HUMAN BIOMARKERS FOR DETERMINING INTRINSIC B-CELL DEFECTS

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of Application No. 61/030,794, filed February 22, 2008.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT The U.S. Government has certain rights in this invention as provided for by contract NIH-R21-AG28586 from the Department of Health and Human Services.

BACKGROUND OF THE INVENTION

This invention relates to human biomarkers (i.e., E47, AID, IgH that has undergone class switching), which characterize the immune potential of B lymphocytes, which produce immunoglobulins (Ig), and their ability to respond to infections by pathogens and vaccines.

Aging affects the humoral immune response in humans both quantitatively and qualitatively with reduced antibody specificity, affinity, and isotype switch (1-3). The changes in the humoral immune response during aging significantly contribute to the increased susceptibility of the elderly to infectious diseases and reduce the protective effects of vaccination. The decreased ability of aged subjects to produce high affinity protective antibody responses against infectious agents or experimental antigens likely also results from additional defects in required T-cell signaling to B cells (4-6). Although B-cell function may suffer from lack of a good T-cell help in aging, intrinsic changes in B cells also occur and have a significant impact on antibody production and somatic mutation (3, 7-9).

During an immune response, B cells can switch the expression of surface Ig from IgM to IgG, IgE, or IgA. This DNA recombination takes place between two switch (S) regions, one located immediately upstream (5 ¹ ) of the μ CH (donor site) and one 5' of one of the other CH regions (γ, ε or α) (acceptor site) to produce IgG, IgE or IgA. The CSR requires chromatin opening of S

regions, recognition and cleavage of the target DNA by an endonuclease and repair and ligation of the cleaved ends (10-15). CSR is extremely important for the humoral immune response, because it generates antibodies of the same specificity, but with different effector functions. In vitro, human naive B cells can be activated to undergo CSR with anti-CD40 or CD40L, together with cytokine signals which can direct CSR to distinct classes. The critical role of CD40- CD40L interaction for CSR is provided by the discovery that mutations in either the CD40 or CD40L (CD154) gene cause the hyper IgM (HIGM) syndrome, HIGM3 or HIGM1 , respectively, characterized by very low levels of serum IgG, IgA, and IgE, with normal or elevated IgM, associated with defective germinal center formation (16).

Human B cell numbers and percentages have been shown to decrease significantly with age (17, 18). It has also been shown that the percentage of IgM memory B cells, which are responsible for the response to S. pneumoniae infection, is significantly decreased whereas that of naϊve B cells is increased in old subjects (19). The reduction in IgM cells has been suggested to cause reduced specific antibody titers in elderly subjects vaccinated against pneumococcal polysaccharides. Others have shown that the percentage of CD27+ memory B cells increases with age, but this increase is not significant (20). Also in the human tonsil, naϊve B cells have been shown to increase with age (21 ). In mice, the number of peripheral B cells is not decreased with age. This discrepancy between humans and mice likely reflects different mechanisms to maintain the number of B cells, possibly by increasing their survival in mice (22). So far, to our knowledge, no one has studied age-related changes in naϊve and memory B cells in mice, probably because good memory B cell markers in the mouse system were not available (cf. CD27 in human).

We have previously shown , only in mice (8, 9) that the E2/\-encoded transcription factor E47 is down-regulated in old splenic B cells, leading to a reduction in AID and in CSR. This intrinsic defect in old splenic B cells gene- rates reduced amounts of switched antibodies. We have extended our study to investigate whether aging also affects antibody production and both E47 and AID expression in B cells isolated from the peripheral blood of human subjects

(18 to 86 years). Results herein show that the expression of E47, AID and CSR progressively decrease with age. We also show that an age-related decline in the number and percentage of switch memory B cells, no change in the percentage but a decline in the number of IgM memory B cells, and an increase in the percentage but not in the number of naϊve B cells, consistent with our data on the decrease seen in CSR in vitro. In addition, we suggest that the subsets of B cells able to class switch in culture are the naϊve and the IgM memory populations. Our results provide possible molecular mechanisms for an intrinsic decrease in B-cell function and the humoral immune response in aging. At the time this invention was made, there was no reasonable expectation from our prior work in mice that such results would be obtained in human subjects.

Therefore, it is an objective of the present invention to provide biomar- kers for determining intrinsic B-cell defects in a humoral immune response of a human subject. A long-felt need is addressed thereby. Other advantages and improvements are described below or would be apparent from the disclosure herein.

SUMMARY OF THE INVENTION The invention provides biomarkers for predicting or determining whether there is an intrinsic defect in the B cells participating in a humoral immune response of a human subject. The subject may have reduced immunocompe- tence because of age (e.g., less than 3 or more than 65 years of age), disease or treatment of a disease, a genetic defect, suppression of the immune system, etc. Although reduction in B-cell expression of a single biomarker may be suffi- cient for predicting or determining whether there is an intrinsic B-cell defect, it could also be required that at least two of the biomarkers or all three (i.e., E47, AID and IgH that has undergone class switching) are reduced.

In particular, a method of the invention may include one or more of the following: obtaining a biological sample, which contains B cells, from a human subject; optionally, isolating B cells from the biological sample (i.e., the method may detect expression from a biological sample that was neither fractionated nor selected for cell subpopulations); optionally, stimulating in vitro B cells to

divide and differentiate at least some B cells being cultured (i.e., the method may detect expression in a biological sample or cell subpopulation that was not stimulated or cultured in vitro) or, alternatively, freshly isolated B cells are analyzed ex vivo without being stimulated during in vitro culture; and detecting expression in the B cells of transcription factor E47, activation-induced cytidine deaminase (AID), or an immunoglobulin heavy chain (IgH) that has undergone class switching to IgG, IgE or IgA. As an alternative to detecting expression of IgH that has undergone class switching, other proxies for the IgH biomarker may be detected in B cells such as episomes produced by DNA recombination, Igγ1 circle transcripts, or somatic mutation. Therefore, only obtaining a biological sample and detecting expression of a biomarker in B cells are required in the method. In some embodiments, antibody production may not be measured. A human subject may be or may have been immunized with a vaccine. The subject may be a resident in a healthcare or assisted living facility, a reci- pient of a solid organ transplant or renal dialysis, afflicted by a chronic cardiovascular or pulmonary disorder, at risk for or affected by autoimmune disease (e.g., type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, especially those with a significant B-cell component involved in pathogenesis), immunocompromised or immunosuppressed, etc. in need of treatment. Alternatively, the subject may be young and needing a series of childhood immunizations. Having an intrinsic B-cell defect in the subject may be used to stage disease or to recommend treatment at an early or late stage of disease pathogenesis.

