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. Author manuscript; available in PMC: 2016 Jul 14.
Published in final edited form as: Cell Rep. 2015 Jul 2;12(2):163–171. doi: 10.1016/j.celrep.2015.06.015

Defective TFH cell function and increased TFR cells contribute to defective antibody production in aging

Peter T Sage 1,2, Catherine L Tan 1,2, Gordon J Freeman 3, Marcia Haigis 4, Arlene H Sharpe 1,2,5,*
PMCID: PMC4504745  NIHMSID: NIHMS699135  PMID: 26146074

Summary

Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here we demonstrate an increase in the ratio of inhibitory T follicular regulatory (Tfr) cells to stimulatory T follicular helper (Tfh) cells in aged mice. Aged Tfh and Tfr cells are phenotypically distinct from those in young mice, exhibiting increased PD-1 expression but decreased ICOS expression. Aged Tfh cells exhibit defective antigen-specific responses, and PD-L1 blockade can partially rescue Tfh cell function. In contrast, young and aged Tfr cells have similar suppressive capacity on a per cell basis in vitro and in vivo. Together these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive Tfr cells combined with impaired function of aged Tfh cells results in reduced T cell dependent antibody responses in aged mice.

Graphical Abstract

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Introduction

It has been widely observed that the extent of humoral immunity, or immunity provided by antibodies, decreases with age in both mice and humans (Goidl et al., 1976; Phair et al., 1978). This decrease in humoral immunity translates into increased frequency and severity of infectious diseases in aged individuals. Furthermore, vaccination of the elderly provides inadequate protection against most infectious diseases, leaving these individuals vulnerable to a number of diseases (Goronzy and Weyand, 2013; Sasaki et al., 2011).

The production of high affinity antibodies results from a complex interaction of B cells with T follicular helper (Tfh) cells in the germinal center (GC) reaction. After differentiation, CXCR5+ Tfh cells migrate to the B cell follicle via gradients of CXCL13 and provide help to B cells via costimulation and cytokine production (Crotty, 2011). Mice lacking Tfh cells, or their key effector molecules, have severely defective antibody production in response to T dependent antigens.

T follicular regulatory (Tfr) cells are a recently defined specialized subset of effector Tregs that inhibit antibody production (Chung et al., 2011; Linterman et al., 2011; Sage et al., 2013; Wollenberg et al., 2011). Tfr cells originate from natural Tregs (Chung et al., 2011; Sage et al., 2013) in contrast to Tfh cells, which develop from naïve CD4+ T cell precursors. Similarly to Tfh cells, Tfr cells express CXCR5, ICOS and PD-1, as well as the transcription factor Bcl6. PD-1 expression on Tfr cells limits both the differentiation and effector function of Tfr cells (Sage et al., 2013). How Tfr cells exert their suppressive effects is not yet clear. We have demonstrated that the ratio of Tfh/Tfr cells is an important factor in humoral immunity and that this ratio dictates the magnitude of antibody responses (Sage et al., 2014a; Sage et al., 2013). Therefore, successful humoral immunity is a delicate balance between stimulatory Tfh cells and inhibitory Tfr cells, and not simply a result of the total number of Tfh cells. Tfr cells appear to be specialized in their suppression of the GC reaction as non-Tfr Tregs do not have the same suppressive capacity (Sage et al., 2014a; Sage et al., 2013; Sage et al., 2014b).

The precise mechanisms leading to poor B cell responses in the aged are not understood. In 1969, Walford used the term immunosenescence to describe the decline in the immune system with age. In the T cell compartment, thymic involution, leading to reduction in the output of naïve T cells in the elderly, is one hypothesized cause of immune system decline (Scollay et al., 1980). Reduced naïve cell output also occurs in the B cell compartment (Miller and Allman, 2003). Additionally, there are alterations in the ability of naïve lymphocytes to become activated and form memory cells (Haynes et al., 2003; Linton and Dorshkind, 2004). Some, but not all, of these changes can be rescued by addition of IL-2, since IL-2 production is attenuated with age (Haynes et al., 1999). There are also increased numbers of natural Tregs in lymphoid organs (but not the blood) (Jagger et al., 2014). It is not yet clear if Tregs from aged individuals are equally or more suppressive compared to Tregs from younger individuals (Nishioka et al., 2006; Raynor et al., 2012).

