried out for an extended length of time such as 3-6 months, 6-12 months, 1-3 years, 3-5 years, 5-10 years, or longer than 10 years after the transplantation. In some implementations, detecting or acquiring an analysis result showing a lower expression level of the set of genes compared to a previous detection or analysis result indicates that the severity of ABMR or CMR has lessened, and the treatment methods may continue administering the directed therapy. In some implementations, detecting or acquiring an analysis result showing that the set of genes (preferably set (iii) or (vi)) are not above respective reference levels indicates that there may no longer be ABMR or CMR, and the treatment methods may discontinue administering the directed therapy. In other implementations, detecting or acquiring an analysis result in a subject showing that the set of genes are once again above respective reference levels indicates reoccurrence of the ABMR or CMR, and the treatment methods may resume administering a directed therapy for management or treatment of ABMR or CMR.

Prognosis/Diagnosis Methods [0110] Various embodiments provide for a method of providing prognosis of kidney allograft outcome, and/or providing diagnosis of transplant rejection, for a human subject having received the kidney transplant, comprising: obtaining a biopsy sample of the kidney transplant from the human subject, and measuring expression levels of each gene in a set of genes in the biopsy sample.

[OHl] In various implementations, the set of genes comprises, or is selected from a group consisting of:

(i) KLRF1, CCL3, CCL4, SH2D1B, and CD160,' or

(ii) KLRF1, FGFBP2, GNLY, and SH2D1B,' or

(iii) KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, and optionally further including IFNG,' or

(iv) /Z70, IL21R, SIRPG, ETV7, STAT1, and CCZS; or

(v) CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,' or

(vi) IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3,- wherein a prognosis or diagnosis of antibody-mediated rejection (AB MR) of the kidney transplant is indicated when the expression levels of each gene in any one of sets (i)-(iii) are above a reference value, and wherein a prognosis or diagnosis of cell-mediated rejection of the kidney transplant is indicated when the expression levels of each gene in any one of sets (iv)- (vi) are above a reference value.

[0112] In some implementations, the prognosis or diagnosis method includes measuring expression levels of each gene in: (iii) KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, optionally further including IFNG,' and in (vi) IL 10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3, wherein a prognosis or diagnosis of antibody-mediated rejection (ABMR) of the kidney transplant is indicated when the expression levels of each (or some) of KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY (and IFNG if measured) are above a reference value, or wherein a prognosis or diagnosis of cell-mediated rejection (CMR) of the kidney transplant is indicated when the expression levels of each (or some) of IL 10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3 are above a reference value.

[0113] In some implementations, these measurement or prognosis methods are performed in combination with a pathology analysis (e.g., histological analysis according to Banff standards) to determine AMBR or CMR.

Systems for Assays [0114] Various embodiments provide for a system for detection or measurement of expression levels of a set of genes indicative of antibody-mediated rejection (AB MR) of kidney allograft, comprising a plurality of reagents, preferably each reagent specifically binding to a different gene in the set or binding to a protein encoded by a different gene in the set, wherein the set of genes comprises, or is selected from a group consisting of, or consists of:

(i) KLRF1, CCL3, CCL4, SH2DIB, and CD160,- or

(ii) KLRF1, FGFBP2, GNLY, and SH2D1B,- or

(iii) KLRF1, CCL3, CCL4, SH2DIB, CD160, FGFBP2, and GNLY, optionally further including IFNG.

[0115] In further implementations, a system for detection or measurement of expression levels of a set of genes indicative of antibody-mediated rejection (AB MR) of kidney allograft, comprising a plurality of reagents, each reagent specifically binding to a different gene in the set or binding to a protein encoded by a different gene in the set, wherein the set of genes comprises, or is selected from a group consisting of, or consists of: a housekeeping gene (e.g., GAPDEL), FWF, or both; and

(i) KLRF1, CCL3, CCL4, SH2DIB, and CD160,' or

(ii) KLRF1, FGFBP2, GNLY, and SH2D1B,' or

(iii) KLRF1, CCL3, CCL4, SH2DIB, CD160, FGFBP2, and GNLY, optionally further including IFNG.

[0116] Other embodiments provide for a system for detection or measurement of expression levels of a set of genes indicative of cell mediated rejection (CMR) of kidney allograft, comprising a plurality of reagents, each reagent specifically binding to a different gene in the set or binding to a protein encoded by a different gene in the set, wherein the set of genes comprises, or is selected from, or consists of:

(iv) /Z70, 11.21 R, SIRPG, ETV7, STAT1, and CCZS; or

(v) CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,- or

(vi) IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0117] In further implementations, a system for detection or measurement of expression levels of a set of genes indicative of cell mediated rejection (CMR) of kidney allograft, comprising a plurality of reagents, each reagent specifically binding to one of the genes in the set or binding to a protein encoded by one of the genes in the set, wherein the set of genes comprises, or is selected from, or consists of: a housekeeping gene (e.g., GAPDH), and

(iv) 7L70, IL21R, SIRPG, ETV7, STAT1, and CCZS; or

(v) CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,- or

(vi) ILIO, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3.

[0118] Additional embodiments provide for a system for detection or measurement of expression levels of a set of genes, which may be indicative of rejection (AB MR and/or CMR) of kidney transplant, comprising a plurality of reagents, each reagent specifically binding to a different gene in the set or binding to a protein encoded by a different gene in the set, wherein the set of genes comprises, or is selected from, or consists of: at least one of:

(i) KLRF1, CCL3, CCL4, SH2D1B, and CD160,- or

(ii) KLRF1, FGFBP2, GNLY, and SH2D1B,- or

(iii) KLRF1, CCL3, CCL4, SH2D1B, CD160, FGFBP2, and GNLY, optionally further including IFNG,' at least one of:

(iv) 7L70, 11.21 R, SIRPG, ETV7, STAT1, and CCZS; or

(v) CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3,- or

(vi) IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3,- and optionally a housekeeping gene.

[0119] Further embodiments provide that a system for detection or measurement of expression levels of a set of genes comprising a plurality of reagents, each reagent specifically binding to a different gene in the set or binding to a protein encoded by each different gene in the set, wherein the set comprises any of (i)-(vi), a housekeeping gene, and any one or more additional genes listed in Table 4, 7, 8, or 12.