Further objectives and advantages aspects of the invention will be apparent to a person skilled in the art from the following description of specific embodiments and the claims, and generalizations thereto.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows age-related changes in B-cell percentages and numbers. B cells isolated from the peripheral blood (PBMC) of subjects of different ages using CD19 magnetic beads, were analyzed for percentage of cells expressing membrane CD19 (Fig. 1A), and absolute number of CD19+ cells calculated

(Fig. 1B). Similarly, the percentages of naive (lgG-/lgA-/ CD27-) B cells, IgM memory (lgG-/lgA-/CD27+) B cells, and switch memory (lgG+/lgA+/CD27+) B cells are shown in Figs. 1 C, 1 E and 1 G, respectively, and the numbers of naϊve (lgG-/lgA-/ CD27-) B cells, IgM memory (lgG-/lgA-/CD27+) B cells, and switch memory (lgG+/lgA+/CD27+) B cells are shown in Figs. 1 D, 1 F and 1 H, respectively. The Pearson's r value for a linear correlation between age and these immune variables, as well as its statistical significance are as follows: B cell numbers: r = -0.68, p = 0.001 ; B-cell percentages: r = -0.64, p = 0.001 ; naϊve B cell numbers: r = 0.19, p = 0.31 ; naϊve B cell percentages r = 0.54, p = 0.002; IgM memory B cell numbers: r = 0.67, p = 0.001 ; IgM memory B cell percentages: r = -0.23, p = 0.220; switch memory B cell numbers, r = 0.726, p = 0.001 ; and switch memory B cell percentages: r = -0.48, p = 0.003. Naϊve B-cell and IgM memory B-cell percentages showed significant quadratic relationships to age. The quadratic, non-linear coefficient betas are, respectively: -2.519 {t- value = -2.561 , p = 0.016) and 2.64 (f-value = 2.279, p = 0.03). Significant differences between young and old are indicated by *(p < 0.05) and ^* *(p < 0.01 ). The solid line refers to linear regression; the dashed line refers to quadratic regression.

Figure 2 shows aging decreases class switch in activated peripheral blood-derived human B cells. B cells (10 ⁶ cells/ml), isolated from the peripheral blood of subjects of different ages using CD19 magnetic beads, were cultured with anti-CD40 (1 μg/ml) and IL-4 (10 ng/ml), for a period of 6 to 7 days. At the end of this period, cells were harvested, RNA was extracted, and sqPCR reactions were performed to evaluate CTs and GAPDH transcripts. Undiluted and 1 :4 diluted RT-PCR from five representative subjects are shown in Fig. 2A. Densitometric analyses (arbitrary units) of CTs, normalized to GAPDH, from 46 subjects of different ages are shown in Fig. 2B. The numbers shown for each sample are percentages of the highest value (randomly chosen) normalized to 100. The Pearson's r value for a linear correlation between age and these immune variables, as well as its statistical significance are r = -0.84, p = 0.001. In Fig. 2C, B cells were cultured as described above. IgG level in culture super- natants were determined by ELISA. The Pearson's r value for correlations

between age and this immune variable, as well as its statistical significance are r = -0.75, p = 0.001. In Fig. 2D, the IgG levels in serum were determined by ELISA. The Pearson's r value for correlations between age and this immune variable, as well as its statistical significance are r = 0.572, p = 0.001 . The solid line refers to linear regression; the dashed line refers to quadratic regression.

Figure 3 shows aging decreases AID mRNA expression in activated peripheral blood-derived human B cells. B cells (10 ⁶ cells/ml), isolated from the peripheral blood of subjects of different ages using CD19 magnetic beads, were cultured with anti-CD40 (1 μg/ml) and IL-4 (10 ng/ml), for a period of 6 to 7 days. At the end of this period, cells were harvested, RNA was extracted, and sqPCR reactions were performed to evaluate AID and GAPDH transcripts. The graph in Fig. 3A shows the amounts of AID mRNA, normalized to GAPDH, from 34 subjects of different ages. These are 34 out of the 46 evaluated by sqPCR (not shown). Values for seven subjects between the ages of 67 and 79 were undetectable (at the x axis). Numbers are percentages of the highest value (randomly chosen) normalized to 100%. The Pearson's r value for a linear correlation between age and these immune variables, as well as its statistical significance are: r = -0.78, p = 0.001. AID mRNA expression showed significant quadratic relations to age. Quadratic, non-linear coefficient betas were the same for qPCR. Alternatively, B cells (10 ⁶ cells/ml) were cultured as above but for only one day (optimal timepoint for E47 mRNA expression). The graph in Fig. 3B shows the amounts of E47 mRNA, normalized to GAPDH, from 34 subjects of different ages. These are 30 out of the 46 evaluated by sqPCR (not shown). The Pearson's r value are: r = -0.75, p = 0.001 . E47 mRNA expression also showed significant quadratic relations to age. The quadratic, non-linear coefficient betas were: -1 .866 (f-va!ue = -2.745, p = 0.009). In Fig. 3C, sqPCR data for E47 and AID were positively correlated. Correlation is significant at the 0.01 level (2-tailed). The solid line refers to linear regression; the dashed line refers to quadratic regression. Figure 4 shows both memory and naive B cells are impaired in their ability to undergo in vitro class switch. Sorted memory (CD19+CD27+) B cells or naive (CD19+CD27-) B cells were stimulated (10 ⁴ cells/200 μl) with anti-

CD40/IL-4, alone or together with F(ab') ₂ fragments of goat anti-human Ig, for a period of five days (at later times, viability in culture significantly dropped off). The graphs in Figs. 4A, 4B, 4C and 4D show amounts of AID mRNA (qPCR) which are normalized to GAPDH ₁ expressed as δCts from four pairs of young (white columns) and old (black columns) subjects. For each pair, results are expressed as percentages of the anti-CD40/IL-4-stimulated memory B cells from young subjects taken as 100. Ages of young #1 , 2, 3 and 4 were 20, 21 , 24 and 23 years and those of old #1 , 2, 3 and 4 were 72, 82, 74 and 71 years. These subjects are not represented in Fig. 3. The differences between anti- CD40/IL-4-stimulated memory B cells from young and old subjects (columns 1 vs. 5) are p < 0.05 for the four pairs, and between anti-CD40/IL-4/lgM-stimu- lated memory B cells from young and old subjects (columns 2 vs. 6) are p < 0.01 (pairs #1 and #3), p < 0.05 (pair #4), and not significant (pair #2). The differences between anti-CD40/IL-4/lgM-stimulated naive B cells from young and old subjects (columns 4 vs. 8) are p < 0.05 for the four pairs. The solid line refers to linear regression; the dashed line refers to quadratic regression.