Although a number of studies have assessed the total CD4+ T cell and Treg populations in the aged, it is still unclear if alterations exist in Tfh and Tfr cells. A previous study found no difference in CXCR5+ cells in aged mice; however, Tfr cells were not examined (Eaton et al., 2004). A recent study found slight increases in Tfh cells in the blood of aged human subjects, but Tfr cells were not evaluated (Zhou et al., 2014). Understanding changes in Tfh and Tfr cells during aging is important because both of these cell types directly interact with cognate B cells and control antibody production.

In this study we compared Tfh and Tfr cell development and function in young and aged mice. We find increases in both Tfh and Tfr cells in aged mice, with a proportionally greater increase in Tfr cells. We also show that Tfh cells from aged mice have defects in antigen-specific B cell stimulation. Aged and young Tfr cells, however, have comparable suppressive capacity. Thus, our studies reveal a mechanism that attenuates antibody responses in the aged: the over-abundance of highly suppressive Tfr cells in aged mice, together with the inability of Tfh cells to effectively respond to specific antigen, results in an overall decrease in B cell responses.

Results and Discussion

Increased Tfr cell Polarization in Aged Mice

To determine if alterations in Tfh and Tfr cells contribute to defective antibody production in aged mice, we first compared antibody production in 2 month old (young) and 20 month old (aged) mice 10 days after subcutaneous (s.c.) immunization with NP-OVA in CFA. Aged mice had greater total serum concentrations of IgM and IgG than young mice; however, NP-specific IgG (but not IgM) antibody titers were significantly lower (Figure 1A). Lower antigen-specific IgG was also seen in aged mice 27 days after immunization (Figure S1B). Together, these data indicate that aged mice produce less antigen-specific antibody following immunization.

Figure 1.

Figure 1

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Increased Tfr Cells in Aged Mice. (a) 2 month (young) or 20 month (aged) mice were immunized s.c. with NP-OVA in CFA and 10 days later sera collected. Total IgM and IgG (top) or NP-specific IgM and IgG (bottom) were analyzed by ELISA. Percentages of CD4+ICOS+CXCR5+FoxP3−CD19− Tfh (b) and CD4+ICOS+CXCR5+FoxP3+CD19− Tfr (c) were analyzed in unimmunized young and aged mice. ILN = inguinal lymph node, (d-f) Representative plots (d) and quantification of Tfh (e) and Tfr (f) cells from the draining lymph nodes (dLNs) of young or aged mice 7 days after s.c. immunization. (g) Quantification of the Tfr contribution to total CD4+CXCR5+ cells (Tfr: Tfh ratio) in the dLN of young and aged mice 7 days after immunization. (h) Quantification of total FoxP3+ cells in the dLN of young or aged mice immunized with NP-OVA. Data are from 5-8 mice per group, and representative of at least three independent experiments. Error bars indicate standard deviation. See also Figure S1-2.

We next determined if Tfh and/or Tfr cells were altered in aged mice. First, we compared Tfh and Tfr cells without immunization. Tfh cells, defined as CD4+ICOS+CXCR5+FoxP3−CD19− cells, were low in percentage in the inguinal lymph node (iLN) and blood of both unimmunized young and aged mice (Figure 1B). The slight increase in Tfh cells in blood of aged mice is likely due to memory cells, since we have shown that blood Tfh and Tfr cells have characteristics of memory cells (Sage et al., 2014a). Blood Tfh cells in aged humans express the memory marker CD45RO (Zhou et al., 2014). Tfr cells, defined as CD4+ICOS+CXCR5+FoxP3+CD19− cells, were similarly low in percentage in the iLN and blood of unimmunized young and aged mice (Figure 1C).

