FIELD OF THE INVENTION

The invention relates to the diagnosis of autoimmune disorders, and pertains to methods, uses, products, biomarkers and kits for diagnosing, identifying or stratifying patients with an autoimmune disorder. In particular, the invention relates to a kit, product, biomarker, use or method for diagnosing or stratifying Sjogren Syndrome.

BACKGROUND TO THE INVENTION

Autoimmune diseases occur when the immune system mounts an undesired response to a self-antigen. These diseases are characterized by the presence of autoreactive antibodies that arise spontaneously. Autoantibodies are generated as a result of dysregulation of the immune system, in particular the germinal centres.

Germinal centres (GCs) are the main sites of generation of high affinity, antibody-secreting plasma cells and Ig class- switched memory B-cells during T-cell-dependent immune responses. Within the GC resides a specialized subset of CD4 ⁺ T cells - the T follicular helper (Tfh) cells - which are essential for GC development and function (1, 2). It is now clear that Tfh cells play a central role in productive vaccine responses, while defects in their formation or function can contribute to immunodeficiency or autoimmunity (3, 4). Dysregulated GC reactions in secondary and tertiary lymphoid organs underlie the generation of self-reactive autoantibodies and many aspects of autoimmune diseases (5,6). GC reactions are orchestrated mainly by follicular helper T (Tfh) cells and T follicular regulatory (Tfr) cells. Tfh cells provide cognate help to B cells, thus promoting their clonal selection and affinity maturation (7).

More recently, the discovery of T follicular regulatory (Tfr) cells, a subset of suppressive regulatory T cells that participate in the GC, added an additional layer of complexity in the biology of GC responses (5-8). Tfr cells are believed to have the potential to regulate and limit the GC reaction assuring antigen-specific antibodies are produced while preventing the generation of auto-antibodies (8). To date, the mechanisms of Tfr cell functions have not been fully elucidated. Tfr cells are generally defined by Bcl-6 ⁺ CXCR5 ⁺ PD-l ⁺ ICOS ⁺ Foxp3 ⁺ and are a distinct subset of thymic Foxp3+ regulatory T cells (Tregs) present in lymphoid tissues. Like the Tfh cell differentiation pathway, Tfr cell-commitment requires both dendritic cell and B cell interactions, as well as CD28, SAP, ICOS, and PD-1 signaling (10, 11, 14). A tight balance between expression of transcription factors Bcl-6 and Blimp- 1 regulates the differentiation of Tfr cells (10). Tfr cells have specialized functions in controlling the magnitude of GC responses and in limiting the outgrowth of non-antigen- specific B cell clones (9, 10). However, the precise mechanisms of Tfr cell suppression remain elusive, although CTLA-4 and regulation of metabolic pathways seem to play a key role (15-17).

Although Tfh and Tfr cells are characterized by their location in lymphoid tissues, an increasing number of studies have described putative circulating counterparts of these cells in peripheral blood. This is particularly relevant for studying the biology of these cells in humans, as access to secondary lymphoid tissues can be limiting. Human blood CXCR5 ⁺ T cells have been established as memory Tfh-like cells, based on the their ability to recapitulate bona fide Tfh cell functions: human blood CXCR5 ⁺ T cells can promote plasmablast differentiation, AID expression and class switch recombination by naive B cells. However, they are phenotypically distinct from tissue Tfh cells and do not express the transcriptional repressor Bcl-6 (18-20). Furthermore, an immunization leading to GC and antibody responses correlates with an increase in the frequency of circulating ICOS ⁺ Tfh cells, suggesting that they indicate ongoing Tfh cell responses in secondary lymphoid tissues (18, 20-23). Human circulating Tfh cells comprise a heterogeneous population concerning their phenotype and the quality of help they provide to B cells (18, 21). In mice, CXCR5 Foxp3 Tfr-like cells were found in peripheral blood after immunization, and shown to represent a circulating counterpart of tissue Tfr cells (13, 14).

Although CXCR5 -expressing Tregs and GC Foxp3 -expressing T cells have been found in humans (11, 24, 25), so far, no study have addressed the biological significance of these putative circulating Tfr-like cells in humans. Human tonsil CD25 CD69- T cells have been shown to directly suppress B cell responses, but the relationship of these putative Tregs to Bcl-6 ⁺ CXCR5 ⁺ PD-l ⁺ ICOS ⁺ Foxp3 ⁺ Tfr cells is unclear (26, 27). Peripheral blood CXCR5 ⁺ Tregs are being studied as circulating Tfr cells in many different human diseases, despite the biological relevance of these cells being unclear (28-32). Additionally, Foxp3 upregulation by non-regulatory human T cells and transient CXCR5 expression by T cells undergoing activation challenge the assumption that peripheral blood CXCR5 ⁺ Tregs are indeed bona fide circulating Tfr cells (33, 34). In the context of diagnostics, particularly in humans, Tfh and Tfr cells are therefore of limited utility as access to secondary and tertiary lymphoid tissues can be limiting. Furthermore, although CXCR5 -expressing Tregs and GC Foxp3 -expressing T cells have been found in humans, no studies have addressed the biological significance of these putative circulating Tfr-like cells.

Tfr cells are thought to play a role in the regulation of GCs, but to date their significance in autoimmune disease remains unclear.

SUMMARY OF THE INVENTION

The present inventors have found that human blood Tfr cells, defined as CXCR5+ Foxp3+ T cells, are generated in peripheral lymphoid tissues as humoral immune responses are established. In contrast to tissue Tfr cells and conventional CXCR5- Tregs, circulating Tfr cells have a na ^' ive-like phenotype. The present inventors have demonstrated that blood Tfr cells are generated following the initial steps, that lead to germinal centre responses being distinct from tissue Tfr cells. Unexpectedly, they have also found a striking increase in the level of circulating Tfr cells in subjects with autoimmune diseases as compared to age- matched healthy donors. The present invention therefore stems from the inventors' surprising discovery that T follicular regulatory (Tfr) cells, particularly a population identified as CD4 ⁺ CXCR5 Foxp3 T, are useful as biomarkers for autoimmune diseases. The invention therefore provides methods of diagnosing autoimmune diseases, biomarkers of autoimmune diseases and kits for the detection of autoimmune diseases.

In a first aspect, the invention provides a method of diagnosing an autoimmune disease in a subject.

In a specific embodiment, said method comprises: obtaining a sample from a subject; detecting the presence or absence of an elevated level of CD4 ⁺ CXCR5+ Foxp3+ T cells in the sample; and diagnosing the patient with an autoimmune disease when the presence of an elevated level of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells in the sample is detected.

In all embodiments of the invention, an elevated level of CD4 ⁺ CXCR5+ Foxp3+ T cells in the sample is a level of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells in the sample from a subject that is at least 1.5 times greater than in a sample from a matched control subject, for example an age- matched healthy donor. For example, the elevated level may be 2, 2.5, 5, 10 or more times greater than in a matched control subject. A matched control may be matched for the species, gender, age and/or any other relevant factor. The elevated level may also be in reference to a reference or standard level of CXCR5 ⁺ Foxp3 ⁺ T cells. For example, the elevated level may be at least 1.5 times greater than a reference level for the subject. For example, the elevated level may be 2, 2.5, 5, 10 or more times greater than the reference level. An elevated level may also be determined by reference to an absolute cut-off value. For example, a level of blood CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells in excess of 15,000/ml, 20,000/ml, 30,000/ml, 50,000/ml or higher may be considered to be an elevated level.

In a further embodiment, the invention provides a method of diagnosing an autoimmune disease in a subject, said method comprising: obtaining a sample from a subject; determining the level of circulating Tfr cells in the blood; and diagnosing the patient with an autoimmune disease when the level of circulating Tfr cells in the blood is elevated. In a specific embodiment, Example 1 demonstrates the unexpected finding that there is an increased frequency of circulating Tfr cells in the exemplary autoimmune disease Sjogren Syndrome (SS) compared to age-matched healthy donors.

In all embodiments of the invention, an elevated level of Tfr cells in the sample is a level of Tfr cells in the sample from a subject that is at least 1.5 times greater than in a sample from a matched control subject, for example an age-matched healthy donor. For example, the elevated level may be 2, 2.5, 5, 10 or more times greater than in a matched control subject. A matched control may be matched for the species, gender, age and/or any other relevant factor. The elevated level may also be in reference to a reference or standard level of Tfr cells. For example, the elevated level may be at least 1.5 times greater than a reference level for the subject. For example, the elevated level may be 2, 2.5, 5, 10 or more times greater than the reference level. An elevated level may also be determined by reference to an absolute cut-off value. For example, an elevated level of Tfr cells in the sample may be present when greater than 20%, 25%, 30%, 35%, 40% or 50% of Tregs in the sample are CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ Tfr cells.

In a further embodiment, the invention provides a method of diagnosing an autoimmune disease in a subject, said method comprising: obtaining a sample from a subject; determining the ratio of Tfr/Tfh cells in the sample; and diagnosing the patient with an autoimmune disease when the ratio of Tfr/Tfh cells is greater than 0.2. The ratio of Tfr/Tfh cells may be greater than 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 or higher.

The ratio of Tfr/Tfh cells in the sample can depend on the method of measurement of the Tfr and Tfh cells. Different gating strategies can give rise to different values for the ratio of Tfr/Tfh ratio. The gating strategy used when determining the Tfr/Tfh ratio in the Examples is as shown in Figure 4. Briefly, cells were gated on CD4 ⁺ T cells, wherein the CD4 ⁺ cells were stained for CD25 and Foxp3. Cells that were CD25 ⁺ Foxp3 ⁺ (double positive) and CD25- Foxp3- (double negative) were gated. Double positive cells were stained for CD4 and CXCR5. CD4 ⁺ CXCR5 ⁺ cells were gated to isolate CXCR5 ⁺ CD25 ⁺ Foxp3 ⁺ CD4 ⁺ Tfr cells. Double negative cells were stained for CD45RO and CXCR5. CD45RO ⁺ CXCR5 ⁺ cells were gated to isolate CXCR5 ⁺ CD45RO ⁺ CD25-Foxp3-CD4 ⁺ cells Tfh cells.

Alternatively, when the ratio of Tfr/Tfh cells is measured according to the method described in Verstappen et al (Arthritis Rheumatol. 2018 Mar 13. doi: 10.1002/art.40488) the threshold for diagnosing a patient with an autoimmune disease is 0.1. In this case, the different method of measurement returns a different value for the ratio of Tfr/Tfh cells when compared to the methods used in the Examples section of the present invention.

An increased Tfr/Tfh ratio is associated with subjects who have serum autoantibodies. The invention therefore also provides a method of identifying subjects with serum autoantibodies comprising obtaining a sample from a subject; determining the ratio of Tfr/Tfh cells in the sample; and diagnosing the patient with an autoimmune disease when the ratio of Tfr/Tfh cells is greater than 0.2. The ratio of Tfr/Tfh cells may be greater than 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 or higher.

An increased Tfr/Tfh ratio is associated with subjects who have specific histopathological changes associated with an autoimmune disease. The invention therefore also provides a method for stratification of subjects with the same underlying autoimmune disease into groups, wherein the groups are characterized by presence of histopathological changes. For example, when the autoimmune disease is Sjogren Syndrome (SS) the histopathological changes may include focal sialedenitis in salivary gland biopsies, or ectopic lymphoid structures in inflamed tissues. The invention provides method for stratification of subjects with the same underlying autoimmune disease into groups comprising: a obtaining a sample from a subject; determining the ratio of Tfr/Tfh cells in the sample; and stratifying the subject with an autoimmune disease in different groups based on the ratio of Tfr/Tfh cells. For example, the subjects may be stratified into groups based on whether the ratio of Tfr/Tfh cells is greater than 0.2 or less than 0.2. The subjects may also be stratified into bands based on the ratio of Tfr/Tfh cells. For example, subjects may be stratified into groups that have a ratio of Tfr/Tfh cells from 0.2 to 0.3, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6 or greater than 0.6. In a further embodiment, the groups may be 0.2 to 0.5, 0.5 to 0.8 and greater than 0.8. The greater the ratio of Tfr/Tfh cells, the greater the likelihood of the presence of histopathological changes.