Figure 5 shows class switch in subsets of B cells. B cells from three young subjects were sorted into IgM memory, switch memory, and naϊve B cell subsets and stimulated (10 ⁴ cells/200 μl) with anti-CD40/IL-4, alone or together with F(ab') ₂ fragments of goat anti-human Ig, for a period of five days. Columns show the amounts of AID mRNA (qPCR) which are normalized to GAPDH. Results (mean ± SE) are expressed as percentages of the IgM memory B cells taken as 100%.

Figure 6 shows that the in vitro AID response of B cells to vaccination decreases with age. Regression coefficients before and after vaccination were, respectively: -0.61 and -0.88 (CpG in Fig. 6A); -0.79 and -0.97 (Ag1 in Fig. 6B): -0.83 and -0.94 (Ag2 in Fig. 6C); -0.84 and -0.97 (Ag3 in Fig. 6D). All regression p values are < 0.05.

Figure 7 shows that the serum hemaglutinin inactivation (HI) response to vaccination decreases with age. Regression coefficient before and after vaccination were, respectively: -0.20 and -0.60 (vaccine in Fig. 7A); 0.07 and -0.023 (Ag1 in Fig. 7B); -0.19 and -0.36 (Ag2 in Fig. 7C); 0.09 and -0.40 (Ag3 in Fig.

7D). p values of the regressions before and after vaccination were, respectively p = 0.23 and p < 0.05 (vaccine); p = 0.84 and p = 0.25 (Ag1 ): p = 0.05 and p = 0.06 (Ag2); p = 0.81 p = 0.05 (Ag3).

Figure 8 shows relative AID gene expression in B cells of AID. Subjects were grouped as at risk for type 1 diabetes, new onset of type 1 diabetes, or long term afflicted with type 1 diabetes. Healthy subjects are the control group.

Figure 9 shows relative IgH gene expression in B cells that have undergone class switching. Subjects were grouped as at risk for type 1 diabetes, new onset of type 1 diabetes, or long term afflicted with type 1 diabetes. Healthy subjects are the control group.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

In a specific embodiment, a B-cell defect in a humoral immune response of a human subject may be predicted or determined. B cells may be obtained from the human subject. The obtained B cells may or may not be stimulated during in vitro culture; thus, they may be freshly isolated B cells to be analyzed directly ex vivo (i.e., no stimulation during in vitro culture) or stimulated B cells to be analyzed after in vitro culture. B cells may or may not be isolated from other cell types prior to analysis. Analysis of the B cells involves at least detecting expression of transcription factor E47, activation-induced cytidine deaminase (AID), or an immunoglobulin heavy chain (IgH) that has undergone class switching to IgG, IgE or IgA. Reduced expression of E47, AID, IgH that has undergone class switch (or a combination of at least two or all three) as compared to a control indicates that there is a B-cell defect in the humoral immune response.

The human subject may be less than six months, less than one year, less than two years, less than three years, less than four years, less than five years, more than 50 years, more than 55 years, more than 60 years, more than 65 years, more than 70 years, more than 75 years of age, or any range of ages therebetween.

The control may be expression of E47, AID or IgH (or a combination of at least two or all three) in B cells obtained from a human aged more than five

years, more than ten years, more than 20 years, less than 50 years, less than 40 years, less than 30 years, or any range of the ages therebetween. The control may determined by ex vivo or in vitro analysis.

A biological sample may be obtained from the human subject. The biolo- gical sample may be blood (preferably with added anticoagulant such as EDTA, citrate, oxalate, or heparin); mononuclear cells derived therefrom by centrifuga- tion (preferably involving at least density gradient medium) or chromatography (preferably involving at least affinity medium for lymphocytes or a subpopula- tion thereof such as CD19+); or cells isolated from bone marrow, lymph node, spleen, or thymus from the human subject. A preservative may be added to the sample to stabilize nucleic acids, proteins, or both of B cells.

B cells may be stimulated through their CD40 or IL-4 signaling pathway, or both. Isolated B cells may be stimulated during in vitro culture with at least antibody specific for a B-cell receptor (e.g., CD40, surface Ig, a Toll-like recep- tor or TLR, a cytokine receptor), a ligand for the B-cell receptor (e.g., CD40L, cognate antigen, CpG, dsRNA, lipopolysaccharide, other pathogen associated molecular pattern or TLR ligand), a cytokine (e.g., TNF-α, IL-4, IL-5, IL-6), or any combination thereof.

Expression of E47, AID or IgH that has undergone class switching may be detected by measuring RNA or protein using conventional techniques such as run-on transcription, nuclease protection, Northern blotting or real-time PCR, DNA protection or gel retardation, Western blotting, and immunoassay such as ELISA. But the technique used for measurement of nucleic acids or proteins would not need to be specific for this analysis, any known in the art or later developed may be used. Instead of measuring RNA or protein of IgG, IgE or IgA, one or more proxies of class switching (e.g., episome produced by DNA recombination, Igγ1 circle transcript, or somatic mutation) may be detected instead. The class switching and/or somatic mutation of antibody produced by B cells may be reduced as compared to control similar to that for E47 or AID. The human subject may be or may have been immunized with at least one vaccine (e.g., normally prescribed dose, increased dose, with an adjuvant). The subject may be immunized on the same day as obtaining B cells from the

subject, or within one week (before or after) of obtaining B cells from him or her. The vaccine may be therapeutic or prophylactic. For example, the subject may have been or would be immunized with one or more vaccines: e.g., diphtheria, influenza, measles, meningococcal, mumps, pertussis, pneumococcal, rubella, tetanus, varicella, and zoster vaccines. The subject may have been or would be immunized with at least a carbohydrate or proteinaceous antigen that induces a humoral immune response against a pathogen (e.g., bacterium or virus) or a cancer (e.g., neoantigen or tumor antigen).

For a human subject predicted or determined to have a B-cell defect in the humoral immune response, the subject may be in need of treatment. An increased dose of vaccine may be administered to the subject as compared to a control (e.g., two doses); an adjuvant may be administered with the vaccine (e.g., aluminum salts such as AIOH or AIPO ₄ , CpG or dldC, modified lipid A such as MF59, saponins such as QS21 , a squalene emulsion); or both. Elderly human subjects have compromised humoral and cellular immune responses, that lead to reduced protection to infectious agents and to vaccination. Currently available vaccines suboptimally protect the elderly population. The capacity to class switch the Ig heavy chain is critical to the effectiveness of humoral immune responses in mice and humans. We have previously shown in mice that the E2/\-encoded transcription factor E47, which regulates many B cell functions is down-regulated in old splenic B cells. This leads to a reduction in the activation-induced cytidine deaminase (AID), which is known to induce class switch recombination (CSR) and Ig somatic hypermutation (SHM). The old activated murine B cells also have less AID and less switched antibodies. We have extended our study here to investigate whether aging also affects antibody production, E47 and AID expression in B cells isolated from the peripheral blood (PB) of human subjects (18 to 86 years). Our results obtained with activated CD19+ B cells show that the expression of E47, AID and Igγ1 circle transcripts progressively decrease with age. We also show an age-related decline in the percentage of switch memory B cells (lgG+/lgA+/CD27+), an increase in that of naive B cells (lgG-/lgA-/CD27-) for most individuals and no

decrease in that of IgM memory cells in PB, consistent with our data on the decrease seen in CSR in vitro.