Next, we compared the generation of Tfh and Tfr cells in young and aged mice upon antigenic challenge. We immunized young and aged mice with NP-OVA s.c., and measured Tfh and Tfr cell percentages in the draining lymph node (dLN), spleen, and blood 7 days later. Tfh cells were increased 3-fold by percentage in the dLN of aged mice compared to young mice after immunization (Figure 1D-E). Similar increases in dLN Tfh cell percentages, but not necessarily total numbers, were found 14 and 27 days after immunization in aged mice (Figure S1A-B). Increased Tfh percentages were also found in the blood and spleen of aged mice (Figure 1E and S1C-D). Serum cytokines levels were similar in immunized young and aged mice (Figure S1E), indicating that differences in Tfh and antibody responses are not due to altered systemic cytokines. Percentages of PD-1hi GC-Tfh, ICOShi Tfh cells, GL7+ GC-Tfh, and Bcl6hi Tfh were also increased in the dLN of aged mice (Figure S2A-D). Therefore, Tfh cells are present in aged mice after immunization.

Tfr cells also were increased sharply in the dLNs, blood and spleen of aged mice compared to young mice after immunization (Figure 1D,F and Figure S1A-D). We have previously shown that increases in the Tfr:Tfh ratio, or the proportion of CXCR5+ cells in the GC that are FoxP3+, correlates with suppression of antibody responses (Sage et al., 2014a; Sage et al., 2013). This ratio, calculated as Tfr cells as a percentage of total CD4+CXCR5+ cells, was markedly increased in the dLN of aged mice (Figure 1G). This increase in Tfr percentages of the total CD4+CXCR5+ pool is partially due to increased total Tregs (which are Tfr precursors) in aged mice (Figure 1H). Thus, in response to an antigenic stimulus, there is an accumulation of Tfr cells which results in an increase of the Tfr:Tfh ratio in dLN.

Aged Tfr cells express more PD-1 and less ICOS, and accumulate in Peyer’s Patches

Next we determined if young and aged Tfh and Tfr cells were phenotypically distinct. We found substantial increases in the level of PD-1 expression on both Tfr and Tfh cells in aged mice (Figure 2A). Greater PD-1 expression may inhibit Tfr (and Tfh) differentiation, suppressive function and/or maintenance, since PD-1 suppresses both the differentiation and suppressive capacity of Tfr cells (Sage et al., 2013). Increased PD-1 expression was not unique to Tfh and Tfr cells in aged mice, because CD4+CXCR5− cells also had slightly elevated PD-1 expression.

Figure 2.

Figure 2

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Aged Tfr cells express more PD-1, less ICOS, and accumulate in Peyer’s patches. Comparison of PD-1 expression (a) and ICOS expression (b) on young and aged Tfr cells. Expression of PD-1 (a) and ICOS (b) was analyzed on CD4+ICOS−CXCR5− (CXCR5−), CD4+ICOS+CXCR5+FoxP3−CD19− (Tfh) and CD4+ICOS+CXCR5+FoxP3+CD19− (Tfr) cells from dLN of 2 (young) or 20 (aged) month old mice immunized with NP-OVA s.c. 7 days previously. Shaded histogram indicates CXCR5− population. (c-e) Comparison of intracellular Bcl6 expression (c), cell death by activated caspase staining (d) and cell cycling by Ki67 staining (e) in young and aged Tfr and Tfh cells and other populations as in (a). (f) Comparison of FoxP3 expression in FoxP3+CXCR5− and Tfr cells from dLNs of young or aged immunized mice. (g) Tfr and Tfh cells in Peyer’s patches (PP). Flow cytometric analysis of total ICOS+CXCR5+ cells in unimmunized young and aged mice (left) and quantification of Tfr cells as a percentage of ICOS+CXCR5+ cells (right). (h, i) Comparison of ICOS (h) and FoxP3 (i) expression on young and aged Tfr cells from PP of unimmunized mice. Data are from 5 mice per group and are representative of at least two experiments. Error bars indicate standard error.