This embodiment of the invention provides a clinician with the ability to stratify subjects using a blood test, thereby avoiding the need to use biopsies, which carry associated risks. The present invention therefore provides a less risky and minimally invasive test for the stratification of subjects with autoimmune disorders. In a specific embodiment of this aspect of the invention, the invention provides a clinician with the ability to stratify subjects with Sjogren Syndrome using a blood test, thereby avoiding the need for salivary gland biopsies which is the conventional method currently used to stratify Sjogren Syndrome.

This aspect of the invention also provides a "companion diagnostic" test to identify a subset of subjects that are most likely to require a particular therapeutic treatment following diagnosis. For example, therapeutic agents that are likely to be of use in subsets of subjects who have a higher ratio of Tfr/Tfh cells and/or active disease include therapies targeting T cell - B cell interactions, such as, therapies targeting T cells, B cells, co-stimulation/immune checkpoints, antigen presentation, or cytokines affecting T or B cell function. Some suitable therapies are set out below. The provision of this companion diagnostic allows for personalised medicine, wherein the patient is identified as likely to benefit from a particular therapy based on their ratio of Tfr/Tfh cells and/or disease state. In certain disclosed embodiments, the patient is treated following the diagnosis or stratification performed according to the invention.

In the present invention, the Tfr cells are CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells. The Tfr cells may be CD4 ⁺ CXCR5 ⁺ PD-1 ^+/- ICOS ^+/- Foxp3 ^+/- CD25 ^+/ T cells. In the present invention Tfr cells may also be CXCR5 ⁺ PD-1 ^+/- ICOS ^+/- CD 127- CD25 ^+/ CD4 ⁺ T cells. In the present invention, the Tfh cells may be CXCR5 ⁺ CD45RO ⁺ Foxp3- CD25- CD4+ T cells. In the present invention, Tfh cells may be CXCR5 ⁺ CD25 CD127 ⁺ CD4 ⁺ T cells. The Tfh cells may be PD-1 ⁺ ICOS ⁺ and/or PD-1 ⁺ CXCR3 GC-like cells.

An elevated level of PD-l ⁺ ICOS ⁺ Tfh cells in a subject may also be used to stratify disease state. For example, an elevated level of PD-1 IC0S ⁺ Tfh cells in a subject is indicative of higher autoimmune disease activity. An elevated level of PD-1 IC0S ⁺ Tfh cells in a subject can be defined as greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more CD4 ⁺ cells also being PD-1 IC0S ⁺ . Alternatively, an elevated level of PD-1 IC0S ⁺ Tfh cells in a subject can be defined as greater than 100 cells/ml, for example greater than 150, 200, 250, 300, 400 or more PD-l ICOS ⁺ Tfh cells/ml in a sample. In a specific embodiment, the sample is a blood sample.

In the context of the invention, "stratification" may also refer to the identification of specific subject populations who will benefit from different approaches to the monitoring and treatment of their autoimmune disease. In the case of Sjogren syndrome, stratification refers to the identification of subjects with an elevated Tfr/Tfh ratio. These subjects are more likely to have histopathological changes including tissue infiltration with ectopic lymphoid tissue, and are more likely to suffer from complications of Sjogren syndrome, including lymphoma, and are more likely to have high levels of autoantibodies. Stratification of Sjogren syndrome also relates to the identification of subjects with more active disease. These subjects have an elevated level of PD-l ⁺ ICOS ⁺ Tfh cells. Disease activity can be measured using the ESSDAI scale, a clinical index designed to measure disease activity in patients with primary Sjogren syndrome. Moderately active disease is usually defined as an ESSDAI >5. An exemplary schematic of the stratification of Sjogren Syndrome is provided in Figure 54.

The present invention also provides a method to identify different subsets of subjects with the same underlying autoimmune condition. For example, in a specific embodiment, the invention provides a method of identifying subgroups of subjects with Sjogren Syndrome, comprising determining the Tfr/Tfh ratio in said subject and detecting presence or absence of an elevated level of PD-1 IC0S ⁺ Tfh cells in said subject. A Tfr/Tfh ratio of greater than 0.2 and a level of PD-1 IC0S ⁺ Tfh cells greater than 100 cells/ml can identify different groups of subjects: (1) Group 1 subjects have an elevated Tfr/Tfh ratio and an elevated level of PD- 1 IC0S ⁺ Tfh cells; (2) Group 2 have a normal Tfr/Tfh ratio and an elevated level of PD- 1 IC0S ⁺ Tfh cells; (3) Group 3 have an elevated Tfr/Tfh ratio and a normal level if PD- l ⁺ ICOS ⁺ Tfh cells; and (4) Group 4 have both Tfr/Tfh ratio and a level of PD-l ⁺ ICOS ⁺ Tfh cells within the normal range. The identification of these groups of subjects is depicted in Figure 51.

In a specific embodiment, the ratio of Tfr/Tfh cells in the sample refers to one of: the ratio of CXCR5 ⁺ CD25 ⁺ Foxp3 ⁺ CD4 ⁺ T cells to CXCR5 ⁺ CD45RO ⁺ CD25 Foxp3-CD4 ⁺ T cells; the ratio of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells to CXCR5 ⁺ Foxp3- CD4 ⁺ T cells; the ratio of CXCR5 ⁺ PD-1 ^+/- ICOS ^+/ - Foxp3 ⁺ CD25+ ^7- CD4 ⁺ T cells to CXCR5 ⁺ CD45RO ⁺ PD-1 ^+/- ICOS ^+/- Foxp3- CD25 CD4 ⁺ T cells; or the ratio of CXCR5 ⁺ CD127 CD25 ⁺ CD4 ⁺ T cells to CXCR5 ⁺ CD45RO ⁺ CD127 ⁺ CD25-CD4 ⁺ T cells.

The sample may be any suitable tissue sample. Suitable samples include blood, plasma, lymphoid tissues, and other tissues with tertiary lymphoid organs. In all embodiments of the invention, the sample may be an ex vivo sample and all methods may be carried out ex vivo, for example in vitro. The subject may be an animal subject, preferably a mammalian subject. For example, in certain embodiments, the subject is a mouse, a rat, a guinea pig, ovine, bovine, porcine, or primate. Typically the subject is a human.

In all embodiments disclosed herein, diagnosis of an autoimmune condition using methods according to the invention may optionally be followed by a step of treating the subject with a suitable therapy for ameliorating the autoimmune disease. In particular, therapeutics that target the T-B interaction are envisaged for use in the invention. Specific therapeutic agents include: B-cell depleting therapies (e.g., rituximab); anti-CD22 antibodies (e.g. Epratuzumab); LTbetaR-Ig (e.g. Baminercept); Rebamipide; glucocorticoids; anti-BAFF mAb (e.g. Belimumab); anti-CD40 mAb; anti-CD28; CTLA4-Fc Ig (e.g abatacept and belatacept); low-dose IL-2; anti IL-2, IL-2 immune complexes, or modified IL-2 molecules; Iguratimod; JAK inhibitors; anti-TNF mAb; anti-IL6/IL-6R; cathepsin S antagonists; cyclophosphamide; azathioprine; cyclosporine; mycophenolate mofetil; cyclophosphamide; hydroxychloroquine or methotrexate.

In a second aspect, the invention also provides biomarkers for autoimmune diseases. A bio marker is a measurable indicator of the severity or presence of the disease state.

In one embodiment, the invention provides the use of CD4+ CXCR5+ Foxp3+ T cells as a biomarker for humoral activity and/or a biomarker for autoimmune disease. The invention also provides the use of the ratio of Tfr/Tfh cells as a biomarker of increased germinal centre activity and/or an autoimmune disease and/or autoimmune disease activity or severity. The invention further provides the use of PD-1 IC0S ⁺ Tfh cells as a biomarker of autoimmune disease and/or autoimmune disease activity or severity.

In one embodiment, the invention provides the use of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells as a biomarker for autoimmune disease. In the disease state, the level of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells is elevated. An elevated level of CD4+ CXCR5+ Foxp3+ T cells in the sample is a level of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells in the sample from a subject that is at least 1.5 times greater than in a sample from a matched control subject, for example an age-matched healthy donor. For example, the elevated level may be 2, 2.5, 5, 10 or more times greater than in a matched control subject. A matched control may be matched for the species, gender, age and/or any other relevant factor. The elevated level may also be in reference to a reference or standard level of CXCR5 ⁺ Foxp3 ⁺ T cells. For example, the elevated level may be at least 1.5 times greater than a reference level for the subject. For example, the elevated level may be 2, 2.5, 5, 10 or more times greater than the reference level. An elevated level may also be determined by reference to an absolute cut-off value. For example, an elevated level of CD4 ⁺ CXCR5 ⁺ Foxp3 ⁺ T cells in the sample may be present when greater than 20%, 25%, 30%>, 35%, 40% or 50% of Tregs in the sample are CXCR5 ⁺ Foxp3 ⁺ Tregs. In a further embodiment, the present invention provides the use of the ratio of Tfr/Tfh cells as a biomarker to identify subgroups of disease state in subjects with autoimmune diseases, and in particular in subject with Sjogren Syndrome. In a specific embodiment, Example 7 demonstrates that peripheral blood Tfr/Tfh ratio correlates with salivary gland infiltration by lymphocytes, providing an indicator of these pathological changes in SS patients. The present inventors have surprisingly found that the ratio of Tfr/Tfh is predictive of ectopic lymphoid structure activity, the presence of autoantibodies and levels of disturbed germinal centre activity.

In a specific embodiment, a Tfr/Tfh ratio of greater than 0.2, for example greater than 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 is indicative of pathological changes in Sjogren Syndrome including ectopic lymphoid structure activity, salivary gland infiltration by lymphocytes, focal sialoadentitis, autoantibody production and/or disturbed germinal centre activity.

The predictive nature of the ratio of Tfr/Tfh cells in a subject with Sjogren Syndrome can avoid the need for a salivary gland biopsy. This provides the advantage of reducing the risk to the patient and provides the clinician with a minimally invasive test for identifying patients with certain characteristics and who will respond to certain therapeutic approaches.

In a particular embodiment, an increased Tfr/Tfh ratio is a biomarker of ectopic GC responses in target organ of Sjogren Syndrome. Subjects with an ectopic GC response in a target organ are suitable subjects for treatment with therapies targeting T-B interactions, such as therapies targeting T cells, B cells, co-stimulation/immune checkpoints, antigen presentation, or cytokines affecting T or B cell function. Thus, this invention may aid in clinical stratification of Sjogren's syndrome patients. Specific therapeutic agents include: B- cell depleting therapies (e.g., rituximab); Epratuzumab (anti-CD22); Baminercept (LTbetaR- Ig); Rebamipide; glucocorticoids; anti-BAFF mAb (Belimumab); anti-CD40 mAb; anti- CD28; CTLA4-Fc Ig (e.g abatacept and belatacept); low-dose IL-2; anti IL-2, IL-2 immune complexes, or modified IL-2 molecules; Iguratimod; JAK inhibitors; anti-TNF mAb; anti- IL6/IL-6R; cathepsin S antagonists; cyclophosphamide; azathioprine; cyclosporine; mycophenolate mofetil; cyclophosphamide; hydroxychloroquine or methotrexate.

The invention therefore provides a method of identifying subjects suitable for treatment with therapies targeting T-B interactions comprising determining the Tfr/Tfh ratio; identifying subjects with an elevated Tfr/Tfh ratio; and optionally providing said subjects with a therapy targeting T-B interactions.

The present inventors have also found that circulating PD-1 ⁺ ICOS ⁺ T cells are elevated in subjects with an autoimmune disease. The invention therefore provides the use of PD- 1 IC0S ⁺ Tfh cells as a biomarker of autoimmune disease and/or autoimmune disease activity or severity.

The presence of PD-l ⁺ ICOS ⁺ Tfh cells and/or the level of PD-l ⁺ ICOS ⁺ Tfh cells in a subject may be determined as a stand-alone diagnostic test or as a stand-alone biomarker of autoimmune disease. The presence of PD-1 IC0S ⁺ Tfh cells and/or detecting an elevated level of PD-1 IC0S ⁺ Tfh cells in a subject may be used in combination with other diagnostic methods and biomarkers of the invention. In a particular embodiment, detecting presence or absence of PD-l ⁺ ICOS ⁺ Tfh cells and/or detecting presence or absence of an elevated level of PD-1 IC0S ⁺ Tfh cells in a subject may be used in combination with determining the Tfr/Tfh ratio in said subject. In a particular embodiment, Example 7 demonstrates a striking increase in PD-l ⁺ ICOS ⁺ Tfh cells in SS.