EXAMPLES B lymphocytes purified from peripheral blood of human subjects aged primarily over 65 years, which were stimulated in vitro with various stimuli (e.g., anti-CD40 plus IL-4), have suboptimal response as measured by Ig class switch (both at the protein and DNA recombinant levels), AID (activation- induced cytidine deaminase, the enzyme necessary to accomplish not only class switch recombination (CSR) but also somatic hypermutation (SHM), necessary for the optimal quality of all Igs), and E47 (a transcription factor known to regulate AID). We also describe the unique primers used to optimally amplify E47 and AID by semi-quantitative and quantitative (real time) RT-PCR. We have previously published similar results in mice, but this is the first demon- stration of (1 ) intrinsic B-cell defects in aged humans and, more specifically, (2) specific combinations of biological markers to easily classify the "goodness" of a human B-cell response.

Subjects Peripheral blood mononuclear cells (PBMC) were isolated from healthy human volunteers of different ages (18 to 86 years) after appropriate signed consent. Initial experiments to optimize the conditions for cell isolation and culture were performed with spleen cells from human cadavers (20 to 56 years) from the Department of Surgery, Division of Transplantation of the University of Miami Miller School of Medicine. Cadaveric spleen cells were used to optimize the conditions for B cell preparation and culture. The subjects participating in the study were screened for diseases known to alter the immune response or for consumption of medications that could alter the immune response. In particular, the following categories were excluded: established diagnosis of diabe- tes; one or more of the following co-morbid conditions including malignancy (subjects without a recurrence in the last five years have been allowed), congestive heart failure, cardiovascular disease (unstable, less than six

months), chronic renal failure, significantly impaired renal or hepatic function, autoimmune diseases, infectious disease, recent (less than three months) trauma or surgery, pregnancy, or documented current substance and/or alcohol abuse. The psychological status of all subjects was within normal limits. Partici- pants were 46 healthy subjects (mean age = 51.3; SD = 19.2). There were 25 females and 21 males in this sample. Two were Asian, 14 were Black, and 30 were White (13 identified themselves as Hispanic Whites). We have achieved highly significant differences between samples from young and old subjects, even before reaching the number of 46 subjects. Statistical analysis (f-test) showed that there were no differences between male and female subjects on any of the parameters measured in this study (all p values > 0.60). Similarly, no differences were noted between subjects of different ethnicities or races in ANOVA analyses (all p values > 0.20).

B cell enrichment

PBMC were collected by density gradient centrifugation on LSM lymphocyte separation medium (ICN Biomedicals). Cells were then washed three times with medium (RPMI 1640). B cells were isolated from the PBMC as follows. Briefly, cells were washed three times with medium (RPMI 1640) and incubated for 20 min at 4°C with 20 μl/10 ⁷ cells of anti-CD19 microbeads (Miltenyi Biotech) according to the manufacturer's MiniMacs protocol. Cells were then purified using magnetic columns. At the end of the purification procedure, cells were found to be almost exclusively (>85%) CD19 ⁺ by cytofluori- metric analysis: Comparable numbers of CD19+ cells were obtained after negative selection. After the isolation procedure was ended, cells were maintained in serum-free medium for one hour at 4°C in order to minimize potential effects of anti-CD19 antibodies on B cell activation and our parameters are measured 24 hr or later.

Cell sorting

To evaluate which cell subset was responsible for class switching in the whole CD19 ⁺ B cell population, PBMC were sorted into memory (CD19 ⁺ CD27 ⁺ )

B cells or naive (CD19 ⁺ CD27 ^" ) B cells. Cells were separated with a FACS ARIA cell sorter (BD Biosciences). Cell preparations were typically >98% pure.

S cell culture B cells were cultured in complete medium (RPMI 1640, supplemented with 10% fetal calf serum, 10 μg/ml gentamicin, 2 x 10 ^"5 M β-mercaptoethanol, and 2 mM L-glutamine). Cells (1 x 10 ⁶ /ml of complete medium) were stimulated in 24-well culture plates with purified anti-human CD40 Abs (BD Pharmingen 555587, 1 μg/ml), alone or together with recombinant human IL-4 (Biosource PHC0044, 10 ng/ml), for a period of 1 to 7 days. At the end of this time period, cells were harvested, RNA was extracted from cells for semiquantitative (sq) or quantitative (q) RT-PCR, and supernatants were collected for ELISA. In four of the elderly subjects (> 65 years), total PBMC were stimulated because of extremely low numbers of B cells. Only B cells were stimulated by this proce- dure.

Memory or naϊve B cells were stimulated (10 ⁴ cells/200 μl) in round- bottom 96-well plates with anti-CD40/IL-4, alone or together with F(ab')2 fragments of goat anti-human Ig (2 μg/ml, Jackson lmmunoresearch 109-006-006) for 1 to 7 days.

Flow cytometry

After magnetic enrichment, B cells (3-5 x 10 ⁵ /tube) were stained with APC-conjugated anti-CD19 (BD Pharmingen 555415) (all volumes were 20 μl) for 20 min at 4°C. To detect naive (lgG-/lgA-/CD27-) B cells, IgM memory (IgG- /lgA-/CD27+) B cells, or switch memory (lgG+/lgA+) B cells, CD19+ cells (3-5 x 10 ⁵ /tube) were stained with PE-conjugated anti-CD27 (1 :40 diluted, BD Pharmingen 555441 ), biotin-SP-conjugated ChromPure human IgG (Jackson lmmunoresearch 009-060-003), and 20 μl biotin-SP-conjugated ChromPure human IgA (Jackson lmmunoresearch 009 -060-011 ). After washings, biotin-conjugated antibodies were revealed with APC-conjugated streptavidin (1 :40 diluted, BD Pharmingen 554067). Samples of 3-5 x 10 ⁵ cells were analyzed on an LSR flow cytometer (BD Biosciences) using logarithmic amplification. For four-color

analysis, controls were included in every experiment to determine background fluorescence.