The costimulatory molecule ICOS is essential both for the differentiation of Tfr cells and the differentiation/maintenance of Tfh cells. Tfh cells from the dLN had lower ICOS expression in aged mice compared to young mice (Figure 2B). Tfr cells from the dLN had much lower expression of ICOS in aged mice than young mice. Decreased ICOS expression may impact both Tfh and Tfr cell function. This phenotype was unique to Tfh and Tfr cells, as ICOS expression did not differ on CD4+CXCR5−FoxP3− cells nor CD4+CXCR5−FoxP3+ cells from the dLN after immunization.

Bcl6 is thought to be the master transcription factor for Tfh cells, and is essential for Tfr cell differentiation (Linterman et al., 2011; Nurieva et al., 2009). Bcl6 expression levels were similar in young and aged Tfh and Tfr cells, demonstrating that altered Bcl6 expression in Tfh and Tfr cells is not responsible for increased Tfh and Tfr expansion in aged mice (Figure 2C). However, both CD4+CXCR5−FoxP3− and CD4+CXCR5−FoxP3+ populations in aged mice had significantly higher levels of Bcl6 compared to young mice, suggesting that higher Bcl6 expression in Tfr and Tfh precursor cells may lead to increased differentiation.

Next, we assessed cell death due to previous reports that aged Tregs had decreased expression of the proapoptotic molecule BIM, which may lead to enhanced Treg survival (Chougnet et al., 2011). Inhibition of cell death could be one potential explanation for increases in Tfr cells in the aged mice. However, cell death, as measured by active caspase staining with VAD-FMK was unchanged between young and aged Tfh and Tfr cells (Figure 2D). Therefore, we investigated if increased cell cycling could explain the increase in Tfr or Tfh cells. Expression of the cell cycle marker Ki67, was significantly reduced (Figure 2E), indicating that increased cell cycling is not the reason for the greater percentages of Tfr or Tfh cells in aged mice. The increased Bcl6 expression on Tfh and Tfr precursors in aged mice, combined with the lack of significant differences in cell death and lower cell cycling, suggests that over-representation of Tfr and Tfh cells in aged mice may be due to increased Tfr and Tfh cell differentiation.

Since FoxP3 expression levels may control Treg suppression and stability (Sakaguchi et al., 2013; Williams and Rudensky, 2007), we also compared FoxP3 expression in Tfr cells from young and aged mice. Tfr cells (as well as total Tregs) from immunized aged mice had slightly attenuated FoxP3 levels compared to young mice (Figure 2F), suggesting that Tfr cells in aged mice may have defective suppressive capacity. Together, these data indicate that Tfr cells in aged mice are phenotypically distinct from Tfr cells in young mice, with increased PD-1, but reduced ICOS and FoxP3 expression, which may alter their effector functions and/or stability.

Besides dLN and blood, Tfh and Tfr cells can also reside in Peyer’s patches (PP) of the gut where they regulate IgA production (Kawamoto et al., 2014; Sage et al., 2014b; Tsuji et al., 2009). We found a substantial population of Tfh cells in PP of unimmunized young and aged mice, and similar percentages (Figure 2G and data not shown). There also was a considerable population of Tfr cells in the PP of both young and aged mice (Figure 2G), but a proportional increase in Tfr cells in the total CXCR5+ CD4+ T cell pool in PP of aged mice compared to young mice (Figure 2G). This overrepresentation of Tfr cells in PP may lead to lower IgA production and contribute to a change in microbiota in the gut of aged animals since altered Tfr:Tfh ratios may change microbiota (Kawamoto et al., 2014). ICOS expression was significantly attenuated on PP Tfr cells, and reduced to a lesser degree on PP Tfh cells of aged mice (Figure 2H). FoxP3 expression however, was similar in aged and young Tfr cells in PP (Figure 2I). Taken together, these studies indicate that Tfr cells are proportionally increased in PP as well as dLN cells of aged mice; this increase may alter T cell dependent antibody production in aged mice.