Biomarkers may be detected in any sample from a subject. In a particular embodiment of the invention, the biomarker is detected in a blood sample, a plasma sample, a tissue sample such as skin, tonsil, lymph nodes, spleen, or other organs with tertiary lymphoid structures. In one particular embodiment, the sample is a blood sample.

In a third aspect, the invention provides kits for the detection of the biomarkers disclosed herein and kits for carrying out the methods of diagnosis of autoimmune diseases as described herein.

The kits of the invention may comprise 2 or more, for example 3 or more, 4 or more, 5 or more, 6 or more reagents for the detection of Tfr and Tfh cells. In a particular embodiment, kits comprise antibodies for the detection of Tfr and Tfh cells, including at least 2 of the following: anti-CD4 (optionally, OKT4, BioLegend), anti-CD45 (optionally, HBO, BioLegend), anti-CD45RO (optionally, UCHL1, BioLegend), anti-CXCR5 (optionally, J252D4, BioLegend), anti-Foxp3 (optionally, PCH101, eBioscience), anti-PD-1 (optionally, EH12.2H7, BioLegend), and/or anti-ICOS (C398.4A, BioLegend). Kits may also optionally include instruction for use.

In a fourth aspect, the invention provides a system for assessing whether a subject has an autoimmune disease the system comprising: detection means able and adapted to detect in a sample, optionally a blood or plasma sample, the ratio of Tfr/Tfh and/or the level of Tfr and/or Tfh cells; and a processor able and adapted to determine from the detected ratio and/or level of cells an indication of the patient having an autoimmune disease. The system optionally contains a data connection to an interface, particularly a graphical user interface, capable of presenting information.

In any aspect of the invention, the autoimmune disease may include autoimmune haematological disorders (including e.g. haemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, scleroderma, Wegener granulomatosis, Churg-Strauss syndrome, dermatomyositis, myasthenia gravis, psoriasis, Steven- Johnson syndrome, celiac disease, autoimmune inflammatory bowel disease (including e.g. ulcerative colitis and Crohn's disease), Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, diabetes, e.g. juvenile diabetes (diabetes mellitus type I), Bechts Disease, Sjogren Syndrome, psoriasis and psoriatic arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies, and glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy), asthma and other inflammatory airways diseases including an autoimmune component, thyroiditis (Hashimoto disease), inflammatory conditions of the central nervous system, and similar autoimmune disorders.

In specific embodiments of the invention described herein, the autoimmune leads to the formation of ectopic lymphoid structures, such as those see in rheumatoid arthritis and Sjogren Syndrome. In a particular embodiment, the autoimmune disease is Sjogren Syndrome.

In specific embodiments of the invention described herein, including methods, kits and biomarkers, the subject is a human, the sample is a blood sample and the autoimmune disease is Sjogren Syndrome. In a fifth aspect, the invention provides screening methods for the identification of agents that can alter the Tfr/Tfh ratio.

In one embodiment, the invention provides a method of screening for an agent that can alter the Tfr/Tfh ratio comprising the steps of: obtaining a first baseline sample from a subject; administering an agent to a subject; obtaining a second sample from a subject subsequent to administering the agent; and comparing the Tfr/Tfh ratio in the first and second samples. The method may further optionally comprise the step of selecting an agent that increases or reduces the Tfr/Tfh ratio.

In a sixth aspect, the invention provides methods of treatment of autoimmune diseases comprising administering an agent to normalise the Tfr/Tfh ratio. The invention also provides the use of an agent that normalises the Tfr/Tfh ratio for the treatment of an autoimmune disease. The agent may have been identified using the screening method of the previous embodiment.

In particular, the invention provides methods of treating Sjogren syndrome and methods of screening for agents that alter the Tfr/Tfh ratio in subjects with Sjogren syndrome.

GENERAL DEFINITIONS

Expression of cell surface markers may be determined, for example, by means of flow cytometry and/or FACS for a specific cell surface marker using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art) to determine whether the signal for a specific cell surface marker is greater than a background signal. The background signal is defined as the signal intensity generated by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker. For a marker to be considered positive the specific signal observed is typically more than 20%, preferably stronger than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000%, 10000% or above, greater relative to the background signal intensity. Alternative methods for analysing expression of cell surface markers of interest include visual analysis by microscopy using antibodies against cell-surface markers of interest. In the present invention Tfr cells, Tfh cells, PD-1 TCOS CD4 ⁺ T cells and CXCR5 Foxp3 CD4 ⁺ T cells can be detected by flow cytometry. In specific embodiments, Tfr cells are CXCR5 ⁺ Foxp3 ⁺ CD4 ⁺ T cells, and are identified by flow cytometry using anti-CXCR5 antibodies and anti-Foxp3 antibodies. In another embodiment, Tfh cells are CXCR5 ⁺ CD45RO cells and are identified by flow cytometry using anti-CXCR5 antibodies and anti- CD45RO antibodies. In another embodiment, the PD-l ⁺ ICOS ⁺ CD4 ⁺ T cells can be identified by flow cytometry using anti-PD-1 antibodies and anti-ICOS antibodies.

Specific antibodies that can be used in the present invention include: anti-CD4 (OKT4, BioLegend), anti-CD45 (HI30, BioLegend), anti-CD45RO (UCHL1, BioLegend), anti- CXCR5 (J252D4, BioLegend), anti-Foxp3 (PCHlOl, eBioscience), anti-PD-1 (EH12.2H7, BioLegend), and anti-ICOS (C398.4A, BioLegend).

As used herein, the term "positive" and "+" when used in regard to a marker means that, in a cell population, more than 20%, preferably more than, 30%, 40%, 50%, 60%, 70%, 80%, 90%) 95%), 98%), 99%) or even all of the cells express said marker.

As used herein, "negative" or "-" as used with respect to markers means that in a cell population, less than 20%, 10%, preferably less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % or none of the cells express said marker.

As used herein with respect to a single cell, the term "positive" and "+" mean that the marker is expressed at a detectable level and the term "negative" or "-" mean that the marker is not expressed at a detectable level. When detected using flow cytometry and/or FACS, typically a positive signal is more than 20%>, preferably stronger than 30%>, 40%>, 50%>, 60%>, 70%>, 80%, 90%, 100%, 500%, 1000%, 5000%, 10000% or above, greater relative to the background signal intensity.

As used herein with respect to a single cell, the term "positive" and "+" with reference to multiple markers means that the cells expresses all of the positive markers at a detectable level. With respect to a cell population, the positive" and "+" with reference to multiple markers means that the cell population is made up of more than 20%, preferably more than, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 98%, 99% cells that express each of said markers.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Blood Tfr cells are indicators of ongoing humoral activity. Frequency of total Tregs, Tfr cells and CXCR5- Tregs in peripheral blood of Sjogren syndrome patients (SS) and age-matched healthy donors (HD) (n=25, unpaired Student t-test with Welch's correction for variance). Representative plots (left) and pooled data (right). Bars represent SEM.

Figure 2: a) Blood Tfr:Tfh ratio in SS patients and HD (n=25, unpaired Student t-test with Welch's correction for variance). Bars represent SEM. b) Blood Tfr:Tfh ratio in SS patients with and without serum autoantibodies (anti-SSA/Ro52, anti-SSB/Ro60, and anti-SSB/La) (n = 25, unpaired Student t-test). Bars represent minimum and maximum values (in box and whiskers graphs).

Figure 3: a) Variation of blood Tfr:Tfh ratio accordingly to c-reactive protein (CRP). Analysis by linear regression, b) Variation of blood Tfr:Tfh ratio accordingly to disease severity score (ESSDAI). Analysis by linear regression.

Figure 4: Activated programmed death 1 -positive (PD-1 ⁺ ) inducible costimulator-positive (ICOS ) follicular helper T (Tfh) cells in peripheral blood indicate disease activity in primary Sjogren's syndrome (SS). Frequency of CXCR5 ⁺ CD45RO ⁺ CD25 FoxP3 CD4 ⁺ Tfh cells, CD25 ⁺ FoxP3 ⁺ CD4 ⁺ Treg cells, and CXCR5 ⁺ CD25 ⁺ FoxP3 ⁺ CD4 ⁺ T follicular regulatory (Tfr) cells in peripheral blood of patients with primary SS (n = 16) and healthy donors (HD) (n = 16). Shown are representative plots (left) and pooled data (right). Symbols represent individual subjects; bars show the mean ± SEM. Significance was determined by

Student's unpaired t-test with Welch's correction for variance.

Figure 5: a) Blood Tfr celkTfh cell ratio in SS patients (n = 16) and healthy donors (n = 16). b) Frequency of PD-l ICOS ⁺ Tfh cells in peripheral blood of SS patients (n = 14) and healthy donors (n = 13). Shown are representative plots (left) and pooled data (right), c) Frequency of PD-1 CXCR3 Tfh cells in peripheral blood of SS patients (n = 14) and healthy donors (n = 13). Shown are representative plots (left) and pooled data (right). Symbols represent individual subjects; bars show the mean ± SEM. Significance was determined by

Student's unpaired t-test with Welch's correction for variance in a) - b); by Student's unpaired t-test in c).

Figure 6: a) Distribution of CCR6 ⁺ CXCR3 Tfhl7-like cells, CCR6 CXCR3 ⁺ Tfhl-like cells, and CCR6 CXCR3 Tfh2-like cells in peripheral blood of SS patients (n = 16) and healthy donors (n = 16). b) Heatmap representation of correlation of the CXCR5 Treg cell:Tfh cell ratio, the Tfr celkTfh cell ratio, peripheral blood Tfr cells, PD-1 ⁺ CXCR3 Tfh cells, PD-l ⁺ ICOS ⁺ Tfh cells, and Tfh cells with serum autoantibody titers (antinuclear antibodies [ANAs], anti-SSA/Ro 52, anti-SSA/Ro 60, anti-SSB), rheumatoid factor (RF; IU/ml), serum electrophoresis gamma fraction levels (gm/dl), C-reactive protein (CRP) level (mg/dl), erythrocyte sedimentation rate (ESR; mm/hour), and disease activity measured by the European League Against Rheumatism Sjogren's Syndrome Disease Activity Index (ESSDAI) in SS patients (n = 16). *=P < 0.05; ** = P < 0.01; *** = P < 0.001 by Pearson correlation test. Significance was determined by Student's unpaired t-test with Welch's correction for variance in a).

Figure 7: Correlation of peripheral blood Tfr celkTfh cell ratio with serum anti-SSA/Ro 60 titer (left) and correlation of blood PD-l ⁺ ICOS ⁺ Tfh cells with serum anti-SSA/Ro 52 titer (center) and ESSDAI score (right) (n = 16). Shown are linear regression lines with interpolated 95% confidence interval curves (broken lines). CU= chemiluminescence units.

Figure 8: Blood Tfr cells show expression of follicular and regulatory markers, a) CXCR5 ⁺ Tfr cells constitute 18.57± 6.55% of Tregs (left) and 0.93± 0.56% of total CD4 ⁺ T cells (middle), representing 9985 ± 9043 cells per mL of blood (right) (n = 42, adult healthy donors), b) Variation of blood Tfr cells frequency (left) and absolute number per ml of blood (right) accordingly to age (age range: 22 - 92 years old), (n = 42, linear regression). Error bars represent SEM.

Figure 9: Expression of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1 Bcl-6, and CD57 by Tfh cells (blue), CXCR5 Tregs (black) and Tfr cells (red), in children blood (top rows) and in tonsils (bottom rows). Na ^' ive CD4 ⁺ T cells were used as control (gray). Representative plots from 6 healthy children. CXCR5 subsets in tonsils were defined as CXCR5 ⁺ ICOS ⁺ cells.