ELISA Human IgGs were titrated in the collected supernatants and sera by

ELISA by a sandwich protocol. Briefly, wells of microtiter plates (Nunc 12-565- 136) were coated (overnight at 4°C) with 100 μl of a purified goat anti-human IgG, F(ab) ₂ capture Ab (Jackson lmmunoresearch 109-006-006) at a concentration of 2 μg/ml in 1x PBS. Reactions were blocked by adding 200 μl of 1 x PBS containing 10% FCS (blocking buffer) to each well for 30 min at room temperature. Wells were then washed thoroughly with 1x PBS containing 0.05% Tween 20 (washing buffer). Serial dilutions of the culture supernatants or an IgG standard (human IgG whole molecule, Jackson lmmunoresearch 009-000- 003) at a concentration of 1120 ng/ml in blocking buffer were added to the wells (100 μl/well), and incubated overnight at 4°C. Wells were washed thoroughly with washing buffer before receiving 100 μl/well of the detecting Ab (anti-human IgG-HRP, BD Biosciences 555788), at a 1 :5000 dilution in blocking buffer. After one hour incubation at room temperature, wells were washed and given 100 μl of the substrate solution (TMB chromogen, Biosource SB01 ). Wells were incu- bated 15 to 20 min at room temperature to allow reactions to develop. Well contents were measured for absorbance at 405 nm.

RNA extraction, reverse transcription, semiquantitative (sq)PCR and quantitative (q)PCR The mRNA was isolated from 0.5x10 ⁴ -10 ⁵ B cells using the μMACS mRNA isolation kit (Miltenyi Biotech) according to the manufacturer's protocol, eluted into 75 μl pre-heated elution buffer and stored at -8O ⁰ C until use. RT- PCR was performed in a Mastercycler Eppendorf machine. Briefly, ten μl of mRNA were used as template for cDNA synthesis in the reverse transcription (RT) reaction.

The sqPCR was used to determine γ1 circle transcripts (CTs). PCR conditions for γ1 CTs (608 bp) were: denaturation at 94°C for 1 min, amplification of

cDNA for 30 cycles, each cycle programmed for denaturation at 94°C for 1 min, annealing at 60 ⁰ C for 1 min, and elongation at 72°C for 1 min followed by a final extension phase at 72 ⁰ C for 3 min. Primers were Iγ1 forward 5'-GGG CTT CCA AGC CAA CAG GGC AGG ACA-3 ¹ (SEQ ID NO:1 ) and Cμ reverse 5'-GTT GCC GTT GGG GTG CTG GAC-3' (SEQ ID NO:2). Conditions and primers for GAPDH have been published previously (8, 9). PCR products were separated on 1.5% agarose gels. Gels were visualized using the Alphalmager Enhanced Resolution Gel Documentation and Analysis System (Alpha Innotech) and images were quantitated using the AlphaEaseFC 32-bit software. During the course of these studies, analysis was switched from sqPCR to qPCR for the measurement of E47 and AID, with results being consistent with both techniques. To perform quantitative qPCR, 2 μl of cDNAs were added to 8 μl of 2x SYBR Green PCR Master Mix (ABI 4367659), 1 μl of 5 μM forward primer, 1 μl of 5 μM reverse primer, and deionized water in a final volume of 20 μl. Reactions were conducted in MicroAmp 96-well plates (ABI N8010560). Calculations were made with ABI software. Briefly, we determined the cycle number at which transcripts reached a significant threshold (Ct) for E47, AID, and GAPDH as control. A value of the target gene (E47 or AID), relative to GAPDH, was calculated and expressed as δCt. Samples are expressed as percentages of the highest δCt value normalized to 100. PCR primers were: E47 forward, 5'-GCG AGG GCA CCC ACT TC-3' (SEQ ID NO:3); E47 reverse, 5'-GGT CCC AGG AAT GTG GAT GA-3' (SEQ ID NO:4); AID forward, 5'-CAC CGC GCG CCT CTA C-3' (SEQ ID NO:5); AID reverse, 5'-CCT TCC CAG GCT TTG AAA GTT-3' (SEQ ID NO:6); GAPDH forward, 5'-CCA GGT TGT CTC CTC CGA CTT-3' (SEQ ID NO:7); and GAPDH reverse, 5'-CCA GCG TCG AAG GTG GAA-3' (SEQ ID NO:8). Primers for E47 were designed with the ABI 7300 software and were selected according to the best penalty score. A set of primers for AID (AID forward, 5'-GCC ACG TGG AAT TGC TCT TC-3' SEQ ID NO:9; AID reverse, 5'-AGC GGC CAG GGT CTA GGT-3' SEQ ID NO:10) was initially designed with ABI 7300 software and selected according to the criteria of having the best penalty score. These primers were not optimal, however, in amplifying small amounts of AID such as those found in B cells from elderly

people. Therefore, we chose primers for AID (23) but used different conditions, presented here and also checked these for a low penalty score. Conditions for both E47 and AID were: 50 ⁰ C for 2 min, 55°C for 10 min, 95°C for 15 sec (40 cycles), and 60 ⁰ C for 1 min. Because the primers for CTs gave a high penalty score, we measured CTs by sqPCR only.

Assessment of multiple gene expression levels

Gene expression levels of activation-induced cytidine deaminase (AID) and immunoglobulin heavy chain (IgH) that has undergone class switching in the peripheral blood was monitored by using a custom-made TaqMan low- density array with a 7900HT fast real-time PCR system (Applied Biosystems). Individual TaqMan assays for these genes was pre-loaded in triplicate into the array's 384 wells of a microfluidic card. Control genes (i.e., 18S rRNA and β- actin) were also included on the card. Several different cDNA samples can be analyzed at the same time using the card in a multiplex assay. Total RNA was extracted from whole blood collected into PAXgene Blood RNA tubes (Qiagen). First strand cDNA was synthesized from total RNA using a Superscript III First- Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). cDNA converted from 500 ng of total RNA for each sample was used in the assay. A control sample from a pool of RNA from 22 human tissues (Applied Biosystems) was used for each array to minimize variations between arrays. The control is preamplified from very small quantities of the cDNA using TaqMan PreAmp Master Mix kit (Applied Biosystems). In this way, the same control sample can be used in all of the arrays involved in the study. Data are expressed as the fold change in gene expression normalized to endogenous genes and relative to the control.

Statistical analyses

Before conducting the main analyses, we examined the data for normality of the distribution and outliers. Four outliers were observed but there was no discernible pattern to these outliers. Outliers were scores with more than two standard deviations above and below the mean. For these four variables, the score was changed to the mean score in order to ensure a normal distribution

for conducting the parametric statistical analyses. Linear correlations between age and the following immune parameters were calculated: B-cell percentages and numbers, B-cell subset percentages and numbers, CTs ₁ AID, E47, and IgG in culture supernatants. Because the graphical representations of these rela- tionships had a non-linear appearance, we also examined non-linear (i.e., quadratic) relationships between age and immune parameters. That is, a non-linear relationship might postulate that middle aged are higher than expected by linear curve. Values are shown in each figure legend.