Aged Tfh cells Have Less Antigen-specific Stimulatory Capacity

We next investigated Tfh cell function in aged mice since immunosenescence has been postulated to cause cell-intrinsic defects in effector cells in aging. We first used antigen-non-specific in vitro B cell class switch recombination assays that we developed (Sage et al., 2014a; Sage et al., 2014b; Sage and Sharpe, 2015). These assays sensitively assess cell-intrinsic Tfh effector function separately from Tfh differentiation and do not depend on the antigen specificity of the Tfh cells. We sorted Tfh cells as CD4+ICOS+CXCR5+GITR−CD19− from dLNs of young (2 month) or aged (20 month) mice that were immunized with NP-OVA s.c. 7 days previously. Importantly, GITR expression was comparably low on aged and young Tfh cells (Figure S3A). These Tfh cells were cultured with young CD19+ B cells (also isolated from dLNs) along with anti-IgM and anti-CD3. Surprisingly, young and aged Tfh cells similarly stimulated B cells to class switch to IgG1 (Figure 3A). We next examined expression of the GC and activation marker GL7 on B cells because GL7 expression is a sensitive marker for B cell activation in these assays (Sage et al., 2014a). GL7 was similarly upregulated on B cells when either young or aged Tfh cells were in the cultures (Figure 3A). Intracellular Bcl6 and Ki67 expression also were similar in young and aged Tfh cells following culture with B cells (Figure 3B). These unexpected findings suggest that young and aged Tfh cells have comparable stimulatory capacity in these antigen non-specific assays. Thus, although many defects have been seen in the CD4+ T cell compartment with advancing age, Tfh cells from young and aged mice appear to have similar cell intrinsic capacity to participate in B cell help.

Figure 3.

Figure 3

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Aged Tfh cells have defective capacity to stimulate antigen-specific antibody production. (a) Non-antigen specific antibody stimulation assays. CD4+ICOS+CXCR5+GITR−CD19− (Tfh) cells sorted from 2 (young) or 20 (aged) month old mice immunized with NP-OVA s.c. 7 days previously were cultured with CD19+ B cells from dLN of similarly immunized young mice along with anti-CD3 and anti-IgM for 6 days. B cells were surface stained for GL7 and intracellularly stained for IgG1. Plots are pre-gated on CD19+IA+ B cells. (b) Ki67 and Bcl6 staining of Tfh cells from assays as in (a). (c) Cells sorted as in (a) were cultured with NP-OVA for 6 days, and IgG1+GL7+ cells analyzed as in (a). (d) Ki67 and Bcl6 staining of young or aged Tfh cells from assays as in (c). (e) Aged Tfh cells were cultured with young B cells and NP-OVA as in (c) in the presence of anti-PD-L1 or isotype control. (f-g) Comparison of B cell responses mediated by young and aged Tfh cells in vivo. Young or aged Tfh cells were sorted from mice immunized with NP-OVA 7 days previously and transferred to CD28−/− recipients that were immunized. 10 days later dLNs were analyzed for GC B cells (f) and plasma cells (g), and (h) serum for NP-specific antibody titers. Data are from replicate wells (a, c, e), and representative of at least two (e) or three (a,c) experiments. In vivo experiments are from 10 pooled mice per condition and transferred into a single mouse recipient, and representative of three independent experiments. See also Figure S3.

The similar stimulatory function of young and aged Tfh cells was perplexing given the defective antibody production in immunized aged mice. We hypothesized that Tfh cells from aged mice may be functional, but exhibit antigen-specific defects. To test this hypothesis, we adapted our in vitro assays to assess antigen-specific responses. We cultured young B cells with young or aged Tfh cells (sorted from dLNs of NP-OVA immunized mice) along with NP-OVA for 6 days and measured B cell class switch recombination. Aged Tfh cells stimulated B cells to undergo class switch recombination slightly less well to IgG1 and substantially less well to IgG2a, compared to young Tfh cells (Figure 3C). Moreover, a slightly lower percentage of aged Tfh cells expressed Bcl6 and Ki67 expression compared to young Tfh cells (Figure 3D). Therefore, in these antigen-specific in vitro stimulation assays, aged Tfh cells showed modest deficits in B cell help, leading to a small reduction in antibody production by the B cells.