Figure 10: a) Immunofluorescence microscopy of formalin- fixed paraffin-embedded human tonsils stained for DAPI (blue), CXCR5 (yellow), CD4 (red) and Foxp3 (green). Top, middle and bottom outlined areas indicate top, middle and bottom enlarged areas on the right, respectively. Data are representative of tonsil sections from 5 healthy children. (E) Blood Tfr:Tfh ratio in adult blood, children blood (tonsil donors) and in tissues (tonsils). Black and red dots represent blood and tonsil results, respectively (n = 42 for adults and n = 6 for children, Student t-test). Error bars represent SEM.

Figure 11: Blood Tfr cells are a distinct subset of suppressive Tregs. a) Schematic representation of in vitro suppression assay. FACS-sorted 25 x 10 ³ CXCR5 CD25- CD127 ⁺ CD4 ⁺ Tconv cells were co-cultured with 25 x 10 ³ CXCR5 CD25 CD127 CD4 ⁺ Tregs or CXCR5 CD25 CD127 CD4 ⁺ Tfr cells under stimulation by anti-CD3 (1 μg/mL), in presence of 10 ⁵ irradiated (2500 rad) allo-PBMC. After 5 days cells responder cells were analyzed for CTV dilution by flow cytometry. Sorting strategy is described in Fig. 24. b) Proliferation of Tconv cells without Tregs or in the presence of either CXCR5- Tregs or Tfr cells. Representative plots (left) and pooled data (right) (n = 3, each with technical triplicates, One-way AN OVA with post-test Turkey's Multiple Comparison). Error bars indicate SEM. (ns= not significant).

Figure 12: a) Suppression curve of CXCR5- Tregs and Tfr cells in different ratios, using the same conditions described in Figure 7 (n = 1, with technical triplicates, Two-way ANOVA). b) Stability of Foxp3 expression by sorted CXCR5- Tregs and CXCR5 ⁺ Tfr cells after 5 days of in vitro culture under aCD3/aCD28 (1 μΕ/well) stimulation. Percentage (left) and cell number (right) (n = 5, each with technical triplicates, Student t-test). Error bars indicate SEM. (ns= not significant).

Figure 13: a) Relative expression of Foxp3 and CXCR5 by sorted Tconv, Tfh cells, CXCR5- Tregs and Tfr cells from blood, by real-time RT-PCR. Gene expression normalized to housekeeping genes (B2M, G6PD and ACTB) (n = 2, each with technical duplicates, Student t-test). b) Expression of Foxp3, CD25, CTLA-4 and CXCR5 by sorted CXCR5 Tregs and Tfr cells at baseline (dO) and after 5 days of in vitro culture under aCD3/aCD28 (1 μΕ/well) stimulation. Representative histograms of 3 independent experiments, each one with technical triplicates. Error bars indicate SEM. (ns= not significant).

Figure 14: Blood Tfr cells do not show specialized humoral regulatory capacity, a) Proliferation of CXCR5 ⁺ CD25-CD127 ⁺ CD4 ⁺ Tfh cells without regulatory T cells or in the presence of either CXCR5- Tregs or Tfr cells after 5 days of in vitro culture as described in Figure 7a. Representative plots (left) and pooled data (right) (n = 3, each with technical triplicates, One-way ANOVA with post-test Turkey's Multiple Comparison), b) Schematic representation of suppression co-culture assay. FACS-sorted 25 x 10 ³ CXCR5 ⁺ CD25- CD127 ⁺ CD4 ⁺ Tfh cells (or CXCR5 CD25 CD127 CD4 ⁺ Tconv cells) were co-cultured for 5 days with 25 x 10 ³ CXCR5 CD25 CD127 CD4 ⁺ Tregs (or CXCR5 CD25 CD127 CD4 ⁺ Tregs) under stimulation by SEB (1 μg/mL) and in the presence of 30 x 10 ³ CD27- IgD ⁺ CD19 ⁺ naive B cells. Error bars indicate SEM. (ns = not significant).

Figure 15: Upregulation of CD38 and downregulation of IgD by na ^' ive B cells (top) and proliferation of Tfh cells by CTV dilution (bottom) without Tregs or in the presence of either CXCR5- Tregs or Tfr cells. Representative plots (left) and pooled data (right) (n = 5, each with technical triplicates, One-way ANOVA with posttest Turkey's Multiple Comparison). Error bars indicate SEM. (ns = not significant).

Figure 16: a) Suppression curve of CXCR5- Tregs and Tfr cells in different ratios, using the same conditions described in Figure 10b (n = 1, with technical triplicates, Two-way ANOVA). b) ELISA determination of for IgA, IgM and total IgG in supernatants after 10 days of in vitro co-culture performed as described in (a), but using SEB (1μg/mL) + SEA (lOg/mL) + SEE (lOng/mL) + TSST-1 (lOng/mL) as superantigen stimulation, (n = 3, each with technical triplicates, One-way ANOVA with post-test Turkey's Multiple Comparison). (E) In vitro migration of 75 x 10 ³ sorted Tconv, Tfh, CXCR5- Tregs, and Tfr cells towards a CXCL13 gradient (2 μ/mL), expressed by chemotaxis index (n = 3, each with technical triplicates, One-way ANOVA with post-test Turkey's Multiple Comparison). Error bars indicate SEM. (ns = not significant).

Figure 17: Blood Tfr cells are immature but are not committed as such in the thymus, a) Backgate of CXCR5- and CXCR5 ⁺ Tregs accordingly to CD45RO and Foxp3 expression, b) Expression of D45RO, CD45RA, CCR7, CD62L, HLA-DR and CD27 by Tfr cells (red) and CXCR5 Tregs (black) in blood.

Figure 18: a) Expression of Ki-67 by CXCR5 ⁺ Tregs and CXCR5 Tregs in blood (n = 22, Student t-test). b) CD45RO ⁺ CCR7- effector-memory (EM), CD45RO ⁺ CCR7 ⁺ central- memory (CM) and CD45RO-CCR7 ⁺ na ^' ive subsets of Tfr cells and CXCR5 Tregs in adult blood. Representative plots (left) and pooled data (right) (n = 22, Student t-test). Tfh cells are represented in blue, CXCR5- Tregs in black and Tfr cells in red. Bars represent SEM.

Figure 19: a) Variation of CD45RO-CCR7 ⁺ na ^' ive Tfr cells and CXCR5- Tregs frequency in blood accordingly to age (n = 22, linear regression), b) Expression of CXCR5 by Foxp3 ⁺ CD4 ⁺ thymocytes (n = 4). c) Expression of CXCR5 by cord blood Foxp3 ⁺ CD4 ⁺ T cells (n = 3). (H) Expression of CD45RO, CCR7 and ICOS by cord blood Tregs (n = 3).

Figure 20: Blood Tfr cells are lymphoid tissue derived Tfr precursors. CD45RO ⁺ CCR7- EM, CD45RO ⁺ CCR7 ⁺ CM and CD45RO-CCR7 ⁺ naive subsets of Tfr cells and CXCR5 Tregs in children blood (top) and in tissues (bottom). Representative plots (left) and pooled data (right) (n = 6, Student t-test). Tfh cells are represented in blue, CXCR5- Tregs in black and Tfr cells in red. CXCR5 subsets in tonsils were defined as CXCR5 ICOS cells. Bars represent SEM.

Figure 21: Blood Tfh and Tfr cells from X- linked Agammaglobulinemia (BTK-deficient) patients, compared to sex and age-matched healthy donors. Representative plots (left) and pooled data (right) (n = 5, Student t-test). Bars represent SEM.

Figure 22: a) Model of CXCR5 ⁺ follicular helper and regulatory cells T cells generation and recirculation in humans, upon antigen stimulation. Tfh cells in red and Tfr cells in blue.

Figure 23: Blood Tfr / Tfh ratio is a biomarker of ectopic GC activity in Sjogren syndrome. Weight and infiltration by CD45 ⁺ live cells, CD4 ⁺ T cells and CD19 ⁺ B cells of salivary gland tissue of SS patients and controls (CTR). Representative plots (left) and pooled data (right) (n = 13 for SS and n = 6 for CTR, Student t-test). Bars represent SEM. Figure 24: a) ICOS PD-1 CD4 T cells in salivary gland tissue of SS patients and controls. Representative plots (left) and pooled data (right) (n = 13 for SS and n = 6 for CTR, Student t-test). b) heatmap representation of correlation between peripheral blood Tfh cells, Tfr cells, and Tfr/ Tfh ratio (Figure 1) and salivary gland infiltration by CD45 ⁺ live cells, CD4 ⁺ T cells, ICOS ⁺ PD-l ⁺ CD4 ⁺ T cells, and CD19 ⁺ B cells (n = 13, Pearson, r). Bars represent SEM.

Figure 25: a) Relationship between peripheral blood Tfr cells (top) and Tfr / Tfh ratio (bottom) with salivary gland infiltration by CD4 ⁺ T cells, ICOS ⁺ PD-l ⁺ CD4 ⁺ T cells and CD19 ⁺ B cells (n = 13, linear regression with interpolated 95% confidence interval curves), b) Peripheral blood Tfr / Tfh ratio and histological diagnosis of salivary gland biopsy (n = 25, One-way ANOVA with post-test Turkey's Multiple Comparison) (LI, lymphocytic infiltration; FSA, focal sialoadenitis). Bars represent SEM.

Figure 26: Blood Tfr cell:Tfh cell ratio identifies pathologic lymphocytic infiltration in the target organ of SS. A, Frequency and absolute numbers of CD45 ⁺ hematopoietic cells in minor salivary gland (MSG) biopsy samples from patients with primary SS (n = 14) and patients with non-SS sicca syndrome (non-SSS) (n = 6). Shown are representative plots (left) and pooled data (right). Values are the mean ± SEM. Significance was determined by Student's unpaired t-test.

Figure 27: B, Frequency and absolute numbers of CD4+ T cells and CD19 ⁺ B cells in MSG biopsy samples from SS patients (n = 14) and patients with non-SS sicca syndrome (n = 6). Shown are representative plots (left) and pooled data (right). Values are the mean ± SEM. Significance was determined by Student's unpaired t-test with Welch's correction for variance.

Figure 28: a) Frequency and absolute numbers of PD-l ⁺ ICOS ⁺ T cells in MSG biopsy samples from SS patients (n = 10) and patients with non-SS sicca syndrome (n = 6). Shown are representative plots (left) and pooled data (right), b) Heatmap representation of correlation of peripheral blood Tfh cells, PD-l ⁺ ICOS ⁺ Tfh cells, PD-1 ⁺ CXCR3 Tfh cells, Tfr cells, the Tfr cell:Tfh cell ratio, and the CXCR5 Treg cell:Tfh cell ratio with salivary gland infiltration by CD45 ⁺ hematopoietic cells, CD4 ⁺ T cells, PD-l ⁺ ICOS ⁺ CD4 ⁺ Tcells, and CD19 ⁺ B cells in patients with primary SS (n = 14). *=P < 0.05; ** = P < 0.01 by Pearson correlation test. For a) values are the mean ± SEM. For a) significance was determined by Student's unpaired t-test with Welch's correction for variance.

Figure 29: Correlation of peripheral blood Tfr celkTfh cell ratio with salivary gland infiltration by CD45 ⁺ hematopoietic cells, CD4 ⁺ Tcells, PD-l ⁺ ICOS ⁺ CD4 ⁺ T cells, and CD19 ⁺ B cells(n= 14). Shown are linear regression lines with interpolated 95% confidence interval curves (broken lines).

Figure 30: Identification, by immunohistochemistry, of CXCR5 FoxP3 ⁺ Tfr cells (arrows) within focal sialadenitis containing CD20 ⁺ B cells in MSG biopsy samples from patients with primary SS. Far left image shows focal sialadenitis containing CD20 ⁺ B cells (brown). The 3 other images show double immunohisto chemical staining for CXCR5 (purple/red) and FoxP3 (brown). Boxed areas in images are shown at higher magnification in immediately adjacent images at right. Pooled data at far right show a bimodal distribution as there are patients with less than 4 Tfr cells per mm and patients with more than 10 Tfr cells per mm ² (n = 16). Symbols represent individual subjects; bars show the mean ± SEM.