Switch memory B cells decrease whereas the percentage of naive B cells increase with age

We analyzed the composition of the peripheral B-cell pool in 46 subjects of different ages (18 to 86 years): i.e., numbers and percentages of total CD19+ B cells, as well as numbers and percentages of naive (lgG-/lgA-/CD27-) B cells, IgM memory (lgG-/lgA-/CD27+) B cells, or switch memory (lgG+/lgA+) B cells. Percentage and absolute number of CD19+ B cells from peripheral blood mononuclear cells (PBMC) are shown in the Figs. 1A and 1 B, respectively. We decided to analyze lgG+/lgA+ cells as switch memory B cells because, in addition to the lgG+/lgA+/CD27+ population, a fraction of lgG+/lgA+/CD27- B cells also expresses mutated IgV region genes, increased expression of the costimu- latory molecules CD80 andcentages of total CD19+ B cells decrease with age, consistent with data previously published (19, 25). The percentage of naive B cells increases with age (Fig. 1 C) at least for most of the subjects tested (such that the linear curve accounted for greater amounts of variance than the quad- ratic function, though both were statistically significant). Due to the decrease in number and percentage of CD19+ cells, the absolute number of naϊve B cells is not significantly different in young and old (Fig. 1 D). The percentage of IgM memory B cells are not statistically different between young and old (Fig. 1 E, linear curve), but show an increase in old age compared to the middle aged group with the quadratic curve. The absolute number of IgM memory B cells decreased with age (Fig. 1 F). With age, switch memory B cells decreased in both percentage (Fig. 1 G) and absolute number (Fig. 1 H). The significant

decrease in switch memory B cells and the increase in percentage of naϊve and IgM memory B cells (from middle to old age) suggest an intrinsic defect in the ability of old B cells to undergo CSR. Therefore, we wanted to investigate whether CSR was decreased in CD19+ B cells stimulated in vitro with anti- CD40/IL-4, a stimulus leading to IgG production.

Aging down-regulates CSR in activated peripheral blood-derived human B cells B cells were stimulated in vitro with anti-CD40/IL-4. CSR was measured by γ1 circle transcripts (CTs). Briefly, CSR generates an extra-chromosomal reciprocal switch DNA recombination product, which includes the intronic heavy chain promoter 5' of the targeted C _H gene, the DNA segment between Sμ and the targeted S region, and Cμ and is referred to as the CT (26). Fig. 2A and 2B show that these transcripts are reduced in B cells from elderly subjects. We have evidence that in the CD19+ B cell population, only IgM memory B cells switch in response to anti-CD40/IL-4 as shown below and as expected from other's results (27). Naϊve B cells require an additional B cell receptor (BCR) stimulus (anti-μ). We also looked at germline transcripts and found that they are present in B cells from young and elderly subjects at a similar extent, similar to that as we have seen in the murine system (9). Thus, our results indicating less CSR products suggest that the defect in aged B cells occurs at the CSR event and is not due to problems with accessibility, nor with the cytokine signaling pathway that leads to γ1 accessibility, as we have shown in murine B cells (9).

We also measured IgG production by ELISA of culture supernatants after 7 days stimulation with anti-CD40/IL-4. Fig. 2C shows that IgG levels in culture supernatants decrease with age and, as expected, parallel the data shown in Fig. 2B. Conversely, when we measured IgG levels in serum, we found them increased with age (Fig. 2D) as others have shown consistently (17, 28). We have done a few experiments with splenic B cells obtained from cadaveric donors (four subjects, aged 22-52) together with peripheral blood- derived B cells (six subjects, aged 22-53), and shown a similar pattern of IgG production in cultures of anti-CD40/IL-4-stimulated B cells (the 52 year old subject had about 14 of the IgG of the 22 year old subject as seen in the peri-

pheral blood-derived B cells, not shown). Therefore, our peripheral blood data are representative of peripheral B lymphocytes.

Aging down-regulates AID in activated peripheral blood-derived human B cells Because the reduction in CSR in old B cells could be due to reduced levels of AID, as- already shown in murine B cells (3, 9), we investigated the levels of AID transcripts in anti-CD40/IL-4-stimulated CD19+ B cells from subjects of different ages. Preliminary results indicated that stimulation with anti-CD40/IL-4, and to a lesser extent with IL-4, induced AID mRNA expression in peripheral blood-derived B cells from both young and elderly subjects. The level of AID expression was lower in old as compared with young B cells stimulated not only with anti-CD40/IL-4 (Fig. 3A), but also with IL-4. Human B cells require the activation of both CD40 and IL-4 signaling pathways to undergo optimal AID expression and likely Ig class switch (29). Fig. 3A shows that aging decreases AID mRNA expression in anti-CD407IL-4-stimulated CD19+ B cells from subjects of different ages, as determined by sqPCR and qPCR, respectively.

Aging down-regulates the E2A-encoded transcription factor E47 in activated peripheral blood-derived human B cells

To gain insight into the mechanisms underlying AID regulation, we analyzed the levels of expression of the E2/\-encoded transcription factor E47 in peripheral blood-derived B cells. Preliminary results determined optimal dose response and kinetics and indicated that stimulation with anti-CD40/IL-4, and to a lesser extent with anti-CD40 alone, induced E47 mRNA expression in peripheral blood-derived B cells from both young and elderly subjects. IL-4 alone was unable to induce E47 mRNA expression. Fig. 3B shows that aging decreases E47 mRNA expression in anti-CD40/IL-4-stimulated CD19+ B cells from subjects of different ages, as determined by qPCR. E47 and AID progressively and similarly decrease with age. The statistically significant correlation between E47 and AID is shown in Fig. 3C. Results show that as E47 increased, AID also increased (r = 0.80, significant at the 0.01 level, 2-tailed).

Higher E47 was also associated with higher CTs (r = 0.65, significant at the 0.01 level, 2-tailed, not shown). Likewise, there was a positive significant association between AID and CTs where greater AID was associated with greater CTs (r = 0.79, significant at the 0.01 level, 2-tailed, not shown).