[0120] In some aspects, the system is for detection or measurement of transcription levels of the set of genes, wherein each of the plurality of reagents specifically binds to a different one of the genes in the set, and each of the plurality of reagents comprise a polynucleotide fully complementary to at least a portion of a different one of the genes in the set. Primers or fully complementary polynucleotide sequences of a known gene can be devised through one or more tools, such as Primer-BLAST. In some implementations, nucleic acid probes for quantifying expression of a gene are obtained from vendors like Thermofisher for a TaqMan assay, such as assay IDs: Hs01044622_ml for detection o KLRFl, Hs00234142-ml for CCL3, Hs00237011-ml for CCL4, Hs01592483-ml for SH2D1B, Hs00199894-ml for CD160,- and Hs00961622-ml for IL10, Hs00222310-ml for IL21R, Hs03043809-ml for SIRPG, Hs00903229-ml for ETV7, Hs01013996-ml for STAT1, and Hs04187715-ml for CCL8. In some implementations, each gene probe is placed in a different well in a multi-well plate for qPCR to quantify the expression of the gene in an aliquot of a sample.

[0121] In some aspects, the system is for detection or measurement of protein expression levels of the set of genes, wherein each of the plurality of reagents specifically binds to a protein encoded by a different one of the genes in the set. For example, in a microwell device/plate for an enzyme-linked immunosorbent assay (ELISA), capture antibodies specific for each of the proteins encoded by the gene set can be immobilized in the device, and upon binding by target proteins in the sample, detection antibodies for the target proteins can be added to the device for signal amplification and/or quantification. ELISA antibody pairs for a gene can be searched for or is available in different vendors. In other implementations, protein expression levels are analyzed via immunohistochemistry with antibodies available by vendors.

[0122] Further embodiments provide the systems are assay systems, which may comprise an assay surface such as a chip, array, or fluidity card, or well plate, or the like, and the reagents specifically binding to a different gene in the set or binding to a protein encoded by each different gene in the set.

[0123] Exemplary reagents or molecules which specifically bind the marker (gene or polypeptide encoded by the gene), e.g., binding ligand, include but are not limited to antibodies, aptamers and antibody derivatives or fragments.

Samples

[0124] In various embodiments, the methods and/or the systems disclosed herein are suitable for a biopsy sample/ specimen of a graft kidney (e.g., a kidney allograft), or a transplant biopsy. For example, a biopsy is performed with a small gauge needle (optionally facilitated with a bioptic device) to remove one or more tiny pieces of kidney guided by ultrasound pictures, so as to obtain a biopsy or specimen of the transplanted kidney. Preferably, adequate renal cortex is obtained/sampled in graft biopsies for pathologic observations (e.g., histological assessment). Banff criteria define adequate cortical sampling as a specimen that contains at least 10 glomeruli and two arteries. Preferably at least two tissue cores are obtained, preferably at some distance from each other, to optimize sampling of pathologic processes such as rejection and infection, which may be focal in the allograft. [0125] In some aspects, the biopsy sample or specimen is of a kidney allograft after transplantation into the recipient. In another aspect, the biopsy sample or specimen is of a kidney allograft before transplantation into the recipient. In some aspects, the biopsy sample or specimen is of a kidney allograft obtained at 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, or at one or more of these times points, after the kidney allograft is transplanted into the recipient. In further aspects, the biopsy sample or specimen for the methods and/or the assay systems is a fresh kidney allograft specimen obtained from a kidney transplant recipient, which is used in the methods or the assay systems within 2 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, or 3 days from time of biopsy collection, or directly used without freezing. In other aspects, the biopsy sample or specimen for the methods and/or the assay systems is a frozen specimen, which is stored for 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 2-5 years, 5-10 years, 10-15 years, or 15-20 years, or longer, until measurement in the methods or the assay systems.

[0126] In various aspects, a reference value of an expression level of a gene is the expression level of the gene in a sample obtained from an individual that does not have a pathology of rejection, or an individual not showing any symptoms of rejection, of a kidney transplant.

[0127] In other aspects, a reference value of an expression level of a gene, for the expression level of the gene in a sample obtained from an individual having a pathology/diagnosis/prognosis of ABMR, is the expression level (or averaged expression level) of the gene in a sample (or a pool of samples) obtained from an individual not having ABMR (or a pool of individuals not having ABMR). In one aspect, a reference value of an expression level of a gene, for the expression level of the gene in a sample obtained from an individual having a pathology/diagnosis/prognosis of ABMR, is the expression level (or averaged expression level) of the gene in a sample (or a pool of samples) obtained from an individual with no rejection of the kidney transplant (or a pool of individuals with no rejection of the kidney transplant). In another, a reference value of an expression level of a gene, for the expression level of the gene in a sample obtained from an individual having a pathology/diagnosis/prognosis of ABMR, is the expression level (or averaged expression level) of the gene in a sample (or a pool of samples) obtained from an individual with cell-mediated rejection of the kidney transplant (or a pool of individuals with cell-mediated rejection of the kidney transplant). [0128] In some aspects, a reference value of an expression level of a gene, for the expression level of the gene in a sample obtained from an individual having a pathology/diagnosis/prognosis of CMR, is the expression level (or averaged expression level) of the gene in a sample (or a pool of samples) obtained from an individual not having CMR (or a pool of individuals not having CMR). In one aspect, a reference value of an expression level of a gene, for the expression level of the gene in a sample obtained from an individual having a pathology/diagnosis/prognosis of CMR, is the expression level (or averaged expression level) of the gene in a sample (or a pool of samples) obtained from an individual with no rejection of the kidney transplant (or a pool of individuals with no rejection of the kidney transplant). In another, a reference value of an expression level of a gene, for the expression level of the gene in a sample obtained from an individual having a pathology/diagnosis/prognosis of CMR, is the expression level (or averaged expression level) of the gene in a sample (or a pool of samples) obtained from an individual with antibody -mediated rejection of the kidney transplant (or a pool of individuals with antibody-mediated rejection of the kidney transplant).

[0129] Additional examples of biopsies or biological samples include but are not limited to body fluids, cells in body fluids, free RNA and protein in body fluids such as but not limited to urine, whole blood, and/or plasma. In other aspects, biopsy samples are extracted nucleic acid components or protein components from kidney allograft.

Detection/Measurement techniques

[0130] In various embodiments, measurements or detection of gene expression can be performed by extracting RNA/DNA from tissue specimens, obtaining polypeptides (including proteins) from the biological sample, measuring UV absorption with a spectrophotometer, gel electrophoresis coupled with biochemical or luminescent quantification, and/or whole/partial genome amplification. In some embodiments, the expression levels are mRNA transcription levels, which can be quantified in one or more techniques such as PCR or electrophoresis. In various implementations, when using qPCRto quantify gene expression by measuring the level of mRNA, total RNA needs to be extracted from the experimental sample and the mRNA is required to be converted into complementary DNA (cDNA) through reverse transcription, and then used as the template for the qPCR reaction. Techniques for RNA extraction, reverse transcription, and performing qPCR assay are generally known in the art, such as described in review paper PLoS One, 2018, 13(5):e0196438.

[0131] In some embodiments, the expression levels are protein expressions levels measured using one or more of these techniques. EXAMPLES

[0132] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

EXAMPLE 1. Analysis of mRNA Gene Transcripts Associated with Rejection in Kidney Allograft Biopsies.

[0133] Quality and Quantity of RNA Extracted from Biopsy Samples

[0134] Among 133 biopsies included in this study, RNA extracted from randomly selected 50 biopsies were tested for quantity and quality. The average total RNA yield was 27.5+33.9 ng, and the average RIN was 2.89+1.22 (Fig. 2). In pilot experiments, we found that mRNA transcripts of - 50% of tested genes might not be detected in a biopsy RNA sample if the Ct value of reference gene (GAPDH) in the final RT-qPCR was higher than 28. Based on this, we determined that all 133 biopsies included in this study had the Ct value of GAPDH below 28 in their extracted RNA for further analysis.

[0135] AMR- and/or CMR-Specific Gene Identification and Related Gene Scores in a Discovery Cohort

[0136] Thirty-eight biopsies from 38 patients (discovery cohort 1, Fig. 1) were used for the preliminary screening of all 101 candidate genes (Table 4). These biopsies included 5 with active/chronic active AMR, 5 mixed rejection of chronic active AMR plus acute CMR, 8 active/ chronic active AMR plus either borderline acute CMR (n=6) or non-acute chronic active CMR (n=2), 10 acute CMR, and 10 No Rejection. When all 5 groups are compared by KW test, 52 genes showed p<0.1. These 52 genes were then tested in the additional 95 biopsies.

[0137] To identify the genes associated with significant AMR or CMR activity among these 52 genes, we analyzed a new set of 69 biopsies containing 30 of 38 biopsies in discovery cohort 1 (discovery cohort 2, Fig. 1) with definitive pathologic findings and divided into 4 groups (30 active/chronic active AMR, 14 mixed rejection of active/chronic active AMR plus acute CMR, 14 acute CMR, and 11 no rejection). The mRNA transcripts levels of the 52 tested genes were compared among the 4 groups by multiple comparison analysis (Table 7). Among these 52 genes, a total of 9, 11 and 6 were identified as AMR, CMR and mixed rejection (AMR+CMR)-associated genes, respectively. An additional 18 genes were identified as being associated with any rejection type (AMR, or AMR+CMR, or CMR) (Tables 7 and 8). The remaining 8 genes evaluated did not exhibit a significant association with any rejection type.

[0138] With this information, we made further selections among these AMR or CMR- associated genes to generate AMR or CMR gene scores for active rejection. Five AMR- associated genes (KLRF1, CCL3, CCL4, SH2D1B, and CI) 160) were selected to generate the AMR gene score, since each of these 5 genes demonstrated expression levels that were at or near significances for predicting AMR and AMR+CMR compared to CMR and the no rejection groups. Specifically, the gene expression of a gene in the AMR samples, or of a gene in the CMR samples, were compared to the gene in no-rejection samples, to identify a difference that has a P value lower or close to significance (p<0.05). This is based on both multiple comparisons analysis of the 4 biopsy groups and binary comparison (AMR & AMR+CMR vs. CMR & no rejection) (Fig. 3A-3E, Tables 7&8). By similar criteria, 6 CMR-associated genes (IL10, IL21R, SIRPG, ETV7, STAT1, and CCL8) were selected to generate the CMR gene score (Fig. 3F-3K). Therefore, of the 24 AMR and 13 CMR-associated candidate genes initially selected for testing (Table 4), only SH2D1B and KLRF1 from the AMR candidate genes (8%), and IL21R and SIRPG from the CMR candidate genes (15%) were included as component genes of the final gene scores for AMR and CMR, respectively.

[0139] We then analyzed receiver operating characteristic (ROC) curves to assess the performance of the 2 gene sets for diagnosis of AMR or CMR in the 69 biopsies from discovery cohort 2. The area under curve (AUC) for the AMR gene set for detecting AMR including AMR+CMR is 0.889 (Fig. 4A-4B), with the best cutoff level (determined by Youden’s index) for diagnosing AMR at 0.49 and above (sensitivity: 84.1% and specificity: 92.0%). The AUC for the CMR gene set for detecting CMR including AMR+CMR is 0.872 (Fig. 4C-4D), with the best cutoff level for diagnosing CMR at 0.92 and above (sensitivity: 71.4% and specificity: 92.7%).

[0140] Performance of Gene Scores in a Test Cohort

[0141] The gene scores were then calculated for the independent 56 biopsies in the test cohort (Fig. 1). The gene score is the averaged gene expression level of the set/panel of genes for the sample type; for example, the gene score for AMR is the average gene expression level of the five AMR-associated genes, whereas the gene score for CMR is the average gene expression level of the six CMR-associated genes. Here, pathologic findings demonstrated inflammation but contained features that were nondeterminative for active/ chronic active AMR and/or acute CMR by Banff 2017 criteria. When the AMR gene set scores were analyzed and correlated with pathologic findings (Table 9), 12 of 20 (60%) with active/chronic active AMR, 6 of 12 (50%) with chronic (inactive) AMR, and all 16 (100%) with suspicious active/chronic active AMR (Table 3), had diagnostic AMR gene scores above 0.49 (Fig. 5 A). Of the remaining 8 biopsies, none demonstrated AMR activity by pathologic and clinical assessments, and all AMR gene scores were below the 0.49 cutoff.

[0142] We then correlated biopsies featuring a pathologic diagnosis of CMR, intermediate or nondeterminative findings with the CMR gene set scores. For the 56 biopsies in the test cohort (Table 10), 8 of 9 (89%) with acute CMR, 9 of 34 (26%) with borderline acute CMR, none of 3 with non-acute chronic active CMR, demonstrated CMR gene scores above the 0.92 cutoff (Figure 5B). The remaining 10 biopsies did not show pathologic features of acute CMR, but pathologic and clinical assessments indicated either chronic AMR or were suspicious active/chronic active AMR. 2 of 10 biopsies (20%) demonstrated CMR gene scores above the 0.92 cutoff and another one was very close (0.88).

[0143] Due to the elevated CMR gene scores in the 3 CMR-free biopsies described above, we then investigated whether AMR activity could contribute to elevation of CMR gene scores in the absence of pathologic findings for any type of CMR including borderline acute CMR. We then analyzed data from 125 biopsies from discovery cohort 2 and test cohort to assess this question. We identified 88 biopsies which had either acute CMR (n=37) or complete absence of CMR activity (11 with no rejection, 40 with any type of AMR) based on their pathologic features and examined their CMR gene scores (Fig. 6A). Of 37 biopsies with acute CMR, 28 (76%) had diagnostic CMR gene scores above 0.92. Among the other 51 CMR-free biopsies, 11 no rejection biopsies all had CMR gene score below 0.92, but among the remaining 40 CMR-free AMR+ biopsies, 5 (13%) had diagnostic CMR gene scores above 0.92. When CMR-specific histologic lesions were compared to biopsies with diagnostic (>0.92, n=5) vs. non-diagnostic (<0.92, n=35) CMR gene scores, no difference was found in terms of either interstitial inflammation (i3 [n=l] or iO [n=39]) or minimal tubulitis (t<l for all 40 biopsies, p>0.99 by MW test) (Fig. 6B). We then determined that the 5 biopsies with diagnostic CMR gene scores all had diagnostic AMR gene scores above 0.49, and their AMR gene scores were also significantly higher than those 35 biopsies with non-diagnostic CMR gene scores (Fig. 6C). Correlation analysis confirmed a significant relationship (p=0.67, p<0.001) between AMR and CMR gene scores in these 40 CMR-free AMR+ biopsies (Fig. 6D).

[0144] In this study, we performed an external validation of genes whose mRNA transcript levels were likely associated with renal allograft rejection, immune activation or inflammation that likely contribute to rejection. Samples were taken from our pathology archives, some of which were nearly a decade old. Despite the relatively low quantity of total RNA extracted from these biopsies, their RIN (1.2-6.9) indicated the RNA quality was in a range similar to RNA extracted from RNAlater-preserved biopsies in a published study (RIN: 1.0-8.8). In recognition of the possible degradation of mRNA in frozen biopsies, we developed protocols to assess the quantity and integrity of mRNA in all biopsies included in this study. This quality control allowed for the generation of quantitatively meaningful gene expression data, allowing detection of significant difference in mRNA transcript levels among the study groups. The feasibility of using archived frozen biopsies in our study had its unique advantages in terms of sample collection, storage, availability and accessibility for molecular diagnosis of renal allograft rejection, compared with previously reported studies. Another advantage of our study protocol is the comparatively low requirement of biopsy total RNA input (as low as 5 ~ lOng). Among the 50 biopsies randomly chosen for RNA yield measurement, the total RNA obtained from 31 biopsies (62%) was less than 20ng and within 20 to 50ng from an additional 11 (22%). This is significantly lower than the usual RNA input requirement of at least lOOng by the Nanostring nCounter system which has been used by multiple studies of renal allograft gene expression using FFPE tissues. However, it is important to note that there have been no direct comparisons of the current mRNA assessment methodologies or inter-laboratory comparisons of mRNA obtained from renal biopsies by different techniques to determine quality assessments and quality control in confirming or supporting pathologic diagnosis.

[0145] Before our studies it was considered challenging to differentiate AMR, CMR and no rejection by gene transcripts. Our study demonstrated that the expression levels of multiple genes in renal allograft biopsies from our patient cohort were associated with pathologic diagnosis of either AMR or CMR. Among the 5 component genes of our AMR gene score, SH2D1B and KLRF1 (identified as AMR-associated genes), and CD 160 (an ADCC- associated gene) all play major roles in natural killer (NK) cell activation and function. CCL3 and CCL4 are CC chemokines involved in inflammatory processes by recruiting various inflammatory cells such as macrophages and NK cells. Thus, the identified 5 gene transcripts in biopsies are consistent with the previous finding of NK cells as key player in AMR and can be used as valuable biomarkers. Surprisingly, none of the 5 endothelial-specific genes (DARC, ECSCR, PECAM1, NOS3, and VWF), all previously reported as AMR-associated genes, showed any significant correlation with AMR in our patient cohort. This could be due to immune modulatory treatments received prior to biopsy that likely alter gene expression profiles. Understanding the molecular and cellular mechanisms of AMR in renal transplant patients remains a work in progress, and multiple types of immune cells and tissues likely play important roles in the pathogenesis of AMR. Most of the AMR biopsies in this study were from HLA-sensitized patients who received desensitization therapy prior to transplant, usually with IVIG + rituximab (either with or without plasma exchange), and then induction therapy posttransplant with lymphocyte-depleting agents. These treatments could impact gene expression and result in observations different than previously reported by other groups. Although we did not see significant expression of a number of reported AMR-specific genes in our study, this could be more thoroughly validated by exchange of blinded samples among laboratories performing molecular analysis of renal biopsy tissue. This would increase robustness of molecular diagnostics and improve the accuracy of the overall assessment of allograft stability and reveal potential targets for therapeutic intervention. In addition, it is also conceived to have an assessment of the immunomodulatory therapies given to each patient pre- and posttransplant.

[0146] The final selected 6 component genes of CMR gene set revealed a number of T cell and/or innate cell-associated genes important in activation, including cytokine or chemokine signaling (IL10, CCL8, and IL21R . T cell adhesion to other immune cells including antigen-presenting cells (SIRPG), and transcription factors (STAT1, ETV7) as significant predictors of CMR. More stringent criteria were applied in selecting the component genes of the CMR gene set compared to the AMR gene score, as stated in the methods. These findings indicate that the cellular and molecular mechanisms of CMR might be simpler than AMR with less variation of gene expression signatures among different patient cohorts. We eventually selected only 6 component genes for the CMR gene score by using more stringent criterion to improve its performance in the discovery cohort with higher AUC.

[0147] Another important finding in this study was the demonstration that the AMR gene set scores exhibited significant utility for detecting AMR activity in 16 biopsies suspicious for active/chronic active AMR (Fig. 5A). They all had significant MVI (g+ptc>2), but most of them lacked a positive C4d and/or DSA, or had acute or borderline acute CMR without glomerulitis (Table 3). All 16 biopsies had diagnostic AMR gene scores above 0.49 (1.20+0.55), strongly indicative of significant AMR activity. The test cohort also included 12 biopsies with the chronic but not active AMR and 6 of these had diagnostic AMR gene scores above 0.49 (1.47+0.65), while 6 did not (0.19+0.11), with no difference in MVI between these two subgroups (g+ptc: 3.0+2.0 vs. 2.8+1.7, p=0.96 by MW test) (Fig. 9A), indicating the presence of significant AMR activity in half of these biopsies. The detection rate for AMR activity in chronic AMR biopsies by our gene set score in this cohort (50%) is very similar to a previous study (58%), thus providing further supporting evidence for the utility of molecular diagnostic tools in detecting AMR activity that may be missed by strict adherence to the Banff 2017 diagnostic criteria. This has critical importance for clinicians since mRNA gene score refinements of Banff diagnostics that improve accuracy of AMR diagnosis allow for a more rational approach to use or avoidance of immune modulatory treatments.

[0148] Among all 125 biopsies included in discovery cohort 2 and test cohort, 64 met Banff 2017 criteria for active/chronic active AMR. Fifteen of these 64 biopsies (23%) had nondiagnostic AMR gene scores below 0.49 (0.22±0.10) (Fig. 9B). Here, an apparent failure to confirm Banff diagnostics may be related to early rejection treatments with immune modulatory agents or very mild AMR activity as indicated by minimal MVI (g+ptc=l), late stage AMR associated with end-stage grafts, or simply the failure in detecting AMR-specific immune activation with our gene set in select patients or settings. Overall, the sensitivity of our AMR gene score for detecting AMR activity still requires verification in larger biopsy/patient cohorts.

[0149] In this study, we found that significant AMR activity may be associated with significant CMR-specific immune activation even when histologic lesions are nondiagnostic for any type of CMR, as 5 of the 40 CMR-free AMR biopsies (13%) had diagnostic CMR gene scores above 0.92 (Fig. 6A). This is not surprising as AMR occurrence might be accompanied by graft-targeting T cell activation. Previous reports indicated that allo-antibody binding to endothelial cells on the graft induces expression of HLA antigens and various adhesion molecules on the endothelial cells, which attract and activate T cells. A recent study of molecular classifiers for CMR diagnosis using FFPE renal allograft biopsy tissues based on RNA-seq platform also showed a very high detection rate (above 60%) of CMR activity in samples categorized as solely AMR from the publicly available datasets. Our data shows that molecular diagnostic tools such as gene sets may help clinicians to recognize and treat early- stage CMR in patients lacking diagnostic pathology findings, particularly when an AMR diagnosis is evident. Finally, three biopsies with non-acute chronic active CMR in the test cohort all had non-diagnostic CMR gene scores (0.66±0.15) (Fig. 5B). Therefore, the significance of CMR activity in non-acute chronic active CMR biopsies remains in question.

[0150] In conclusion, by analyzing mRNA transcript levels of AMR and/or CMR candidate genes in frozen archived biopsies, we identified multiple genes that are specific to either AMR or CMR diagnosis in the biopsies of our patient cohort. Comprehensive gene scores calculated from the mRNA transcript levels of these specific genes exhibited significant utility for detecting AMR or CMR activity, especially when pathologic and clinical findings were ambivalent.

Materials, reagents, and techniques

[0151] Study Subjects and Study Outline

[0152] Archived frozen biopsies (a number of 133 biopsies) obtained from 87 kidney transplant recipients who were transplanted between June 2000 and June 2017 were used for this study (Tables 1 and 2). The biopsies were performed between January 2008 and November 2018.

[0153] Table 1. Demographic and clinical information of 87 patients ’*.

^# 87 patients had 89 transplants from which 133 biopsies were obtained and tested in this study.

¹ Data of 89 transplants. ² Data of 133 biopsies.

³ Average of PRA class I and class II.

Table 2. Breakdown of all 133 biopsies based on their pathology diagnosis of AMR and CMR status.

^# Biopsies included in the discovery cohort 2.

[0154] Of 87 patients, 2 received re-transplants, and the biopsies from both transplants for both patients were included in this study, considered as samples from independent transplants for each patient. Of all 89 transplants, 55 had only 1 biopsy, while the other 34 transplants had 2 or more biopsies (2.3±0.5, range from 2-4) included in this study. The time intervals between 2 biopsies from the same transplant were 16.4±11.6 months, and longer than 3 months in about 90% of the cases.

[0155] We initially analyzed 101 genes whose mRNA transcript levels were previously reported to be associated with pathologic features of renal allograft rejection or with immune activation, inflammation and/or tissue injury, which contribute to rej ection. Using biopsies with definitive pathology diagnosis of AMR, AMR+CMR, CMR or no rejection, we identified AMR or CMR-associated genes. The definitive pathology diagnosis of AMR includes active AMR and chronic active AMR; and the definitive pathology diagnosis of CMR includes acute CMR. Comprehensive gene scores specific for either AMR or CMR were then generated, based on the mRNA transcript levels of identified genes, and evaluated in biopsies whose pathology findings contained components nondeterminative for either active AMR or acute CMR, including suspicious AMR, chronic (inactive) AMR, borderline acute CMR, and chronic active CMR not meeting the Banff criteria for acute CMR (non-acute chronic active CMR).

[0156] Pathologic Assessments of Biopsies

[0157] Pathologic assessments and Banff scores of histologic lesions for all the biopsies were made and assessed by one expert pathologist following the Banff 2017 classification with three exceptions, including diagnosis of borderline acute CMR, rejection diagnosis based on intimal arteritis (v), and evaluation of CMR activity in biopsies diagnosed as chronic active CMR. In addition, 16 biopsies were diagnosed as suspicious active/chronic active AMR based on pathohistologic findings of microvascular inflammation (MVI) and C4d, as well as patients’ clinical history of DSA and biopsy-proven rejection (Table 3). Finally, 12 biopsies were diagnosed as chronic AMR, but not chronic active AMR, per Banff 2017. These biopsies all had either transplant glomerulopathy or peritubular capillary basement membrane multilayering, confirming chronicity of AMR, but they also all had negative C4d, while absence of MVI and DSA did not meet requirements for chronic active AMR diagnosis per Banff 2017.

[0158] Candidate Genes

[0159] We initially assessed 101 candidate genes (Table 4) for preliminary screening in a smaller discovery cohort of 38 biopsies. Here, 24 candidate genes with specificity for AMR and 13 CMR-associated genes were identified using biopsies from renal transplant recipients. We also identified 14 genes associated with chronic AMR- and 14 acute rejection-associated genes identified in previous studies using PBMC from renal transplant recipients stimulated in vitro in mixed lymphocyte cultures (MLR) or with anti-HLA (DSA) antibody dependent cellular cytotoxicity (ADCC). Nineteen candidate genes were identified in ADCC assays while 8 candidate genes were CD8 T memory cell-associated genes. In addition, we selected 6 genes associated with inflammation and three genes associated with IL-6 cell activation and signaling for analysis.

[0160] RNA Extraction, Reverse Transcription, Pre-amplification and RT-qPCR [0161] 5 sections (5 pm per section) of each frozen biopsy were used for total RNA extraction with RNeasy Micro Kit (Qiagen, Hilden, Germany). Due to the restricted specimens and low RNA yield from possible degradation in these archived frozen biopsies, all the RNA extracted from each biopsy was used in the subsequent experiments of reverse transcription, pre-amplification and then the final RT-qPCR for each tested gene. [0162] Quality Control for Biopsy RNA Samples

[0163] Among all 133 biopsies included in this study, RNA quality and quantity were tested for RNA extracted from 50 biopsies. These 50 RNA samples were submitted for measurement of RNA yield and RNA integrity number (RIN), on an Agilent Bioanalyzer 2100 using the Agilent RNA 6000 Pico kit following the manufacturer’s protocol.

[0164] Panel Reactive Antibody (PRA), Anti-HLA Antibody Specificity and DSA [0165] PRA and anti-HLA antibody class I and class II antibody assays were performed at Cedars-Sinai Medical Center HLA Laboratory (ASHI & CLIA approved) using methods previously described by Reinsmoen, N.L., et al., in Transplantation, 2008. 86(6): p. 820-5. Anti-HLA antibodies were detected by either flow quick screen or single antigen Luminex bead assay (Luminex, Austin, TX) in serum samples.

[0166] Data Analysis

[0167] Non-parametric Mann-Whitney U test (MW test) was performed to test the statistical significance if only 2 groups were compared for difference, otherwise multiple comparisons analysis was performed after non-parametric Kruskal-Wallis H test (KW test). Identification of AMR-association for a candidate gene required significantly higher mRNA transcript level in the AMR group than CMR and no rejection groups at once in both pairwise comparisons by multiple comparisons analysis. (The five AMR-associating genes in the set are all higher than CMR and no rejection groups.) If 0.05<p<0.10 was obtained in one of these 2 pairwise comparisons, binary comparison (AMR & AMR+CMR vs. CMR & no rejection) by MW test must have p<0.05. The similar criteria were also applied to identify CMR-, AMR+CMR-, and rejection-associated genes. The gene score for AMR (or CMR) diagnosis was calculated as further described in the paragraph(s) below (Zhang et al., in Transplantation, 2017. 101(10): p. 2419-2428).

[0168] For correlation analysis, Spearman’s Rank Correlation test was performed. Most statistical analysis was performed by Prism 8.0 (GraphPad Software, La Jolla, CA). For comparison of categorical variables, Fisher’s exact test was performed by RStudio 1.2.5001 (RStudio, Inc., Boston, MA). Unless specified, p value <0.05 was considered statistically significant.

[0169] Study Subjects: Of 89 transplants from 87 patients, 23 (26%) were both PRA class I and class II <10% at transplant, 57 (64%) were PRA either class I or class II >10%, and the HLA sensitization status of the other 9 transplants (10%) were unknown. The desensitization protocols used for ABO compatible transplant in HS patients and also for ABO incompatible (ABO-i) transplant in non-HS patients have been reported. Briefly, a standard protocol for HS patients consisted of 2 doses of IVIg (2g/kg) one month apart with one dose of rituximab (1g) in between. The protocol for ABO-i transplant consisted of one dose of rituximab (1g) two weeks prior to initiation of 5-7 sessions of plasma exchange followed by one dose of IVIg (2g/kg). The combination of both protocols was used for HS patients who received an ABO-i transplant. If a negative or acceptable crossmatch was achieved and/or the anti -blood group titer became <1:8 after desensitization, patients proceeded to transplantation. Most patients received induction therapy with lymphocyte depleting agent (alemtuzumab or anti -thymocyte globulin) and some with anti-IL-2 receptor antibody (Table 1). Maintenance immunosuppression consisted of calcineurin inhibitor (tacrolimus or cyclosporine A), mycophenolate mofetil (or everolimus for 2 patients) and steroids. The target levels were dependent on the type of induction as reported elsewhere. All patients received anti-viral prophylaxis with ganciclovir (1.25 mg/kg daily) while inpatient, and then valganciclovir or acyclovir post-transplant for 6 months depending on a risk for viral infection.

[0170] Pathology Diagnosis of Biopsies: Pathology diagnosis and Banff scores of histologic lesions for all the biopsies were made and assessed by one expert pathologist following the Banff 2017 classification with three exceptions as follows. Firstly, per the option given in the Banff 2017 classification, borderline/suspicious for acute TCMR was diagnosed retaining the threshold of at least minor interstitial inflammation (il) in the setting of minimal tubulitis (tl), while for biopsies with more significant tubulitis (t>2), borderline acute TCMR was diagnosed with iO or il. This was done as minimal tubulitis without interstitial inflammation (tl iO) was very common in biopsies with any type of ABMR or suspicion of ABMR in our patients. Secondly, in the Banff 2017 classification, intimal arteritis may be used as a criterion for the diagnosis of either ABMR or TCMR activity, which creates confusion. In this study, v>l lesion was only found in biopsies with tubulointerstitial inflammation of i>l and t>l; therefore, the diagnoses of acute TCMR was made based on the v>l lesion. The last is concerning the Banff 2017 criteria for chronic active TCMR. Due to the continuing debate on TCMR activity in biopsies diagnosed as chronic active TCMR (Banff 2017), to ensure that the TCMR-specific genes identified in this study reflect disease activity, we required cases meeting criteria for chronic active TCMR to also meet criteria for acute TCMR (t>2 and i>2 or v>l) to be considered as active TCMR.

[0171] RNA extraction, reverse transcription, pre-amplification and RT-qPCR: The total RNA extracted from each biopsy was submitted to generate cDNA using High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA), and then the cDNA templates of each biopsy was pre-amplified with the pooled TAQMAN® Gene Expression Assays (Applied Biosystems/Thermo Fisher Scientific) of all the 101 genes in addition to the reference gene of GAPDH with Human GAPD Endogenous Control (Applied Biosystems/Thermo Fisher Scientific), following the standard protocols with TAQMAN® PreAmp Master Mix (Applied Biosystems/Thermo Fisher Scientific). The pre-amplification product was then submitted for RT-qPCR for each tested gene using TAQMAN ® Gene Expression Assay (Table 3) as well as GAPDH, and TAQMAN ® Gene Expression Master Mix (Applied Biosystems/Thermo Fisher Scientific) on QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific). Total RNA prepared from a normal healthy individual’s PBMCs stimulated with phorbol 12- myristate 13 -acetate and ionomycin was included at each RT-qPCR run in the entire study and used as the reference RNA. Because of the high sequence similarities between XCL1 and XCL2 (98%), and between CCL4, CCL4L1 and CCL4L2 (96% between CCL4 and CCL4L1, 100% between CCL4L1 and CCL4L2), the selected assays for XCL1 and CCL4 will detect two and three transcripts, respectively. The expression level of each gene was first normalized to GAPDH using ACt (cycle threshold) method for each RNA sample respectively, and then presented as relative quantity (RQ) to the expression level of the same gene in the reference RNA sample (also normalized to GAPDH) by calculating AACt.

[0172] Data analysis: For the statistical analysis involving more than 2 groups, multiple comparisons analysis was performed by the following procedure. Non-parametric Kruskal- Wallis H test (KW test) was first performed to compare all groups, followed by uncorrected Dunn’s tests for multiple comparisons to obtain p values of each pairwise comparison. After that, multiple comparisons were corrected by controlling the False Discovery Rate (FDR) at 0.05, to determine if each pairwise comparison was a “discovery” with statistically significant difference, and then the FDR-adjusted p values for all pairwise comparisons were reported. For controlling the FDR, the “Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli” was used. The gene score for ABMR (or TCMR) diagnosis was calculated by the following procedure. Briefly, the RQ of each gene in each biopsy was first divided by the average RQ of the same gene among all 133 biopsies included in this study for normalization. The ABMR (or TCMR) gene score of each biopsy was calculated as the arithmetic mean of the normalized RQ values of the selected ABMR (or TCMR) genes from that biopsy. The criteria for selecting component genes to calculate gene scores are as follows: for ABMR gene score, the FDR-adjusted p values of pairwise comparisons between ABMR (or ABMR + TCMR) vs. TCMR (or No Rejection) groups must be all <0.10 when FDR is set at 0.10; for TCMR gene score, those between TCMR (or ABMR + TCMR) vs. ABMR (or No Rejection) groups all <0.05 when FDR is set at 0.05. The best cutoff level of ABMR (or TCMR) gene score for ABMR (or TCMR) diagnosis was selected at the value with the maximal Youden’s index (sensitivity+specificity-1).

[0173] Abbreviations:

AMR, antibody-mediated rejection

ABO-i, ABO incompatible

ADCC, antibody-dependent cellular cytotoxicity

AUC, Area under curve

CCL3, chemokine (C-C motif) ligand 3

CCL4, chemokine (C-C motif) ligand 4

CCL8, chemokine (C-C motif) ligand 8

CCL4L1, chemokine (C-C motif) ligand 4-like 1

CCL4L2, chemokine (C-C motif) ligand 4-like 2

CD8, cluster of differentiation 8

CD 16a, cluster of differentiation 28 a

CDC, complement-dependent cytotoxicity

CMR, T cell-mediated rejection

Ct, cycle threshold

CTLA4, cytotoxic T-lymphocyte-associated protein 4

DARC, atypical chemokine receptor 1 (Duffy blood group)

DSA, donor specific antibody

ECSCR, endothelial cell surface expressed chemotaxis and apoptosis regulator

ETV7, Ets variant 7

FDR, false discovery rate

FFPE, formalin-fixed paraffin-embedded g, glomerulitis

GAPDH, glyceraldehyde 3 -phosphate dehydrogenase

HLA, human leukocyte antigen i, interstitial inflammation

IL 10, interleukin 10

IL21R, interleukin 21 receptor

IL-6, interleukin 6

KLRF1, killer cell lectin-like receptor subfamily F, member 1

KW test, Kruskal-Wallis H test MVI, microvascular inflammation MW test, Mann-Whitney U test NK cells, natural killer cells NO S3, nitric oxide synthase 3 PBMC, peripheral blood mononuclear cells PEC AMI, platelet/endothelial cell adhesion molecule 1

PRA I, panel reactive antibody class I PRA II, panel reactive antibody class II ptc, peritubular capillaritis

RT-qPCR, real time quantitative polymerase chain reaction RIN, RNA integrity number

RQ, relative quantity

ROC, Receiver Operating Characteristic SH2D1B, SH2 domain containing IB SIRPG, signal-regulatory protein gamma STAT1, Signal transducer and activator of transcription 1 t, tubulitis v, intimal arteritis

VWF, von Willebrand factor

XCL1, chemokine (C motif) ligand 1

XCL2, chemokine (C motif) ligand 2

Table 3. Pathology findings, clinical information and AMR gene scores of 16 biopsies with suspicious active/chronic active AMR.

Table 4. One hundred and one (101) candidate genes tested in the preliminary screening with the discovery cohort 1 of 38 biopsies.

¹ The type of rejection the candidate genes are associated with based on a published study of renal allograft biopsies.

² The type of rejection the candidate genes are associated with based on a published study of renal allograft biopsies.

³ The type of rejection the candidate genes are associated with based on a published study of PBMC from renal transplant patients.

⁴ The type of rejection the candidate genes are associated with based on a published study of PBMC from renal transplant patients.

* Comparison of 38 biopsies in 5 different groups (AMR, AMR+CMR, AMR+ borderline CMR, CMR, and No Rejection by pathology diagnosis) by KW test.

Genes with names in bold fonts were selected for further analysis based on p<0.1 by KW test.

Table 5. Demographic and clinical information of 38 biopsies from 38 patients in the preliminary screening of 101 candidate genes.

* Including 2 biopsies with chronic active CMR not meeting Banff criteria for acute CMR.

^# cgla and cglb were converted into 1.0 and 1.5 respectively for calculation.

Table 6. Demographic and clinical information of 69 biopsies with definitive pathology diagnosis in the discovery cohort 2 from 58 patients.

Table 7. Fifty-two (52) tested genes and their statistical analysis results in 69 biopsies of discovery cohort 2.

* Rejection indicates any type of rejection, either AMR, CMR or mixed rejection of AMR+CMR.

** Comparing discovery cohort 2 of 69 biopsies with definitive pathology diagnosis in 4 different groups (AMR, AMR+CMR, CMR, and No Rejection) by KW test

^# FDR-adjusted p value (FDR=0.05) for pairwise comparison by multiple comparisons analysis of all 4 different groups.

^## FDR-adjusted p value (FDR=0.10) for pairwise comparison by multiple comparisons analysis of all 4 different groups.

Table 8. Forty-four (44) genes identified with rejection specificity in 69 biopsies of discovery cohort 2.

* Rejection indicates any type of rejection, either AMR, CMR or mixed rejection of AMR+CMR.

** Comparison between 2 groups by MW test as indicated in the left column. Table 9. Demographic and clinical information of 56 biopsies categorized by AMR activity in the test cohort from 41 patients.

¹ Average of PRA class I and class II. Table 10. Demographic and clinical information of 56 biopsies categorized by CMR activity in the test cohort from 41 patients.

¹ Average of PRA class EXAMPLE 2. Additional assessment of genes to calculate gene scores.

[0174] We completed analysis of gene expression data of 71 genes from 349 biopsies (200 patients) in total. 136 biopsies with definitive pathology diagnosis from 136 patients were selected for discovery and validation cohorts.

[0175] There were 71 biopsies in discovery cohort (21 ABMR, 13 ABMR+TCMR, 12 TCMR, 25 no rejection), and 65 biopsies in validation cohort (21 ABMR, 8 ABMR+TCMR, 11 TCMR, 25 no rejection).

[0176] Results showed there were 9 genes whose expression levels were associated with pathologic diagnosis of ABMR, and 4 of them were selected for gene score calculation, i.e., KLRF1, FGFBP2, GNLY, and SH2D1B, (Fig. 7A-7C). There were 9 genes whose expression levels were associated with pathologic diagnosis of TCMR, and 6 of them were selected for gene score calculation, i.e., CTLA4, SIRPG, ADAMDEC1, IL21R, CD8A, and TNIP3, (Fig. 8A-8C). Over 20 additional genes were identified as associated with any rejection. [0177] After merging with the gene sets identified and selected for gene score calculations in Examples 1 and 2, there are 7 genes associated with ABMR, namely KLBF1, CCL3, CCL4, SH2D1B, CD 160, FGFBP2, and GNLY,' and 10 genes associated with TCMR, namely, IL10, IL21R, SIRPG, ETV7, STAT1, CCL8, CTLA4, ADAMDEC1, CD8A, and TNIP3. [0178] Observations of ABMR gene score:

[0179] ABMR-associating gene score showed a very decent specificity in the additional test cohort, but the sensitivity appeared to be affected by various factors, including minimal MVI (g+ptc=l), strong immune suppression, and rejection treatment. Pulse steroids, rituximab, tocilizumab, clazakizumab all may have significant effects on expression of ABMR- associating genes.

[0180] For late-stage grafts (within 3-4 months to graft failure), the majority of biopsies with (+) ABMR pathology findings showed (-) ABMR-associating gene scores, confirming the findings by Halloran’s group, while the same finding was not observed for TCMR-associating gene scores in late-stage grafts with TCMR.

[0181] Among all 349 biopsies included in this study, 10 of 14 (71%) ABMR biopsies (including chronic inactive ABMR and suspicious ABMR) from late-stage grafts had (-) ABMR gene scores. (-) ABMR or TCMR gene scores are close to the diagnostic threshold.

[0182] Strong immune suppression within 2-3 weeks immediately after transplantation, often wiped out any (+) gene expression signatures for both ABMR and TCMR. Similar findings were also observed within 2-3 days right after rejection treatment with pulse steroids, but seemingly with less frequency and intensity. [0183] In the test cohort, 29 of 74 (39%) biopsies with active/chronic active ABMR had (-) ABMR gene scores. Among these 29 biopsies, 4 (5%) either had MVI=1 or barely met the criteria for active ABMR; 2 more (3%) were from within 10 days after transplantation; 5 more (7%) were from late-stage grafts; 6 more (8%) were done immediately after pulse steroids or after extensive ABMR treatment (Ritux, TCZ or CLAZA); 3 more (4%) were from patients who had very stable renal function both before and after biopsies with/without ABMR treatment. For the remaining 9 biopsies (12%) with (+) ABMR pathology but (-) ABMR gene scores, no good explanation could be identified.

[0184] Meanwhile, biospies with (-) ABMR pathology but (+) ABMR gene scores were usually from patients who had extensive history of ABMR, or only TCMR history with very short graft survival, and in some cases, no rejection pathology findings or only borderline TCMR but with (+) DSA.

[0185] Close to 50% of the chronic inactive ABMR biopsies, and about 2/3 of the suspicious ABMR biopsies had (+) ABMR gene scores in the test cohort.

[0186] Observations of TCMR gene score:

[0187] TCMR gene score showed good sensitivity in the test cohort ([+] in 22 of 27 [81%] definitive TCMR biopsies [t>2 and i>2], plus 2 more cases very close to being [+]).

[0188] TCMR-free ABMR biopsies often showed (+) TCMR gene scores. In the test cohort, 9 of 22 TCMR-free biopsies (41%) with active/chronic active ABMR, 6 of 24 TCMR- free biopsies (25%) with either chronic inactive ABMR or suspicious ABMR, had (+) TCMR gene scores.

[0189] Biopsies with inflammation unrelated to rejection (GN, bacterial/viral infections, etc.) also often showed elevated TCMR gene scores.

Table 11. Performance of Gene Scores (GS) in Biopsies from the Test Cohort.

(+) ABMR or TCMR gene scores, shown in bold;

(-) ABMR or TCMR gene scores, but close to the diagnostic threshold, shown in italics,

Infections appeared to have less effect on ABMR gene expression than TMCR gene expression.

Table 12. Candidate Genes in Biopsy Gene Expression Study

EXAMPLE 3.

[0190] Figure 10A and 10B show the result from 31 ABMR biopsies compared to 32 no-rejection biopsies. The 31 ABMR biopsies were determined via pathology assessment. VWF was used as an internal quality control for biopsies, as VWF was expressed by renal epithelial cells.

Table 13. Data shown in figures 10A and 10B.

Table 14-1. Gene score of each gene of interest and of VWF in 31 ABMR biopsies. (Gene score below 0.025 is considered as non-detectable, i.e., five biopsies being VWF non- detectable; VWF was detected by qPCR in (31 -5)/32 = 83.8% of the ABMR biopsies.)

Table 14-2. Gene score of each gene of interest and of VWF in 32 no-rejection biopsies. (Gene score below 0.025 is considered as non-detectable, i.e., three biopsies being VWF non- detectable; VWF was detected by qPCR in (32-3)/32 = 90.6% of the no-rejection biopsies.)

[0191] Each ABMR biopsy was also analyzed according to the Banff scoring system for ‘glomerulitis (g)’, ‘eg’, ‘peritubular capillaritis (ptc)’, ‘C4d’, ‘t’, ‘i’, ‘v’, ‘ci’, ‘ct’, ‘i-IFTA’, and ‘ti’. The ABMR biopsies were categorized by a ‘g+ptc’ score of (1) 0, 1, or 2, denoted as “0 — 2”; (2) 3 or 4, denoted as “3 — 4”; and (3) 5 or 6, denoted as “5 — 6”; and the gene score for each gene of interest (the marker genes for ABMR) in each class of ‘g+ptc’ score was plotted in FIG. 11 A, to evaluate the correlation between a marker gene’s gene score and its ‘g+ptc’ score. For example, figure 11B shows that CCL4 gene score is inversely correlated with the ‘g+ptc’ score, i.e., a biopsy showing ABMR pathology with a higher CCL4 gene score would generally have a lower ‘g+ptc’ score, compared to a biopsy showing ABMR pathology with a lower CCL4 gene score. Figure 11C shows a ‘V’ shape trend of the CCL3 gene score in ABMR biopsies with increasing ‘g+ptc’ score. Generally, the ‘g+ptc’ score is used to indicate the severity of microvascular inflammation (MVI) from a histological standpoint.

[0192] A large pool (as shown in Example 1) of biopsies were assayed, and the gene expression levels were analyzed in JMP® software.

Table 15. The mean gene expression levels of a large pool of biopsies performed via JMP® software.

[0193] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0194] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0195] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).