Figure 31: Blood Tfr celkTfh cell ratio as a marker of primary SS and focal sialadenitis (FSA). a) Blood Tfr celkTfh cell ratio in SS patients (n =16), patients with non-SS sicca syndrome (non-SSS; n = 11), and healthy donors (n =16). b) Receiver operating characteristic (ROC) curves for prediction of SS diagnosis (versus healthy donors and versus patients with non-SS sicca syndrome) based on the Tfr cell:Tfh cell ratio. AUC = area under the curve, c) Blood Tfr celkTfh cell ratio in patients with sicca symptoms (with focal sialadenitis [n = 7] or normal histology/nonspecific lymphocytic infiltration [n = 20]) undergoing minor salivary gland biopsy. In a) significance was determined by Student's unpaired t-test with Welch's correction for variance except when variance was not significantly different between groups (patients with non-SS sicca syndrome versus healthy donors); one-way analysis of variance was used to compare all 3 groups. In c) significance was determined by Mann- Whitney U test due to skewed distribution of values and nondifferent variance according to the Brown- Forsythe test.

Figure 32: a) Odds ratio (OR), P value, and AUC of logistic regression models predicting SS diagnosis (versus healthy donors and versus patients with non-SS sicca syndrome) and focal sialadenitis (versus normal histology or nonspecific lymphocytic infiltration [LI]) based on the Tfr cell:Tfh cell ratio as a continuous variable or as specific cutoffs. The Tfr cell:Tfh cell ratio is transformed by 1 decimal place (x 10 ¹ ) for better interpretation of the OR. Shown are percentages of sensitivity, specificity, and correct classification of patients based on given cutoffs. NA = not applicable, b) ROC curve for prediction of focal sialadenitis diagnosis (versus normal histology/nonspecific lymphocytic infiltration) based on the Tfr cell:Tfh cell ratio.

Figure 33: a) Microscopy of formalin- fixed paraffin-embedded salivary glands stained for CD4 (top) and CD20 (bottom) by immunohistochemistry. Data are representative of salivary gland sections from 25 SS patients, with unspecific lymphocytic infiltration (LI) (left) and focal sialoadenitis (FSA) (right), b) ROC curve analysis of peripheral blood Tfr/ Tfh ratio for SS diagnosis.

Figure 34: a) Gating strategy for identification and analysis of conventional T cells (Tconv), Tfh cells, CXCR5- Tregs and CXCR5 ⁺ Tfr in human peripheral blood, b) Absolute numbers of Tfh cells, total Tregs and Tfr cells (cells/mL of blood) in peripheral blood of Sjogren's syndrome (SS) patients and healthy donors (HD) (n 0 25, Student t-test). c) Blood Tfr/Tfh ratio in SS patients accordingly with treatment regimen (HCQ, hydroxychloroquine; PDN, prednisolone) (n = 25, Student t-test). d) Impact of SS treatment regimen in blood Tfr/Tfh ratio and in CXCR5 MFI. CXCR5 MFI in Tfh (left) and Tfr cells (right) in SS patients accordingly with treatment (n = 25, Student t-test). Error bars represent SEM.

Figure 35: Blood T cell subsets in primary Sjogren's syndrome, a) Frequency and absolute number (per mL of blood) of CD4 ⁺ T cells in primary Sjogren's syndrome (SS) patients (n=16) and healthy donors (HD) (n=16). Unpaired Student T-test. b) Absolute numbers (per mL of blood) of Tfh, Treg, CXCR5 ⁺ Treg (Tfr cells) and CXCR5 Treg cells in SS (n=16) and HD (n=16). Unpaired Student T-test with Welch's correction for variance, c) Mean fluorescence intensity of CXCR5 and Foxp3 in peripheral blood Tfh and Tfr cells in SS (n=16) and HD (n=16). Unpaired Student T-test. d) Blood CXCR5 Treg/Tfh ratio in SS (n=16) and HD (n =16). Unpaired Student T-test. e). Distribution of CCR6 ⁺ CXCR3- Thl7 cells, CCR6-CXCR3 ⁺ Thl cells, and CCR6 CXCR3- Th2 cells in peripheral blood of SS (n=16) and HD (n=16). Unpaired Student T-test. Bars in scatterplots represent SEM. Figure 36: a) Variation of blood Tfh cells (left) and total Tregs (right) frequency accordingly to age (age range: 22 - 92 years old) (n = 42, linear regression), b) Gating strategy for identification and analysis of Tconv, Tfh cells, CXCR5- Tregs and CXCR5 ⁺ Tregs in human tonsils.

Figure 37: Expression of regulatory and follicular markers by adult blood Tfh cells, CXCR5- T regs and Tfr cells. Expression of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1 Bcl- 6, and CD57 by Tfh cells (blue), CXCR5 Tregs (black) and CXCR5 ⁺ Tfr cells (red) in adult blood. Naive CD4+ T cells were used as control (gray). Representative plots from 42 healthy volunteers.

Figure 38: MFI (mean fluorescence intensity) of regulatory and follicular markers by adult blood Tfh cells, CXCR5 T regs and Tfr cells. MFI of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1 Bcl-6, and CD57 by Tfh cells (blue), CXCR5 Tregs (black) and Tfr cells (red) in adult blood (**p<0.01, ***p<0.001, n = 6, one-way ANOVA with post-test Turkey's multiple comparison). Error bars indicate SEM. (ns = not significant).

Figure 39: a) Isotype controls for intracellular stainings by flow cytometry. Isotype controls for intracellular Bcl-6 and Foxp3 staining by flow cytometry, b) Relative expression of Bcl-6 by sorted Tconv, Tfh cells, CXCR5- Tregs and Tfr cells from blood, by real-time RT-PCR. Gene expression normalized to housekeeping genes (B2M, G6PD and ACTB) (n = 2, each with technical duplicates, Student t-test). CD38 ^hl IgD- germinal center B cells sorted from human tonsils were used as positive control, c) Negative control for immunofluorescence microscopy, merge (left) and composite of the four immunofluorescence channels (right). After paraffin removal and antigen retrieval by heat sections of formalin- fixed paraffin- embedded human tonsil were stained with Alexa-Fluor 488 (anti-mouse), Alexa-Fluor 546 (anti-rabbit) and AlexaFluor (anti-Rat) secondary antibodies, without primary antibodies. DAPI was used as nuclei counter staining. Unspecific binding of secondary antibodies and cross-reactivity between secondary antibodies were excluded. Error bars represent SEM.

Figure 40: Sorting strategy for human blood na ^' ive B cells, Tfh cells, CXCR5- T regs and Tfr cells. Sorting strategy for FACS-sort of CXCR5 CD25 CD127 CD4 ⁺ Tfh cell, CXCR5 CD25 CD127 ⁺ CD4 ⁺ Tconv cell, CXCR5 CD25 CD127 CD4 ⁺ Tfr cells, CXCR5 CD25 CD127 CD4 Tregs, and CD27-IgD CD 19 naive B cell populations from peripheral blood (buffy-coats).

Figure 41: a) Purity of FACS-sorted CXCR5 CD25 CD127 CD4 ⁺ Tfr cells, CXCR5 CD25 ⁺ CD127 CD4 ⁺ Tregs. b) MFI of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1 Bcl-6, and CD57 by CXCR5 Tregs (black) and Tfr cells (red) by sorted CXCR5 Tregs and Tfr cells at baseline (dO) and after 5 days of in vitro culture under aCD3/aCD28 (1 μL/well ) stimulation in adult blood (*p<0.05, **p<0.01, ***p<0.001, n =3, one-way ANOVA with post-test Turkey's multiple comparison). Error bars represent SEM. (ns = not significant).

Figure 42: Proliferation of Tfh cells by CTV dilution without Tregs or in the presence of either CXCR5- Tregs or Tfr cells (n = 1, each with technical triplicates), at 1 : 1, 1 :2, 1 :4 and 1 :8 suppressor cell to responder cell ratios. Representative plots of CXCR5- T regs and Tfr cells suppression curves for coculture assay.

Figure 43: Expression of CD45RO, CD45RA and CD31 by human blood Tfh and Tfr cells, a) Expression of CD45RO and CD45RA in adult blood by total Tfh cells and Tfr cells (left) and by CCR7 ⁺ CD45RO- naive Tfh cells (nTfh cells) and by CCR7 ⁺ CD45RO- naive Tfr cells (nTfr cells) (right). Representative plots from 22 healthy donors, b) Variation of CD45RO- CD31 ⁺ na ^' ive Tfr cells and CXCR5- Tregs frequency in blood accordingly to age (n = 22, linear regression).

Figure 44: a) Expression of CD31 by Tfh and Tfr cells in adult blood of healthy donors. Representative plots (left) and pooled data (right) (n = 22, Student t-test). b) Expression of CXCR5, ICOS and PD-1 by cord blood Foxp3 ⁺ CD25 ⁺ Tregs. Representative plots from 3 healthy newborns. Error bars represent SEM.

Figure 45: Effector memory, central memory and na ^' ive subsets of human blood CXCR5- T regs and Tfr cells in SS and BTK patients. CD45RO ⁺ CCR7- effector-memory (EM), CD45RO ⁺ CCR7 ⁺ central-memory (CM) and CD45RO-CCR7 ⁺ na ^' ive subsets of Tfr cells and CXCR5- Tregs in peripheral blood of SS and BTK patients. Representative plots of 25 SS patients and 5 BTK patients. Figure 46: Optimization of enzymatic digestion of salivary gland tissue from SS patients. Impact of different combination enzymes in membrane markers used for identification of cellular components of salivary gland tissue infiltrates, including CXCR5- expressing T cells. Human tonsil fragments from healthy children were used for optimization. Liberase TM (O. lmg/mL), DNAse I (O. lmg/mL), Collagenase P (O. lmg/mL). Representative plots of 2 independent experiments.

Figure 47: Validation of ICOS ⁺ PD-l ⁺ CD4 ⁺ T cells for identification of Tfh cells, a) CXCR5 and ICOS expression by salivary gland CD19 ⁺ B cells and CD4 ⁺ T cells after enzymatic digestion by Liberase TM (O. lmg/mL) plus DNAse I (O.lmg/mL). Representative plots of 25 salivary gland biopsies, b) Microscopy of formalin- fixed paraffin-embedded salivary glands stained for CXCR5 (right), with correspondent negative control (left) by immunohistochemistry. Data are representative of salivary gland sections from 4 SS patients, c) CXCR5 and Bcl-6 expression by ICOS ⁺ PD-l ⁺ CD4 ⁺ T cells from human tonsils. Representative plots from 6 healthy children.

Figure 48: Characteristics of minor salivary gland biopsies of primary Sjogren's syndrome patients. Impact of different enzymes in membrane markers used for identification of cellular components of minor salivary gland tissue infiltrates, including CXCR5 -expressing T cells. Human tonsil fragments from healthy children were used for optimization. Liberase TM (0.1 mg/mL), DNAse I (0.1 mg/mL), Collagenase P (0.1 mg/mL). All enzymes led to significant duction of CXCR5 on PD-1 ⁺ CD4 ⁺ T cells. Representative plots of 2 independent experiments.

Figure 49: a) Weight of salivary gland tissue removed during minor salivary gland (MSG) biopsy in primary Sjogren's syndrome (SS) patients (n=14) and non-Sjogren sicca syndrome (non-SSS) patients (n=6). b) CXCR5 and ICOS expression by salivary gland CD4+ B cells and CD 19+ T cells after enzymatic digestion by Liberase TM (0.1 mg/mL) plus DNAse I (0.1 mg/mL), confirming the loss of CXCR5 staining. Representative plots of 14 MSG biopsies, c) Microscopy of formalin- fixed paraffin-embedded MSG stained for CXCR5 (brown, right), with correspondent negative control (left) by immunohistochemistry. Data are representative of MSG sections from 4 SS patients, d) CXCR5 and Bcl-6 expression by ICOS+PD-1+CD4+ T cells from human tonsils. Representative plots (left) and pooled data (right) (n=6). e) Microscopy of formalin- fixed paraffin-embedded MSG stained for CD20 (brown) in a SS patient with FSA (top) and a SS patient with no pathologic findings (normal, bottom). (G) Frequency of CD4+ T cells, CD19+ B cells, and PD-1+ICOS+ T cells in MSG biopsies of SS (n=14) and non-SSS (n=6), according to histological diagnosis (normal, no infiltration by lymphocytes; LI, unspecific lymphocytic infiltration; FSA, focal sialadenitis). Two-way ANOVA with post-test Bonferroni's Multiple Comparison. Bars on scatterplots represent SEM.

Figure 50: Identification of ectopic lymphoid structures in minor salivary glands of Sjogren's syndrome patients, a) Correlation between SS disease activity as measured by ESSDAI and histological diagnosis (normal, no infiltration by lymphocytes; LI, unspecific lymphocytic infiltration; FSA, focal sialadenitis), b) Microscopy of formalin- fixed paraffin-embedded MSG stained for CD20, CD21 and Bcl-6 in a SS patient with FSA harboring features of ectopic lymphoid structures (ELS) with ectopic germinal centre formation (top) and without ELS (bottom), c) Number of primary SS patients with normal, LI and FSA patients further characterized for the presence of ELS and ectopic germinal centre accordingly to CD21 and Bcl-6 immunohistochemistry. Bars on scatterplots represent SEM.

Figure 51: Flowchart of patient recruitment and selection. DMARDs, disease modifying antirheumatic drugs; TNF, tumor necrosis factor; AECG, American-European Consensus Group criteria for Sjogren's syndrome.

Figure 52: Demographic and clinical characteristics of primary Sjogren's syndrome (SS) and non-Sjogren sicca syndrome (non-SSS) patients.

Figure 53: a) Antibodies used for flow cytometry, b) Correlation between absolute number of Tfr cells per mm2 of minor salivary gland tissue section (Fig. 2F) wit clinical and laboratory parameters, and minor salivary gland infiltration by CD45+, CD4+, CD 19+ and PD- 1+ICOS+ lymphocytes. (n=16, Pearson coefficient, r).

Figure 54: An exemplary stratification scheme for Sjogren Syndrome, where ESSDAI represents disease activity (high or low) and FSA represents focalsialedenitis (ie. infiltration of salivary glands with ectopic lymphoid structure, or, in other words, biopsy with pathological changes). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Germinal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high affinity antibodies. Although the biology of human circulating and tissue Tfh cells has been established, the relationship between blood and tissue Tfr cell defined as CXCR5 ⁺ Foxp3 ⁺ T cells remains elusive. Here, we found that blood Tfr cells are increased in Sjogren syndrome, an autoimmune disease with ongoing GC reactions, especially in patients with high autoantibody titres and ectopic GC activity in target tissue of autoimmune responses. While, blood Tfr cells correlated with humoral responses they lack full B-cell suppressive capacity, despite being able to suppress T-cell proliferation. Blood Tfr cells have a na ^' ive-like phenotype, although they are absent from human thymus or cord blood. Here, we found, these cells were generated in peripheral lymphoid tissues prior to T-B interaction, as they are maintained in B-cell deficient patients. Therefore, blood CXCR5 Foxp3 ⁺ T cells in human pathology can be used as a biomarker for ongoing humoral activity, and can predict the results of diagnostic biopsies in Sjogren Syndrome. Given our results it is anticipated that level of circulating Tfr cells or blood Tfr/Tfh ratio can be useful for diagnosis in other immune-mediated and autoimmune diseases.

Example 1: Blood Tfr cells indicate ongoing germinal center responses

Methods:

Study Design

Samples sizes were estimated based on previous studies and accordingly to each cohort (see human samples). No outliers were excluded. Number of biological and technical replicates is stated in figure legends. Human samples from different conditions were used (see human samples) with appropriate age-matched controls. This experimental study was performed unblinded.

Human samples

Fresh peripheral blood samples were collected from patients referred to Rheumatology Department of Hospital de Santa Maria, Centra Hospitalar Lisboa Norte for salivary gland biopsy due to clinical suspicion of SS. Blood samples were collected on the day of salivary gland biopsy. Salivary gland tissue was divided into two pieces. One was used by Pathology Department for routine diagnostic purposes. The other was processed for flow cytometry. All patients with exclusion criteria for SS (37) or treated with biologic drugs, Disease Modifying Anti-Rheumatic Drugs (DMARDs) or more than 7.5mg per day of prednisolone were excluded. Patients diagnosed with an infectious disease in the previous month were also excluded, as well as those who received any vaccine in the same period of time. Patients were diagnosed as having SS if they met AECG diagnosis criteria (n = 25) (37). Routine c-reactive protein plasma levels (mg/dL) closest to blood collection were used. Age-matched healthy volunteers (from the cohort described below) were used for statistical comparison. Fresh peripheral blood samples were collected from adult healthy volunteers (n = 42). Fresh buffy- coats (blood collection in less than 24 hours) were used for in vitro suppression and co- culture assays. Tonsils and peripheral blood samples were collected from healthy children submitted to tonsillectomy due to tonsil hypertrophy (n = 6). Children with any clinical condition, under any drug treatment or submitted to tonsillectomy due to chronic tonsillitis were excluded. Umbilical cord blood samples were collected from healthy pregnancies during delivery (n = 3). Thymus tissue was collected from children submitted to cardiac surgery due to congenital heart disease who were otherwise healthy (n = 4). Blood samples were also collected from X-linked Agammaglobulinemia (BTK-deficient) patients during routine blood tests (n = 5). All blood samples were collected in in EDTA coated tubes. These studies were approved by the Lisbon Academic Medical Center Ethics Committee (Ref. n.° 505/14). Informed consent was obtained from all adult volunteers, parents or legal guardians.

Cell isolation and flow cytometry

PBMCs were isolated from blood samples by Ficoll-gradient medium (Histopaque-1077, Sigma- Aldrich) using SepMate tubes (StemCell Technologies). Lymphocytes from tonsils and thymocytes were also isolated by Ficoll-gradient medium after mechanical disruption. Before cell sorting, PBMCs from Buffy-coats were enriched for CD4 ⁺ T cells using Mojo Sort Human CD4 T Cell Isolation Kit (BioLegend). The CD4 ⁺ fraction was used for cell sorting of CD4 ⁺ T cell subsets. The CD4- fraction was used for cell sorting of na ^' ive B cells (Figure S3A for sorting strategy). A cell suspension was prepared from salivary gland tissue for flow cytometry analysis. Briefly, salivary gland was cut into small fragments and incubated at 37.°C with Liberase TM O. lmg/mL plus DNAse I O. lmg/mL in RPMI medium for 20 minutes. After washing, fragments were incubated again for 10 minutes incubation with the same enzyme solution. After washing, fragments were vigorously pipetted and filtered to obtain a cell suspension. For flow cytometry cells were stained with anti-Bcl-6 (Kl 12-91 , BD Biosciences), anti-CCR7 (#150503, R&D Systems), anti-CD127 (eBioRDr5, eBioscience), anti-CD 19 (HIB19, BioLegend), anti-CD25 (BC96, eBioscience), anti-CD27 (LG.7F9, eBioscience), anti-CD3 (OKT3, eBioscience), anti-CD31 (WM-59, eBioscience), anti-CD38 (HB-7, BioLegend), anti-CD4 (OKT4, BioLegend), anti-CD45 (HI30, BioLegend), anti- CD45RA (HI 100, eBioscience), anti-CD45RO (UCHL1, BioLegend), anti-CD57 (HNK-1, BioLegend), anti-CD62L (DREG-56, BioLegend), anti-CD69 (FN30, BioLegend), anti-CD8 (RPA-T8, eBioscience), anti-CTLA-A (L3D10, BioLegend), anti-CXCR5 (J252D4,

BioLegend), anti-Foxp3 (PCH101, eBioscience), anti-HLA-DR (G46-6, BD Biosciences), anti-ICOS (C398.4A, BioLegend), anti-IgD (IA6-2, BioLegend), anti-Ki67 (Ki-67, BioLegend), anti-PD-1 (EH12.2H7, BioLegend). For Bcl-6, CTLA-4, Foxp3, and Ki67 intracellular staining, Foxp3 Fix/Perm Kit (eBioscience) was used accordingly to manufacturer instructions. For cell viability staining, Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies) was used. Cell Trace Violet Cell Proliferation Kit (Life Technologies) was used for cell proliferation assessment. Cell sorting was performed in Aria IIu and Aria III instruments (BD Biosciences). Flow cytometry analysis was performed in a LSR Fortessa instrument (BD Biosciences) and further analyzed with FlowJo vlO software (TreeStar).

Cell culture and functional assays

For in vitro suppression assays 25 x 10 ³ CXCR5-CD25-CD127 CD4 ⁺ conventional T cells or 25 x 10 ³ CXCR5 CD25 CD127 CD4 ⁺ Tfh cells were plated with CXCR5 CD25 CD127 CD4 ⁺ Tregs cells or CXCR5 CD25 CD127 CD4 ⁺ Tregs cells in 1 : 1 ratio. Cells were cultured with ^g/mL anti-CD3 (OKT3, eBioscience) in the presence of 10 ⁵ irradiated (2500rad) allo- PBMCs. After 5 days cells were harvested and responder cells were analyzed for CTV dilution by flow cytometry. For TCR stimulation assays 25 x 10 ³ CXCR5 CD25 CD127- CD4 ⁺ Tregs cells and CXCR5 CD25 CD127 CD4 ⁺ Tregs were plated with 1μL/well of anti- CD3/anti-CD28 MACSiBead particles (T Cell Activation Kit, Miltenyi Biotec). For co- culture in vitro suppression assays 25 x 10 ³ CXCR5 ⁺ CD25-CD127 ⁺ CD4 ⁺ Tfh cells were plated with CXCR5 CD25 CD127 CD4 ⁺ Tregs cells or CXCR5 CD25 CD127 CD4 ⁺ Tregs cells in 1 : 1 ratio, in the presence of 30 x 10 ³ CD27-IgD ⁺ CD19 ⁺ naive B cells. Cells were cultured with ^g/mL SEB (Sigma- Aldrich). After 5 days responder Tfh cells were analyzed for CTV dilution, B cells for CD38 upregulation and Tregs for follicular and activation markers. For immunoglobulin measurement 25 x 10 ³ CXCR5 CD25-CD127 CD4 ⁺ Tfh cells were plated with CXCR5 CD25 CD127 CD4 ⁺ Tregs cells or CXCR5 CD25 CD127 CD4 ⁺ Tregs cells in 1 : 1 ratio, in the presence of 30 x 10 ³ CD27-IgD CD19 ⁺ naive B cells. Cells were cultured with ^g/mL SEB (Sigma-20 Aldrich) + lOng/mL SEA (Toxin Technology) + lOng/mL SEE (Toxin Technology) + lOng/mL TSST-1 (Toxin Technology). After 10 days supernatants were collected and immunoglobulin concentration determined by ELISA. Cultures were performed in U-shape 6 wells plates in RPMI medium (RPMI 1640, Life Technologies) supplemented with 10% heat-inactivated Fetal Bovine Serum (Life Technologies), 1% HEPES (Sigma- Aldrich), 1% Sodium Pyruvate (Life Technologies), 1% PenStrep (Life Technologies), 1 and 0.05% Gentamicin (Life Technologies) and in 37.° C, 5% C0 ₂ incubator conditions.

Statistical analysis

Unpaired, paired Student T-test, one-way ANOVA with post-test Turkey's Multiple Comparison, and two-way ANOVA with post-test Bonferroni's Multiple Comparison were used as described. Pearson correlation, linear regression and ROC curve analysis was also conducted for some data. Results are presented as mean ± SD. P values of less than 0.05 were considered statistically significant. GraphPad Prism v5 software was used for statistical analysis. Heatmaps were computed in R studio v3.1.2.

Results:

To address the impact of Tfr:Tfh ratio in human autoimmunity, we studied Sjogren syndrome (SS), a systemic autoimmune disease characterized by the lymphocytic infiltration of salivary and lachrymal glands with formation of ectopic lymphoid structures demonstrating the pathogenic involvement of B-T cell interactions (35, 36). We studied a cohort of 25 patients with recently diagnosed SS, accordingly to American European Consensus Group (AECG) criteria (37), under no immunosuppressive treatment other than prednisolone (less than 7.5 mg per day or equivalent) or hydroxycloroquine (Table 1). Unexpectedly, we found a striking increased frequency of circulating Tfr cells in SS as compared to age-matched healthy donors (Figure 1, Figure 34a,b). Interestingly, this CXCR5 ⁺ Treg subset was specifically increased providing an explanation for the high Treg frequency observed in SS patients (Figure 1). SS patients showed a significant increase in the TfinTfh ratio compared to healthy donors (Figure 2a) (12, 14). Furthermore, among SS patients, the increased Tfr:Tfh ratio is associated with patients with serum autoantibodies (Figure 2b). On the contrary, we found no correlation between high Tfr:Tfh ratio with c-reactive protein or disease activity score (ESSDAI) (Figure 3). Table 1: Clinical characteristics of Sjogren's syndrome (SS) patients. Clinical characteristics of 25 patients with primary (pSS) and secondary (sSS) Sjogren's syndrome. RA, rheumatoid arthritis. SLE, systemic lupus erythematosus. PBC, primary biliary cirrhosis. ESSDAI, EULAR Sjogren's syndrome disease activity index. PDN, prednisolone. HCQ, hydroxychloroquine.

Example 2: Blood and tissue Tfr cells present different follicular and regulatory markers

Methods:

Real-time RT-PCR

Total RNA was extracted and reverse transcribed from FACS-sorted CXCR5 CD25- CD127 ⁺ CD4 ⁺ conventional T cells, CXCR5 CD25 CD127 CD4 ⁺ Tfh cells, CXCR5 CD25 CD127 CD4 ⁺ Tregs cells and CXCR5 CD25 CD127 CD4 ⁺ Tregs using RNeasy Micro Kit (Qiagen) according to manufacturer instructions. cDNA was generated using Superscript III reverse transcriptase (Life Technologies), according to manufacturer instructions. Realtime PCR was set up with Power SYBR Green PCR Master Mix (Applied Biosystems) and performed on ViiA 7 Real-Time PCR System (Applied Biosystems), according to manufacturer instructions. The expression of each gene was normalized to housekeeping genes (B2M, ACTB or G6PD) and calculated by change-in-threshold method (ACT), using QuantStudio Real-Time PCR software vl . l (Applied Biosystems).

Immunofluorescence and immunohistochemistry microscopy

After paraffin removal and antigen retrieval by heat (HIER pH 9, Leica Biosystems) 3μιη sequential sections of formalin- fixed paraffin-embedded human tonsil and salivary gland tissue were used for microscopy studies. For immunofluorescence, tonsil sections were stained with anti-human CXCR5-Alexa-Fluor 488 (J252D4, BioLegend), anti-human CD4 (SP35, CellMarque) and anti- human Foxp3 (PCH101, eBioscience) primary antibodies. Alexa-Fluor 488 (anti-mouse), Alexa-Fluor 546 (anti-rabbit) and Alexa-Fluor (anti-Rat) were used as secondary antibodies, respectively. DAPI was used as nuclei counterstaining. Images were acquired with ZEN 2012 software on a Zeiss LSM 710 confocal point-scanning microscope (Carl Zeiss, Oberkochen, Germany) using a dry plan-apochromat 20x objective (200x magnification) and with a numerical aperture of 0.80. Images were further analyzed using Image-J Fiji software. For immunohistochemistry, salivary gland tissues were stained with anti-human CXCR5-Alexa-Fluor 488, anti-human CD4 and CD20, using DAKO EnVision+ System-HRP labeled polymer. Hematoxylin was used as counterstaining. Images were acquired with NanoZoomer-SQ (Hamamatsu, Japan) using a 20x objective with a numerical aperture of 0.75. Images were further analyzed using NDP.view software.

Results:

To test whether CXCR5 Foxp3 ⁺ Tfr cells in human peripheral blood are circulating counterparts of tissue Tfr cells, we studied peripheral blood from a cohort of 42 healthy volunteers between 22 and 92 years old (mean age 46.76 ± 18.14 years old, 30 females and 12 males). We found that CXCR5 was expressed by 18.57 ± 6.55% of total Tregs (defined as CD4 ⁺ CD25 ⁺ Foxp3 ⁺ T cells) (Figure 8a). The frequency and number of CXCR5 ⁺ Foxp3 ⁺ T cells did not change with aging (Figure 8b, Figure 36a).

As CXCR5 is used to identify human circulating Tfh cells, we compared the phenotype of circulating CXCR5 Foxp3 ⁺ Tfr cells with that of circulating Tfh cells and CXCR5- conventional Tregs. Peripheral blood CXCR5 Foxp3 ⁺ T cells share characteristics with both circulating Tfh cells and CXCR5- Tregs (Figure 9). Taking advantage of routine tonsillectomies performed due to tonsil hypertrophy in otherwise healthy children, we compared the cell phenotype of paired blood and tissue samples from the same child (Figure 9, Figure 36b). We found that circulating Tfh cells were phenotypically distinct from their tissue counterparts, in line with previous reports, especially regarding their PD-1, ICOS and Bcl-6 expression (Figure 9) (18, 19). In a similar way, circulating CXCR5 ⁺ Foxp3 ⁺ T cells were also ICOS-PD- Bcl-6-CD57-, and consequently distinct from tonsil Tfr cells (Figure 9). In addition, we confirmed that these cell populations displayed a similar phenotype in adults (Figure 37, Figure 38). Our results are consistent with murine studies showing that blood and tissue Tfr cells are phenotypically distinct (13). Notably, ICOS was not differentially expressed by Tregs and Tfr cells in tonsils (Figure 9). Bcl-6 expression was not detected in any population by real-time PCR (Figure 39b), consistently with previous reports showing that blood Tfh cells do not express Bcl-6 (19-22, 38). We also confirmed that tissue CXCR5 ⁺ Foxp3 ⁺ T cells are localized within germinal centers, therefore, corresponding to Tfr cells (Figure 10a, Figure 39c). Curiously, we observed different Tfr:Tfh ratios in the blood and tonsils (Figure 10b). Example 3: CXCR5 Foxp3 Tfr cells are a distinct subset of suppressive Foxp3 T cells

Results:

It has been described that CXCR5 expression can transiently occur upon human T cell activation (34, 39, 40). Moreover, human T cells can also transiently express Foxp3 upon in vitro TCR stimulation in a TGF-β dependent manner (33, 41).

To address whether ex vivo CXCR5 ⁺ Foxp3 ⁺ Tfr cells were bona fide regulatory cells, we sorted that cell population, as well as CXCR5- conventional Tregs (Figure 40, Figure 41a), and cultured them with CTV-labeled conventional T cells. Proliferation of responder cells was analyzed after 5 days of soluble aCD3 stimulation (Figure 11a). Blood CXCR5 Foxp3 ⁺ Tfr cells significantly reduced conventional T cell proliferation (Figure l ib, Figure 12a), definitely demonstrating their regulatory function.

Stability of Foxp3 expression is required for the suppressive function of Tregs cells (42). In order to determine whether blood Tfr cells have stable Foxp3 expression, we stimulated sorted Tfr cells and CXCR5- Tregs with anti-CD3/CD28 microbeads for 5 days, in the absence of exogenous IL-2. In the absence of IL-2 Tregs do not survive well in culture.

Under these conditions, both CXCR5- Tregs and Tfr cells retain a similar frequency of Foxp3 ⁺ cells, albeit lower than in the beginning of the culture (Figure 12b). Interestingly, the frequency of recovered live Foxp3 -expressing cells was slightly higher for sorted Tfr cells as compared to CXCR5- conventional Tregs. Next, we analyzed the relative expression of Foxp3 and CXCR5 in sorted conventional T cells, Tfh cells, Tfr cells and CXCR5- Tregs from human blood, by real-time PCR. Although Foxp3 protein expression was lower in Tfr cells than in CXCR5- Tregs (Figure 9, Figure 38), Foxp3 gene expression was similar between the two subsets (Figure 13a). In addition, circulating Tfh cell and Tfr cells also showed comparable CXCR5 gene expression (Figure 13a).

To investigate whether activation of blood Tfr cells triggers upregulation of Foxp3, CD25 and CTLA-4, a phenomena known to be associated with increased Treg suppressive function (43), we analyzed the phenotype of sorted CXCR5 ⁺ and CXCR5- Tregs after 5 days of culture in presence of aCD3/CD28 microbeads. We found an upregulation of Foxp3 and CD25 by Tfr cells, while CTLA-4 was increased in both populations (Figure 13b, Figure 41b). The levels of expression of these markers by blood Tfr cells after activation resembled those from tissue Tfr cells (compare with Figure 9). Importantly, CXCR5 upregulation was not detected in sorted CXCR5- Tregs showing that CXCR5 Tfr cells are a distinct subset of human blood Tregs.

Conclusions:

Our comprehensive evaluation of human Tfr support a model in which blood Tfr cells are generated following the initial steps that lead to germinal centre responses in secondary lymphoid tissues, exiting the tissue prior to interactions with B cells that are required for complete differentiation towards tissue resident Tfr cells.

Although some studies have quantified blood CXCR5 ⁺ Tregs as circulating Tfr cells in different diseases, the human biology of CXCR5 ⁺ Tregs remains elusive (28, 29, 31 , 52, 53). Moreover, most of the literature describe studies where what is defined as blood Tfh cells contain both Tfh and CXCR5 ⁺ Tfr cells, while many others studies identify as Tregs a mixture of bona fide conventional Tregs together with CXCR5 ⁺ Tfr cells. As such, results may be confounded by combining effector and regulatory cell populations. As an example, our cohort of SS patients show an increase in the frequency of Foxp3 ⁺ Tregs, compared with the control population. However, only CXCR5 ⁺ Tfr cells, and not conventional CXCR5- Tregs, are increased in those patients. As a consequence, the apparent increase of Tregs in the blood of SS patients is in fact an increase o f CXCR5 Tfr cells that reflect the ongoing humoral activity. It was the search for an explanation for this apparent counterintuitive observation that led us to establish the ontogeny and function of human circulating Tfr cells.

We found that Tfr cells in tonsils have follicular and regulatory markers and were found within germinal centers, whereas blood Tfr cells do not express ICOS, PD-1, or Bcl-6, apparently diverging these cells from follicular imprinting. Previous studies have described low ICOS and PD-1 expression, and no Bcl-6 expression in human blood Tfh cells (18). In mice, blood Tfr cells have also lower expression of ICOS (13). It was also reported that murine circulating Tfr cells can bypass the B cell zone and do not gain full activation as part of a memory programmed state (13). In line with these studies, the absence of ICOS, PD-1 and Bcl-6 from human blood CXCR5 ⁺ Tfr cells does not exclude their follicular ontogeny.

Example 4: Blood Tfr cells do not preferentially suppress humoral responses

Methods:

ELISA IgA, IgM and total IgG concentration were determined in supernatants from T-B co-culture (as described above) by ELISA using Human ELISA Ready Set Go Kit, according to manufacturer instructions (eBioscience).

Migration Assays

For in vitro chemotaxis assays 75 x 10 ³ CXCR5 CD25 CD127 CD4 ⁺ conventional T cells, CXCR5 CD25 CD127 CD4 ⁺ Tfh cells, CXCR5 CD25 CD127 CD4 ⁺ Tregs cells and CXCR5 ⁺ CD25 ⁺ CD127-CD4 ⁺ Tregs cells were loaded on top wells of HTS Transwell

96-well permeable supports (5μιη pore size) (Corning). Plain RPMI medium (RPMI 1640, Life Technologies) or that supplemented with O^g/mL CXCL13 (Peprotech) was added to the bottom wells of the plate. After 4 hours of incubation (37. °C, 5% C0 ₂ ), filters were removed and cells that migrated to the lower chamber were counted in a LSR Fortessa instrument (BD Biosciences) and further analyzed with Flow Jo vlO software (TreeStar). Chemotaxis index was calculated as the ratio of cells migrating toward CXCL13 and cells randomly migrating.