Both memory and naive B cells show intrinsic defects in class switch with age

We have so far shown that CD19+ B cells lose their ability to undergo in vitro class switch with age and we have shown that there is an intrinsic defect in the expression of E47 and AID. The data shown herein do not clarify whether there is an intrinsic defect in the subsets of memory B cells or if the decrease we saw in class switch depends on the age-related reduction in the numbers of memory B cells. To clarify this point, we sorted naive and memory B cells which were then stimulated in vitro by anti-CD40/IL-4, a stimulus mainly for memory B cells. Sorted cells were also stimulated with anti-CD40/IL-4 and F(ab') ₂ fragments of anti-human IgM, used as surrogate antigen, because naϊve B cells require the activation of the B-cell receptor (BCR) signal transduction to undergo class switch (27). Fig. 4 shows that memory B cells express AID in response to anti-CD40/IL-4, independently from the presence of anti-lgM and differentiated into antibody-secreting cells. We conclude that the subset of memory B cells able to class switch in culture is likely the IgM memory population, although here we only looked at AID, and not also CT. As shown in Fig. 4, naϊve B cells do not express AID in response to anti-CD40/IL-4 and up-regulate AID only in the presence of BCR triggering by anti-lgM. Even under the best conditions of stimulation (anti-CD40/IL-4 and anti-lgM), the response of naϊve B cells was lower than that of memory B cells. Therefore, both IgM memory and naϊve B cells are negatively affected by aging. The old subject in pair #2 (Fig. 4B) is clearly exceptional from the other data in Figs. 3-4. We can conclude from these data on sorted subsets of B cells that the defect we observed in the ability of CD19+ cells to class switch upon in vitro stimulation with anti-CD40/IL- 4 does not simply depend on a reduction in the numbers of memory B cells but is an intrinsic defect of the switched cells (IgM memory in previous experiments

and naϊve and IgM memory here). The percentages naive, IgM memory, and switch memory B cells in the subjects of Fig. 4 are listed in Table I.

The order from left to right 1-4 corresponds to the # of the pair in the Figure. Phenotype (% cells) of old #2 is "young-like", (see text).

Both switch memory and IgM memory B cells have been shown to proliferate and differentiate into plasma cells in response to anti-CD40/IL-4 (27) but this differentiation would not be expected to increase AID at least in the switch memory cell population. We have shown this when naϊve, IgM memory, and switch memory B cells from young subjects were sorted and found that naϊve and IgM memory B cells had comparable levels of AID transcripts, whereas switch memory B cells had four-fold less as compared to the other subsets (Fig. 5). Thus, in unsorted populations, AID expression to anti-CD40/IL-4 is predominantly measuring the IgM memory cell population.

Significant differences in B cell functions were observed between young (18- 30) and old (>65) subjects

The younger age group (subjects from 18-30 years) differed from the older age group (aged greater than 65 years) when analyzed by independent samples f-test for percentage (%) and number (#) (Table II). The groups differed on every variable with the exceptions of the numbers of naϊve B cells and the percentage of IgM memory B cells, which is consistent with the findings above (Figs. 1 B and 1C).

Young: 18-30 years (12 subjects). Old: >65 years (15 subjects). Independent samples Mest were calculated.

The in vitro AID response of B cells to vaccination decreases with age as shown in Fig. 6. B cells (10 ⁶ cells/ml), isolated using CD19 magnetic beads from the peripheral blood of subjects of different ages, before (filled circles) or after vaccination (open circles) with 1 μl of flu vaccine, were cultured with the indicated stimuli, for 7 days. At the end of this time, cells were harvested, RNA was extracted, and qPCR reactions were performed to evaluate AID normalized to GAPDH transcripts. Five μg of CpG was used to stimulate 1x10 ⁶ B cells for the control in Fig. 6A.

Seven subjects (ages 22-71 years) were enrolled to evaluate the age- related effects of influenza vaccination on B cell Ig class switch. The blood from these subjects was drawn immediately before vaccination and 4-6 weeks after vaccination. Five tubes of heparinized blood and 1 tube for serum were drawn from each subject. Serum was used to evaluate by hemaglutination inhibition the response to vaccination to the whole vaccine as well as to the single antigens AG1 , AG2 and AG3. The single antigens included in the 2007-2008 (last year) influenza vaccine were: Flu antigen H1 N1 (A/Solomon lslands/3/2006- like), Flu antigen H3N2 (A/Wisconsin/67/2005), and Flu antigen B (B/Malaysia/ 2506/2004). One hundred μl of blood were used to evaluate by flow cytometry the following parameters: subsets of B cells (naϊve, IgM memory or switch memory B cells); expression of co-stimulatory molecules (CD40, CD80, CD86); and percent of regulatory T cells (CD4+CD25+ or CD8+CD28-). Cells were processed as follows. Briefly, B cells were isolated from the peripheral blood by- Ficoll gradient centrifugation using magnetic beads. Cells (1X10 ⁶ /ml) were stimulated in culture for 7 days with CpG (5 μg/ml), or with the single antigens. AG2 and AG3 (H3N2 and B) were already present in the 2006-2007 vaccine preparation. The antigens were used at the concentration of 1 μg/ml. At the end of the culture period, cells were harvested, and mRNA extracted to evaluate AID expression by real-time PCR. Results show that influenza vaccination induced up-regulation of AID mRNA expression in all subjects, and in response to all antigens, the up-regulation being higher in young than in old subjects. This up-regulation of AID was concomitant with up-regulation of the serum flu- specific IgG response against all antigens in Fig. 6. In human subjects, the serum hemaglutinin inactivation (HI) response to flu vaccination decreases with age in Fig. 7. Serum was collected from subjects of different ages, before (filled circles) or after vaccination (open circles) with 1 μl of flu vaccine, and analyzed by HI assay to evaluate antibody production to vaccine or to the single antigens present in the vaccine preparation. The HI assay is based on the ability of certain viruses or viral components to hemag- glutinate (HA) the red blood cells of specific animal species. Antibodies specific to influenza can inhibit agglutination. HI is useful for the measurement of anti-

body titers of sera. The assay is performed as follows. Sera from subjects un- immunized or flu-vaccinated were pre-treated with receptor destroying enzyme (Denke Seiken) for 20 h at 37 ⁰ C. To inactivate this enzyme, sera were then heated at 56°C for 60 min. Two-fold serial dilutions were done. Diluted serum (25 μl) was incubated with an equal volume of 4 HA units of whole vaccine or each of the HA antigen vaccine's components, for 1 h at room temperature and then 50 μl of a 1.25% suspension of human red blood cell, type O (HO)were added. Whole vaccine and HA antigen components were provided by Glaxo- SmithKline. After 2 h of incubation at room temperature, titers were determined. The results are expressed as reciprocal titer values. A four-fold rise in titer at two different time points pre- to post-vaccination indicates exposure to whole vaccine and to each of the HA antigens.

Figs. 6 and 7 show that AID gene expression is a good biomarker for predicting the effectiveness of flu vaccination. We have also compared gene expression levels by RT-PCR for three different categories of human subjects "at risk" for type 1 diabetes, with new onset of type 1 diabetes, or having long-term type 1 diabetes to healthy human subjects as a negative control. Both AID and IgH that has undergone class switching were measured for gene expression. RT-PCR was done using resting whole blood as the RNA source. No stimulation or culturing was required prior to analysis of gene expression.