Since PD-1 is more highly expressed on aged Tfh cells compared to young Tfh cells, and PD-1 blockade can enhance CD4+ T cell cytokine production and CD8+ proliferation in aged mice (Lages et al., 2010; Mirza et al., 2010), we next tested whether PD-1 pathway blockade could overcome some of the age-related defects in Tfh cell function. Anti-PD-L1 blocking mAb substantially enhanced the stimulatory capacity of aged Tfh cells in antigen-specific B cell stimulation assays (Figure 3E). Thus, aged Tfh cells show defects in activation following antigen-specific stimulation, and PD-L1 blockade can improve the function of the antigen-specific Tfh cells.

Next we determined if aged Tfh cells had less stimulatory capacity in vivo using an adoptive transfer approach. We immunized young or aged mice with NP-OVA, and 7 days later sorted Tfh cells from dLNs and transferred them to young CD28−/− mice (which cannot generate Tfh and Tfr cells) and immunized them with NP-OVA. Aged Tfh cells stimulated markedly less GC B cell differentiation and plasma cell formation, as well as significantly less NP-specific antibody production in vivo compared to young Tfh cells in transfer recipients at day 10 post transfer and immunization (Figure 3F-H). The differences in GC B cells persisted until 20 days after transfer, but plasma cell percentages did not (Figure S3B). Thus, there were much greater defects in the capacity of aged Tfh cells to support B cell responses in vivo compared to in vitro likely reflecting increased requirements for proliferation in vivo than in vitro. In support of this, more young Tfh cells persisted in vivo after transfer compared to aged Tfh cells. (Figure S3C). Also, it is possible that Tfh and/or B cells have a higher threshold for activation in vivo than in our in vitro assays.

Aged Tfr cells Have Potent Suppressive Capacity

We next compared the suppressive function of young and aged Tfr cells, since Tfr cells predominate in aged mice, and appear to be phenotypically distinct from young Tfr cells. First, we cultured young or aged dLN Tfr cells with young dLN Tfh and B cells using in vitro non-antigen-specific assays, and stimulated the cells with anti-CD3 and anti-IgM, as in Figure 3A. Young and aged Tfr cells potently suppressed class switch recombination to IgG1 to a similar extent (Figure 4A-B). Young and aged Tfr cells attenuated GL7 expression on B cells almost to the level of unstimulated B cells and comparably suppressed young Tfh cells, as evidenced by the similar reduction in Ki67 expression in Tfh cells (Figure 4C). In addition, a similarly high proportion of young and aged Tfr cells were in cell cycle in these cultures (Figure 4D). The ratios of Tfh and Tfr cells in these cultures also were roughly similar (Figure S4). Thus, Tfr cells from young and aged mice have similar and potent intrinsic capacity to inhibit B cell responses. Since Tfr cells are greatly expanded in aged mice and these cells have suppressive capacity comparable to young Tfr cells, the increase in Tfr cell proportions in aged mice is a major reason for defective B cell responses in vivo.

Figure 4.

Figure 4

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Aged Tfr cells potently suppress Tfh cell-mediated antibody production. (a) Tfr cell antigen non-specific suppression assays. Sorted CD4+ICOS+CXCR5+GITR+CD19− (Tfr) cells from 2 (young) or 20 (aged) month old mice immunized with NP-OVA s.c. 7 days previously were cultured with young B cells and young Tfh cells and anti-CD3 plus anti-IgM. (b) Quantification of IgG1+GL7+ cells in plots in (a). (c) Intracellular Ki67 staining of FoxP3− Tfh cells and (d) Intracellular Ki67 staining of FoxP3+ Tfr cells from young Tfr (red) or aged Tfr (grey) in cultures described in (a). (e-i) Cells sorted as in (a) were cultured with Tfh and B cells from young mice along with NP-OVA. 6 days later B cells were stained for surface GL7 or intracellular IgG1 (e) or IgG2a (f). B cells were identified as CD19+IA+. CD4+FoxP3− Tfh from cultures as in (e) were stained intracellularly for Ki67(g) or (h) IFNγ and IL17A (e). (i) Young (red) or aged (gray) Tfr cells from cultures as in (e) were stained intracellularly for Ki67. (j) In vivo Tfr cell suppression assay. Young Tfh and young or aged Tfr cells (from mice immunized with NP-OVA 7 days previously) were transferred to CD28−/− mice which were immunized with NP-OVA. 10 days later serum was analyzed for NP-specific IgG. (k) In vivo functional assay of Tfh and Tfr cells from young or aged mice. Total CD4+CXCR5+ICOS+CD19− cells from young or aged mice immunized with NP-OVA 7 days previously were transferred to CD28−/− mice which were immunized with NP-OVA. 10 days later serum was analyzed for NP-specific IgG. Data are from replicate wells and are representative of three independent experiments (a-i), representative of two independent experiments (j), or combined data from three independent experiments (k). See also Figure S4.

Next we analyzed Tfr cell function using antigen-specific in vitro assays. Aged and young Tfr cells had virtually the same ability to suppress class switch recombination to IgG1 when NP-OVA was added to the cultures (Figure 4E). However, there was a significant decrease in suppression of class switched IgG2a+ GL7+ B cells by aged Tfr cells compared to young Tfr cells, albeit this difference of ~0.3% is very minor (Figure 4F). Aged and young Tfr cells similarly suppressed Ki67 expression (Figure 4G) and IFN-γ production by Tfh cells (Figure 4H). In addition, young and aged Tfr cells similarly expressed Ki67 at the end of the suppression assay (Figure 4I). These data suggest that young and aged Tfr cells are similarly stimulated by B cells and have comparable suppressive function.

Next, we compared the ability of young and aged Tfr cells to suppress antigen-specific antibody responses in vivo. We adoptively transferred young Tfh cells along with young or aged Tfr cells (all sorted from immunized mice) to CD28−/− recipients, immunized these recipients with NP-OVA, and assessed antigen-specific antibody levels in the serum 10 days later. Aged Tfr cells suppressed antigen-specific antibody levels to the same degree as young Tfr cells (Figure 4J).

Since aged Tfh cells have defects in B cell stimulation in vivo and aged Tfr cells are equally suppressive in vivo, we next used adoptive transfer approaches to compare the young vs. aged CXCR5+ populations in their endogenous ratios. This approach enables us to determine the consequences of the altered function of aged Tfh cells together with the marked increases in Tfr cells in aged mice on antibody production in vivo. We sorted total CD4+CXCR5+CD19− cells from young or aged mice on day 7 after immunization with NP-OVA, and transferred these cells (preserving their endogenous proportions) into CD28−/− recipients that were then immunized with NP-OVA. Ten days later we assessed antigen-specific antibody levels in serum. There were significantly higher levels of NP-specific IgG in the sera of recipients of total Tfh and Tfr cells from young mice compared to recipients of aged Tfh and Tfr cells (Figure 4K). These results show that defective antibody responses in aged mice are due, at least in part, to the combination of defective Tfh cell function and higher proportions of Tfr cells in aged mice.

Together, our studies provide insights into mechanisms for defective antibody production in aging. We find an over-representation of functionally competent suppressive Tfr cells in aged mice, most likely resulting from enhanced differentiation of Tfr cells. However, expansion of memory Tfr cells may also contribute. In addition, we find that Tfh cells are generated following immunization of aged mice, but these aged Tfh cells fail to elicit strong antigen-specific B cell responses in vivo. Aged Tfh cells express higher levels of PD-1 and PD-1 blockade can improve Tfh cell function Thus, the substantial increase in fully suppressive Tfr cells, combined with the decrease in antigen-specific responses of Tfh cells, results in a significant defect in antibody production in aged mice. Although other mechanisms, such as defects in clonality and/or naïve T or B cell numbers also may contribute, our data point to alterations in Tfh cell activity and Tfr cell proportions as being a key mechanism that impairs antibody production in aging. Therefore, approaches that downmodulate Tfr cells may provide a strategy for improving humoral immune responses in the elderly.

Experimental Procedures

Mice

C57BL/6 mice were obtained from the National Institute of Aging or the Jackson Laboratory. Young mice were 8 weeks old and aged mice were 20 months old. CD28−/− mice were purchased from The Jackson Laboratory. Animal protocols were approved by the Harvard Medical School Standing Committee on Animals.

Immunizations

For standard NP-OVA immunizations, 100μg NP18-OVA (Biosearch Technologies) in a 1:1 H37RA CFA (DIFCO) emulsion was injected s.c. into the flanks of young or aged mice. The spleen, dLN, and blood were harvested 7-27 days later. PP were harvested from unimmunized mice. Serum was isolated from blood using serum separator tubes (BD vacutainer) and NP-specific IgG measured by ELISA as previously described (Sage et al., 2013).

Flow Cytometry

Cells were harvested and stained with directly labeled antibodies (as detailed in Supplement). For intracellular staining, the FoxP3 fix/perm kit was used (eBioscience). For VAD-FMK staining, the Caspglow kit was used (eBioscience). For intracellular cytokine staining, cells were incubated with 1μg/ml ionomycin (Sigma) and 500ng/ml PMA (Sigma) in the presence of Golgistop (BD biosciences) for 4 hours prior to staining.

Adoptive Transfers

For Tfh cell transfers, 10 young or aged mice were immunized and 7 days later dLN were harvested. 2×105 CD4+ICOS+CXCR5+GITR−CD19− Tfh cells were sorted and transferred to CD28−/− mice that were then immunized. 10 days later the dLNs and sera were harvested. For young and aged Tfr transfers, 1×105 Tfh and 2.5×104 young or aged CD4+ICOS+CXCR5+GITR+CD19− Tfr cells were sorted and transferred to CD28−/− mice which were immunized. For total CXCR5+ cell transfers, 7.5×104 young or aged sorted CD4+CXCR5+ICOS+CD19− cells were transferred to CD28−/− mice, which were immunized.

In vitro Suppression Assay

For non-antigen specific Tfh stimulation assays, 3×104 dLN CD4+ICOS+CXCR5+CD19−GITR− Tfh cells from young or aged mice were plated with 5×104 CD19+ young B cells (all purified from dLNs of immunized mice) and 2μg/ml soluble anti-CD3 (2C11, BioXcell) plus 5μg/ml anti-IgM (Jackson Immunoresearch) for 6 days. Antigen-specific assays were performed similarly, except NP-OVA was added to the cultures instead of anti-CD3/IgM. In some assays 20μg/ml of anti-PD-L1 (clone 10F.9G2) or isotype control was added. For Tfr cell suppression assays, 1.5×104 CD4+ICOS+CXCR5+CD19−GITR+ Tfr cells from the dLN of young or aged immunized mice were added to the wells along with Tfh and B cells. Cells were harvested and analyzed 6 days later.

Statistical Analysis

Unpaired Student’s t test was used for all comparisons, data represented as mean +/− SD or SE are shown. P values < 0.05 were considered statistically significant. * P<0.05, ** P<0.005, *** P<0.0005.

Supplementary Material

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2

Acknowledgments

This work was supported by the NIH; BAA-NIAID-DAIT-NIHAI2010085 (to AHS, MH and GF), R37 AI38310 (AHS), and P01 56299 (AHS, GF), 5T32HL007627 (PS), the Glenn Foundation for Medical Research (MH) and the Evergrande Center for Immunologic Diseases (AHS).

Footnotes

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Associated Data

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Supplementary Materials

1
NIHMS699135-supplement-1.pdf (6.7MB, pdf)
2
NIHMS699135-supplement-2.pdf (43.2KB, pdf)

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