Results:

In order to address the function of blood Tfr cells we first investigated if this population could directly suppress Tfh cells. Using a similar in vitro assay used to prove the regulatory capacity of blood Tfr cells, but with sorted Tfh cells as responders, we found that blood Tfr cells strongly suppressed Tfh cell proliferation, however without a specific advantage when compared with CXCR5- Tregs (Figure 14a). Next, to directly assess the impact of blood Tfr cells on B cell activation, we used in vitro T-B co-cultures in presence of SEB superantigen (Figure 15). After 5 days of culture, B cells upregulated CD38 and downregulated IgD only in presence of Tfh cells (Figure 15). Both CXCR5- and CXCR5 ⁺ Tregs impaired the generation of CD38 ⁺ IgD- GC-like B cells. Consistent with our results from suppression assays with Tfh cells (Figure 14a), Tfh cell proliferation was similarly inhibited by CXCR5- and CXCR5 ⁺ Tregs (Figure 15, Figure 16a, and Figure 42a). As expected Tfh cells showed better proliferation responses in co-culture with B cells.

To further address the function of blood Tfr cells on humoral responses we analyzed class switch recombination by naive B cells 10 days after superantigen stimulation. We found that blood Tfr cells, although able to reduce activation of naive B cells and proliferation of Tfh cells as shown before, did not significantly limit class switch recombination by B cells, as no impact on IgA nor IgG production was observed (Figure 16b). On the contrary, CXCR5- Tregs efficiently suppressed humoral responses (Figure 16b). CXCR5/CXCL 13 -dependent migration to GC is critical for suppression of humoral responses by Tfr cells (9, 11) and plasma CXCL13 levels have been correlated to ongoing GC responses in humans (44). To prove that blood Tfr cells were capable to migrate towards a CXCL13 gradient we conducted in vitro chemotaxis assays with sorted populations from human peripheral blood. We found that, although the CXCR5 MFI of peripheral Tfh and Tfr cells was slightly different (Figure 9, Figure 38), both populations shared their ability to migrate towards a CXCL13 gradient, showing functional capacity of blood Tfr cells to enter CXCL13 enriched tissues (Figure 16c).

Conclusions:

Our results show key differences between mice and humans regarding the function of blood CXCR5 Tfr cells: while murine blood Tfr cells appear to be specialized in suppressing antibody production (despite their lower suppressive capacity when compared to tissue Tfr cells) (13, 14, 16, 54), human blood Tfr cells do not have the ability to fully suppress humoral responses.

We found that blood Tfr cells specifically migrated towards CXCL13 gradient, suggesting these cells have the capacity to reach the follicles. Importantly, CXCR5- conventional Tregs did not upregulate CXCR5 upon in vitro activation, further confirming CXCR5 -expressing Tfr cells as a distinctive subset.

Example 5: Blood Tfr cells have distinctive naive-like phenotype

Results:

To explain the surprising observation that blood Tfr cells do not suppress antibody production we hypothesized that this population could represent thymus-derived precursors of Tfr cells not yet fully committed to regulate humoral responses. Indeed, we found that blood Tfr cells were predominantly CD45RO-Foxp3 ^l0 resting Tregs (Figure 17a), expressing high levels of CD45RA, CCR7, CD62L, CD27 and low levels of HLA-DR, reminiscent of a na ^' ive phenotype (Figure 17b). Virtually, all blood Tfr cells were quiescent Ki-67- non- proliferating cells when analyzed ex vivo (Figure 18a). Moreover, circulating Tfr cells were virtually devoid of CD45RO CCR7- effector-memory cells in striking contrast to CXCR5- Tregs, a phenotype more similar to circulating Tfh cells (Figure 18b). While the vast majority of blood Tfh cells were CD45RO CCR7 central-memory cells, consistently with previous reports (18-21), a significant proportion of Tfr cells were CD45RO-CCR7 ⁺ naive cells (Figure 18b). Furthermore, the few CD45RO- Tfh cells did not express high levels of CD45RA indicating that those cells were not really naive, in contrast to Tfr cells (Figure 43a). Therefore, blood Tfr cells constitute a pool of na ^' ive resting cells.

To test whether blood Tfr cells were indeed thymus-derived precursors of tissue Tfr cells we analyzed the frequency of these cells accordingly to age. Contrary to thymic derived na ^' ive Tregs, CD45RO-CCR7 ⁺ na ^' ive Tfr cells did not significantly decrease with increasing age (Figure 19a). In addition, the expression of CD31, a marker used to identify recent thymic emigrants in human blood (45-47), was not specifically enriched in the population (Figure 43b, Figure 44a). Although these observations suggest blood Tfr cells are not a thymic population, this was not conclusive. Therefore, we directly examined CXCR5- expressing T cells in the human thymus and neonatal cord blood. There was not a population of CXCR5 ⁺ Tregs detected in any of those tissues (Figure 19b,c and Figure 44b). Although, CXCR5- expressing Tregs were not found in cord blood, some Foxp3 ⁺ cells expressed CD45RO, suggesting that additional activation signals not present before birth are required to shape a CXCR5 phenotype in circulating Tregs. Consistent with our previous data, ICOS ⁺ Tregs were detected in cord blood, indicating that ICOS cannot be used as a specific follicular marker in circulating human Treg cells (Figure 19d).

Conclusions:

We found that blood Tfr cells have a prominent na ^' ive phenotype. However, they are absent from the thymus and cord blood (where activated Tregs can already be found).

These observations provide compelling evidence that activation signals generated in peripheral lymphoid organs are required to shape a CXCR5 ⁺ phenotype on human Foxp3 ⁺ T cells. Conversely, tissue Tfr cells are almost all CD45RO ⁺ antigen experienced effector cells. Taken together, these observations led us to hypothesize that blood Tfr cells leave lymphoid tissues as immature cells, prior to B cell interaction in T-B border, and full differentiation into Tfr cells. This view was supported by the presence of blood Tfr cells in peripheral blood of patients lacking B-cells due to genetic defects. This finding provides an explanation for the incomplete suppressive function of blood Tfr cells. An important limitation of our study is the difficulty to isolate tissue Tfr cells for functional assays, as CD25 and CD127 are not reliable to identify tonsil Foxp3 -expressing cells. However, the phenotype between blood and tissue Tfr cells is remarkably different in particular with respect to maturation markers.

Example 6: Blood Tfr cells emerge from lymphoid organs before B-cell interaction

Results:

Having demonstrated that circulating Tfr cells did not egress from the thymus, we investigated whether Tfr cells recirculate from secondary lymphoid tissues before being fully committed to tissue Tfr cells.

We compared both CXCR5 ⁺ and CXCR5- Treg subsets from children paired blood and tissue (tonsils) concerning their effector-memory, central-memory and naive composition. We found that CD45RO ⁺ CCR7- effector Tfr cells were present in lymphoid tissues but not in blood, suggesting that effector Tfr cells are selectively retained in tissues, similarly to effector Tfh cells (Figure 20). Therefore, it is unlikely that blood CD45RO- Tfr cells derive from the fully mature tissue Tfr cells that express CD45RO, as the few CD45RA-re- expressing end-stage memory CD4 ⁺ T cells do not become CD45RO- (Figure 46) (48, 49).

Our data suggest that blood Tfr cells are generated in secondary lymphoid tissue prior to full differentiation towards mature Tfr cells. It has been know that full differentiation of follicular T cells requires a two-step process with an initial activation mediated by dentritic cells and a subsequent B cell interaction in the B-T border. We investigated whether blood Tfr cells, given their immature phenotype could be generated before the B cell interactions required for acquisition of terminal differentiation. To investigate this issue we analyzed peripheral blood from X-linked Agammaglobulinemia (BTK- deficient) patients, with a complete absence of CD19 ⁺ cells. We observed a striking decrease in blood Tfh cells in those patients, in line with previous reports (Figure 21) (50). However, frequency of blood Tfr cells were not decreased in B-cell deficiency patients (Figure 21). These observations are conclusive in establishing that blood Tfr cells enter the circulation before B-cell contact, while the majority of blood Tfh cells require B cell interactions. To investigate whether CD45RO ⁺ and CD45RO- blood Tfr cells could discriminate between Tfr cells recirculating before and after B-cell interaction, we analyzed these two populations in peripheral blood of patients with B-cell deficiency, as well as in SS patients. We found no significant differences in CD45RO ⁺ or CD45RO- Tfr cells in these two diseases (Figure 45), suggesting that although CD45RO upregulation occurs irrespective of B-cell interaction on Tfr cells.

Conclusions:

Importantly, our results from vaccination and SS patient cohorts show that blood Tfr cells are indicative of ongoing humoral activity. In SS patients, where ongoing germinal center reactions promote the production of autoantibodies (35, 36), blood Tfr cells were significantly increased (directly contributing to an increased Tfr:Tfh ratio). Although, we expected to find a decrease in this putative humoral suppressive cell population in autoimmune conditions, our results suggest that blood Tfr cells indicate ongoing humoral activity and are not a measurement of suppressive potential. This is in line with recently published reports showing an increase in blood CXCR5 ⁺ Tregs in other autoimmune conditions and infectious diseases (31, 32, 53). Therefore, studies regarding blood CXCR5 ⁺ Tregs in different human settings should be carefully interpreted.

Example 7: Blood Tfr/ Tfh ratio is a biomarker of ectopic GC responses in Sjogren Syndrome

Results:

As Tfr / Tfh ratio was increased in SS patients with the presence of serum autoantibodies, suggesting a disturbed GC activity, we hypothesized Tfr / Tfh ratio is a biomarker of ectopic GC formation in the target tissue of SS. To test this hypothesis, we analyzed salivary gland biopsies of our SS cohort compared to salivary gland tissue of patients referred to salivary gland biopsy not fulfilling any of SS diagnostic criteria (Figure 46). SS patients showed a significant increase in CD45 -expressing hematopoietic cells in salivary gland tissue, as well as B cells (Figure 23). Salivary gland infiltration by CD19 ⁺ B cells was specifically observed in SS patients, without differences in CD4 ⁺ T cell infiltration between SS and controls (Figure 23). As we were not able to directly study CXCR5 -expressing cells due to loss of this chemokine receptor during tissue processing (Figure 46, Figure 47a,b), we analyzed PD- l ⁺ ICOS ⁺ T cells. This subset of T cells is enriched for CXCR5 ⁺ Tfh (Figure 47c) and only present in tissues harboring GC (data not shown). Additionally, a recent report demonstrated that CXCR5 PD-1 TCOS T cells in human non-lymphoid tissues provide help to B cells, similarly to Tfh cells (51). Interestingly, we found a striking increase in this T cell subset in SS (Figure 24a).

We found that peripheral blood Tfr / Tfh ratio correlated with salivary gland infiltration by lymphocytes (Figure 24b), being a good predictor of these pathological changes in SS patients (Figure 25 a). Therefore, our data suggest that peripheral blood Tfr / Tfh ratio is a biomarker of ectopic lymphoid structure activity in SS. Indeed, Tfr / Tfh ratio discriminated SS patients with no histological changes in their salivary gland tissue, patients with unspecific lymphocytic infiltration and patients with diagnostic focal sialoadenitis (Figure 25b, Figure 33a). Additionally, Tfr / Tfh ratio was found to be a potential new diagnostic tool to identify the subgroup of SS patients with the highest level of disturbed GC activity (Figure 33b).

Conclusions:

Our data also demonstrate that peripheral blood Tfr / Tfh ratio is a biomarker of ectopic GC responses in target organ of SS. Therefore, Tfr / Tfh ratio is a putative novel diagnostic tool to identify more suitable SS patients to therapies targeting T-B interactions.

Given that circulating Tfr cells have an immature phenotype, it is not surprising that blood Tfr cells are not fully endowed with suppressive function, because the suppressive capacity of conventional Treg cells have been ascribed predominantly to those cells with a more mature phenotype. The TCR repertoire of Tfr cells is different from Tfh and probably skewed towards auto-antigens (55), it is possible that circulating Tfr cells represent a pool of cells ready to be recruited into subsequent GC responses as they retain the ability to migrate towards CXCL13.

In conclusion, these data support a model in which CXCR5 ⁺ Bcl6- T cells egress from secondary lymphoid tissues during antigen-driven immune responses. While, the frequency of blood Tfh cells is reduced in the absence of B cells, Tfr cells do not require interactions with B cells. Thus, the acquisition of a CXCR5 ⁺ Foxp3 ⁺ phenotype in the tissues precedes access to the follicle, where the cells acquire a fully mature phenotype. As a consequence, circulating Tfr cells represent lymphoid tissue derived Tfr precursors not yet endowed with full B-cell and humoral regulatory function. References:

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