For AID expression in Fig. 8, there was a significant difference between healthy controls and long-term patients. For gene expression of IgH that has undergone class switching in Fig. 9, there was a significant difference between healthy controls and long-term patients. Surprisingly, there was also a nearly significant difference (p = 0.0556) between new-onset and long-term patients.

Figs. 8 and 8 show that assaying for gene expression of biomarkers has utility outside of the context of vaccination. In vitro stimulation and culturing of B cells prior to analysis is not required.

Discussion

We show both quantitative and qualitative changes occurring in human B cells with aging. In particular, we confirm data from other groups showing that B-cell percentages and numbers decrease with age (17-19, 25), whereas the percentage of naive B cells increase with age (19, 25). Moreover, we show for the first time that the number of switch memory B cells decreases with age. Because of the decreased numbers of total B cells with age, the absolute number of switch memory B cells is extremely low in elderly subjects, whereas that of naive B cells is maintained. We also show, as others have reported (19), that IgM memory B cells are reduced in numbers, not in percentage. Therefore, the depletion in both switch memory and IgM memory B cells seems to be a feature of aging human B cells.

The significant decrease in the number of naive T cells and the shrinkage in their TCR repertoire reported in the elderly (30) can both account for the reduced stimulation of memory B cells at older ages in vivo, but likely does not account for the intrinsic B-cell defects seen here.

The decrease in switch memory B cells, the increase in the percentage of naive B cells and the absence of a decrease in the percentage of IgM memory B cells (or an actual increase in some subjects in IgM memory B cells, at least compared to middle age, see quadratic curve, Fig. 1 C) suggest an intrinsic defect in the ability of old B cells to undergo CSR. We have indeed shown in the present study that CSR induced in cultures of whole peripheral blood-derived B cells by anti-CD40/IL-4 is significantly impaired by aging. Our data on sorted subsets of B cells have shown that the defect we observed in the ability of CD19+ cells to class switch upon in vitro stimulation with anti- CD40/IL-4 is not simply depending on a reduction in the numbers of memory B cells (IgM memory/switch), but by an intrinsic defect in AID in both naive and (IgM) memory B cells. This leads to decreased amounts of IgG released in culture supernatants of B cells from old as compared to from the young or adults. But a seeming paradox exists: i.e., the ability of B cells to undergo class switch upon in vitro stimulation with anti-CD40/IL-4 is decreased, but there is a concomitant increase in serum IgG levels, as also others have shown (17, 28).

Both peripheral blood-derived B cells and splenic B cells are less able to produce IgG in response to anti-CD40/IL-4. These facts may be explained by the initial antigenic stimulation being defective in aged B cells. This is a major point of our studies. The accumulation in the sera of IgG, not from these types of cells, likely comes from plasmablasts (many in the bone marrow) (31 , 32) secreting immunoglobulin of suboptimal quality both in affinity and polyclonality (and autoimmune immunoglobulin (33)).

Studies of human V _H 6 and V _K 4 genes from peripheral blood-derived B cells have shown either a decrease or no change in SHM with age (34-38). In the human tonsil, diversity of the Ig repertoire is maintained with aging, although germinal center cells are reduced and naϊve cells are increased (21 ). These seemingly conflicting sets of results likely reflect the levels of chronic exposure to some environmental antigens that activate specific- or cross- reactive B cell clones. B cell clones that were stimulated initially in young subjects may show more SHM whereas those to a "new" antigen may be defective in age, as we suggest here.

We have previously shown in mice (8, 9) that the E2>A-encoded transcription factor E47 is down-regulated in old splenic activated B cells, leading to a reduction in the activation-induced cytidine deaminase (AID) and in CSR. This leads to reduced amounts of switched antibodies. Here we show that E47 is also reduced in human B cells and this leads to reduced AID, CTs and IgG released in culture supernatants. These are novel observations which help to establish the specific deficits in B-cell activity in aged humans and in particular provide molecular mechanisms for reduced Ig class switch seen in aged human subjects. E47 has been shown to be necessary for CSR (39-41 ) and SHM (39) as it transcriptionally regulates the gene for activation-induced cytidine deaminase (AID) (42). Therefore, E47 has a relevant role in all processes generating antibody diversity, such as V(D)J recombination (43-48), CSR and SHM. Our results herein clearly indicate that intrinsic defects in the B cell may have a crucial role in the generation of an effective humoral immune response in sensecence.

In conclusion, our results show an intrinsic defect in the ability of old B cells to undergo CSR. The transcription factor E47 in activated B cells is significantly impaired by aging. This leads to a reduction in AID and, in turn, to less switched antibodies produced by the activated B cells. Although there is a clear and significant decrease in both E47 and AID biomarkers with age, there is also an increase in the variability in the aged population for these two biomarkers (Figs. 3-4) which allows us to pinpoint important defects in the humoral immune response in elderly humans. The population of elderly people worldwide is increasing due to decreased mortality, but their morbidity has not decreased in parallel. Identification of the causes and impact of immunosenescence may offer the possibility for improving recovery from, preventing, or delaying some infectious diseases and improve the quality of life for our aging population.

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Patents, patent applications, books, and other publications cited herein are incorporated by reference in their entirety. In stating a numerical range, it should be understood that all values within the range are also described (e.g., one to ten also includes every integer value between one and ten as well as all intermediate ranges such as two to ten, one to five, and three to eight). The term "about" may refer to the statistical uncertainty associated with a measurement or the variability in a numerical quantity which a person skilled in the art would understand does not affect operation of the invention or its patentability.

All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. A claim reciting "comprising" allows the inclusion of other elements to be within the scope of the claim; the invention is also described by such claims reciting the transitional phrases "consisting essentially of (i.e., allowing the inclusion of other elements to be within the scope of the claim if they do not materially affect operation of the invention) or "consisting of (i.e., allowing only the elements listed in the claim other than impurities or inconsequential activi- ties which are ordinarily associated with the invention) instead of reciting the "comprising" term. Any of these three transitions can be used to claim the invention.

It should be understood that an element described in this specification should not be construed as a limitation of the claimed invention unless it is explicitly recited in the claims. Thus, the granted claims are the basis for determining the scope of legal protection instead of a limitation from the specification which is read into the claims. In contradistinction, the prior art is explicitly exclu-

ded from the invention to the extent of specific embodiments that would anticipate the claimed invention or destroy novelty.

Moreover, no particular relationship between or among limitations of a claim is intended unless such relationship is explicitly recited in the claim (e.g., the arrangement of components in a product claim or order of steps in a method claim is not a limitation of the claim unless explicitly stated to be so). All possible combinations and permutations of individual elements disclosed herein are considered to be aspects of the invention. Similarly, generalizations of the invention's description are considered to be part of the invention. From the foregoing, it would be apparent to a person of skill in this art that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification.