Age-related Defects in CD4 T Cell Cognate Helper Function Lead to Reductions in Humoral Responses

Keywords: aging, B lymphocytes, germinal centers, antibody, vaccines

Animals.

All mice used in this study were bred at the Trudeau Institute Animal Core Facility (Saranac Lake, NY). Intact young (2–4 mo) and aged B10.BR (>20 mo) mice were used in the first set of experiments. Adoptive transfer experiments involved the transfer of antigen-specific CD4 cells into adoptive hosts. Young (2–4 mo) and aged (15–19 mo) as well as TCR Tg mice, on a B10.BR background, were used as the source of donor CD4 T cells. These mice express a Vβ3/Vα11 TCR transgene specific for a peptide fragment of pigeon cytochrome C (PCC; 24). Young (2–4 mo) or aged (20–24 mo) CD4KO mice, backcrossed to B10.Br for 10 generations, were used as the adoptive hosts. All mice were housed in sterilized, Hepa-filtered, individually ventilated caging at the animal facility at the Trudeau Institute until their use. All experimental procedures involving mice were approved by Trudeau Institute Institutional Animal Care and Use Committee.

CD4 T Cell Isolation, Adoptive Transfer, and Immunization.

Lymphocytes were harvested from the spleens and peripheral lymph nodes of young or aged TCR Tg mice. For each experiment, lymphocytes from two young and four aged mice were pooled to generate donor T cell populations. The enrichment of naive Tg CD4 T cell populations by negative selection has been described previously (16). The percent TCR Tg⁺ cells within the resulting enriched CD4 populations were similar for young (97.2 ± 1.9%) and aged (94.5 ± 5.6%) populations. In addition, these experiments have also been performed using FACS sorted Tg⁺CD4 T cells from young and aged donors with similar results. Donor Tg CD4 cells were transferred i.v. into CD4KO adoptive hosts (10⁶/mouse or as indicated). When indicated, donor cells were carboxy fluorescein succinimidyl ester (CFSE) labeled as previously described (16). Control hosts received no CD4 T cells. Mice were immunized i.p. with 200 μg 4-hydroxy-3-nitrophenyl acetyl conjugated PCC (NP-PCC) or PBS in alum. For each experiment, three to five hosts were used in each group and each experiment was performed from two to four times.

In Vitro Stimulation of CD4 T Cells.

Culture conditions for CD4 T cells have been described previously (16). Enriched populations of Tg CD4 T cells from young and aged mice were stimulated with PCC peptide and DCEK-ICAM APC cell line (25) at a 2:1 T/APC ratio in 24-well plates. At the indicated time points, T cells were harvested and examined for CD4, Vβ3 and CD154, or CD28 expression by flow cytometry as described below. Data presented representative of at least three separate experiments.

Immunofluorescent Staining.

Splenocytes from immunized host animals or from in vitro–stimulated cultures of young and aged TCR Tg CD4 T cells were harvested at the indicated time points and individually analyzed. All staining was done at 4°C in PBS with 1% BSA and 0.1% NaN₃ and all samples were treated with Fc blocking antibody (24G2) before staining. The following antibodies were used (all purchased from BD Biosciences): Cy-chrome anti-CD4 (RM4-5), PE or biotin anti-Vβ3 (KJ25), PE anti-CXCR5, PE anti-CD154, FITC anti–mouse IgG1, biotin anti-CD134, and PE anti-CD38. Strepavidin conjugated to allophycocyanin was also purchased from BD Biosciences. FITC-peanut agglutinin (PNA) was purchased from Sigma-Aldrich. NP conjugated to allophycocyanin (NP-APC) was prepared as described previously (26). Immediately before analysis, propidium iodide was added to each sample to allow for identification of viable cells. Flow cytometry was performed using a FACS Calibur flow cytometer (Becton Dickinson) and the data were analyzed with Cell Quest software.

Detection of NP-specific Antibodies.

Serum was collected from all immunized animals. NP-specific IgM and IgG1 was determined by ELISA with NP-conjugated BSA. Isotype-specific antibodies for detection were purchased from Southern Biotechnology Inc. Titers were determined by the last dilution of serum with detectable antibody above background.

Histology.

Spleens were frozen directly in Tissue-Tec OCT (Fisher Scientific) over liquid nitrogen. Frozen tissue blocks were brought to –20°C and 7-μm sections were cut and placed on poly-L-lysine coated slides. Slides were dried at room temperature overnight and then either probed with antibodies or stored at –20°C. Slides were brought to room temperature, fixed in acetone at 4°C for 10 min, and then placed in PBS for 5 min to remove the OCT. All slides were blocked with 5% BSA in PBS for 30 min and then washed before being probed with biotinylated antibodies to various antigens for 30 min in a humidified chamber. Slides were incubated with biotinylated anti-CD4 (L3T4; BD Biosciences) and biotinylated PNA (Vector Laboratories) and were then washed. They were then incubated with streptavidin-Alexa 594, streptavidin-Alexa 488, or streptavidin-Alexa 350 (Molecular Probes) for 30 min, washed, and mounted with Polymount (Polysciences). Sections were also counterstained with Hoechst (Sigma-Aldrich). Slides were viewed with a Zeiss Axioplan 2 microscope and images were recorded with a Zeiss AxioCam digital camera (Carl Zeiss MicroImaging, Inc.) using the Zeiss proprietary software, Axiovision 3.0.6.0. Images, which were originally obtained at 10× magnification, were manipulated in Adobe Photoshop 5.5.

Decreased B Cell Expansion and GC Formation in Aged Mice.

To confirm the findings previously published of reduced antigen-specific B cell expansion and differentiation in aged mice (1, 27), nontransgenic young and aged B10.Br mice were immunized i.p. with NP-PCC/alum and on day 14, the NP-binding population was examined by flow cytometry. In NP-PCC immunized groups, there was a threefold reduction in the expansion of NP⁺ cells in aged mice compared with young (Fig. 1 A). There was also reduced differentiation of these NP⁺ cells in aged mice. Fig. 1 B shows dot plots demonstrating that a significant proportion (63%) of the young NP⁺ cells had differentiated to GC phenotype (CD38^loPNA^hi), while those in aged mice retained a predominantly naive phenotype (CD38^hi PNA^lo; 28). In addition, the NP-specific IgG1 titers in immunized young mice were over 100-fold higher compared with the aged mice (Fig. 1 C). Together, these results demonstrate that there was a statistically significant age-related defect in B cell expansion, differentiation and antibody production. However, the frequency and differentiation state of PCC-specific CD4 T cells in these young and aged mice could not be enumerated or controlled. This is an important issue since the number of naive CD4 T cells decreases dramatically with age and the frequency of antigen-specific CD4 cells is also likely to be affected (29, 30). Therefore, we developed an adoptive transfer model to examine the effect of age on CD4 T cell helper function.

Adoptive Transfer Model to Study In Vivo Cognate Helper Function.

To examine the impact of age on CD4 T cell cognate helper function more precisely, we adoptively transferred AND TCR Tg CD4 cells from young and aged mice into CD4KO recipients. The AND TCR Tg CD4 cells are specific for a peptide of PCC and express a naive phenotype (both cell surface phenotype and functional phenotype) even in very old Tg mice (18). We have extensively characterized the responses of the young and aged AND TCR Tg CD4 T cells and our published results show that the naive Tg CD4 cells from aged mice do not respond to TCR stimulation as well as cells from young mice, resulting in reduced IL-2 production, expansion and differentiation both in vitro and in vivo (16, 18, 20, 31). Therefore, this model is both novel and very advantageous as it allows us to directly compare similar populations of antigen-specific naive CD4 T cells from young and aged mice. It also allows us to eliminate differences in the frequency or antigen experience of CD4 T cells that are likely to occur in intact young and aged mice (29, 30).

AND TCR Tg CD4 cells from young or aged donors were transferred into young or aged CD4KO hosts, which exhibit no endogenous cognate helper function, as shown in Fig. 2. When young Tg CD4 cells were transferred into young NP-PCC–immunized hosts, there was significant expansion and differentiation to GC phenotype (CD38^lo PNA^hi) of the NP-binding population (Fig. 2, A and B, respectively, left panels) on day 14. This indicated that the transferred Tg CD4 cells provided effective cognate help that induced good GC differentiation. Upon CD4 T cell transfer and immunization with PBS (Fig. 2, A and B, middle panels) or upon NP-PCC immunization with no transfer of CD4 T cells (Fig. 2, A and B, right panels), there was no NP-specific expansion or differentiation to GC phenotype. Therefore, in these experiments, all of the observed cognate helper function could be attributed to the adoptively transferred Tg CD4 cells.

Decreased Cognate Helper Function of Aged CD4 Cells.

To determine whether the age of donor CD4 T cells affected expansion and differentiation of NP⁺ B cells, we transferred naive TCR Tg CD4 T cells from young and aged donors into young CD4KO hosts and immunized with NP-PCC. Fig. 2, C and D, shows representative flow cytometry results comparing young and aged donor T cell function on day 14. The percentage of NP⁺ cells generated in hosts that received young donor cells was dramatically increased compared with hosts receiving aged donor cells (Fig. 2 C). In addition, the percentage of NP⁺ cells that had differentiated to a GC⁺ phenotype (CD38^loPNA^hi) was greater in hosts receiving young donor cells (Fig. 2 D).

On days 7, 14, and 21 after immunization, the total numbers and percentages of host NP-binding cells and NP-binding B cells expressing a GC phenotype were determined by flow cytometry. Fig. 3, A and B, shows that at each time point, hosts receiving young CD4 T cells had significantly greater expansion of the NP-binding population compared with those receiving CD4 T cells from aged donors. Fig. 3, C and D, shows that at each time point there is also a significant increase in the percentages and numbers of differentiated GC⁺NP⁺ B cells in hosts receiving young donor cells compared with those receiving aged T cells. In PBS-immunized hosts or in hosts receiving no donor TCR Tg CD4 cells very few GC phenotype cells were evident (unpublished data).

In addition, we examined whether CD25⁺ CD4 regulatory T cells played a role in this age-related reduction in cognate helper function by removing CD25⁺ CD4 T cells by FACSorting before adoptive transfer. When the number of total NP⁺ and NP⁺GC⁺ cells was determined on days 7, 14, and 21, the results were similar to those in Fig. 3. Significant reductions in NP⁺ cell expansion and GC differentiation were observed in hosts receiving aged donor T cells (unpublished data). These results indicate that regulatory CD4 T cells do not play a role in the reduced function of aged CD4 T cells in this model.

In the experiments shown in Fig. 3, A–D, 10⁶ Tg CD4 cells from young or aged donors were transferred into the adoptive hosts. To determine if transferring fewer or more aged CD4 T cells would enhance helper function, the number of transferred cells was titrated from 10³ to 10⁷. Fig. 3 E shows that when 10³ to 10⁶ T cells were transferred, CD4 T cells from young donors always induced significantly more NP-specific GC phenotype cells compared with those from aged donors. Interestingly, when 10⁷ CD4 cells were transferred, both young and aged populations provided greatly reduced levels of cognate helper activity. For the young helper population at the 10⁷ cell concentration, the heightened engagement of CD40 on responding B cells, as a result of increased antigen-specific T cell help, has been shown to ablate GC formation and prematurely terminates an ongoing GC response through an unknown mechanism (32). These results indicate that providing greater numbers of antigen-specific aged CD4 T cells does not lead to better cognate helper function.

Production of NP-specific serum antibodies was also examined in each adoptive host. Fig. 3 F shows that on days 7, 14, and 21 after immunization, there were significantly higher levels of NP-specific IgG1 in the hosts receiving CD4 cells from young mice compared with those receiving cells from aged donors. Interestingly, there was a significant age-related difference in IgM production only on day 7, but not on days 14 or 21 (Fig. 3 G), which was not surprising as IgM production is less dependent on CD4 cognate help compared with IgG1 (33).

In Vivo Migration and Expansion of Donor Tg CD4 T Cells.

Because CD4 T cells from aged donor mice exhibited reduced cognate helper function, the next set of experiments examined whether the in vivo migration and expansion of the aged donor T cells was defective. Fig. 4 shows photomicrographs of splenic sections from young hosts receiving young (Fig. 4 A) or aged (Fig. 4 B) donor T cells. Although the GCs observed in the sections from donors receiving young CD4 T cells were larger and more frequent compared with those receiving aged donor cells, we found no difference in migration of the donor T cell populations. Both young and aged CD4 T cells (Fig. 4, red) migrated similarly and could be found in the B cell follicle (blue) and in the GC (green), with no age-related differences. We also examined the expression of the chemokine receptor CXCR5 by these young and aged donor CD4 T cells by flow cytometry. As shown in Fig. 5 A, we found no difference in CXCR5 expression on the young and aged donor cells. This correlates well with the histology results shown in Fig. 4, since CXCR5 expression is required for appropriate T cell migration into GCs (23).

Because the CD4 T cells from aged mice seem to migrate appropriately, they must lack the ability to provide cognate help once they reach the B cell follicle. Given that CD28 and CD134 (OX40) have been implicated in the cognate functions of CD4 T cells (22, 34), we examined the expression of these molecules on young and aged Tg CD4 T cells after 4 d of culture with Ag/APC. We found no difference in the expression of either CD134 or CD28 on naive (unpublished data) or in vitro activated young and aged Tg CD4 T cells (Fig. 5, B and C). Another molecule that has been shown to play an important role in T–B cognate interactions is CD154 (CD40 ligand), which is expressed by activated helper T cells (33). We examined the expression of CD154 on young and aged Tg CD4 T cells after in vitro stimulation with Ag/APC. CD154 expression occurs in two phases, one very early after TCR stimulation (6 h) and one much later (40–60 h) (35). Fig. 5 D shows that both young and aged Tg CD4 T cells up-regulate CD154 similarly at the early time point, but the second phase of expression is significantly lower on aged cells. As we find that there is a significant reduction in CD154 expression on the aged cells, this could have a dramatic impact on cognate helper function and could account for the results shown in Fig. 3.

Additionally, the in vivo expansion of these donor CD4 populations in young hosts was examined. We have shown previously that the early (through day 6) in vivo expansion of aged CD4 T cells is reduced compared with young cells (17, 20). Fig. 6 A shows that on day 7 after immunization there was significantly greater expansion of donor cells from young mice compared with those from aged mice, but by days 14 and 21, expansion had reached a plateau and similar numbers of young and aged donor cells were recovered. These results could be due to the fact that the young donor effectors had either stopped proliferating or had begun to undergo apoptosis. In either case, this is likely due to host-related factors controlling the number of peripheral T cells. Because these CD4 T cells from young and aged donors were labeled with the fluorescent dye CFSE before transfer, we could also examine the extent of cell division after immunization. Fig. 6 B shows that on days 7, 14, and 21, cells from young donors (top panel) had undergone more rounds of cell division compared with those from aged donors (bottom panel). These results indicate that even though the total cell recoveries were the same on days 14 and 21, there may be other age-related differences in the in vivo–generated CD4 effector populations derived from young and aged donors.

Effect of Host Age on Humoral Responses.

To determine if there were age-related defects in host components, including B cells and follicular dendritic cells (FDCs), we modified our adoptive transfer model such that TCR Tg CD4 T cells from young mice were transferred into young and aged hosts. In these studies, the adoptive hosts were 20 to 24 mo old. On day 14 after immunization, we found no difference in expansion (Fig. 7 A) or GC differentiation (Fig. 7 B) of the NP⁺ populations in young and aged hosts. We also examined other time points and found no significant differences in either the number of NP-specific cells (Fig. 7 C) or the number of GC phenotype cells (Fig. 7 D) in young versus aged hosts. Finally, we compared the cognate function of young versus aged donors transferred into young versus aged hosts in the same experiment. We found no difference in the expansion of NP⁺ B cells when young donor T cells were used, but there was significantly reduced expansion when aged donor cells were used, regardless of host age (Fig. 7 E). The same pattern was also seen for GC differentiation of the NP⁺ B cells (Fig. 7 F). Although it does appear that there was a reduction in the number of NP⁺ and GC⁺ B cells when aged donor T cells were transferred into aged hosts, this difference was not significant. Importantly, this experiment shows that transferring a population of antigen-specific TCR Tg–aged donor cells into aged hosts does not overcome the defect that we observed in intact aged mice as shown in Fig. 1.

As it has been shown that aging negatively impacts B cell Ig class switching (36), we examined serum IgG production and surface IgG expression on NP-specific cells from these young and aged hosts. Fig. 8 A shows that there was no difference in serum titers of NP-specific IgG1 in the young and aged hosts over a period of 3 wk after immunization. Additionally, we also found no difference in serum IgM titers (Fig. 8 B). To examine whether similar proportions of NP-specific cells in the young and aged hosts were undergoing class switching, we examined surface IgG1 expression by flow cytometry. Fig. 8 C shows representative histograms indicating that similar percentages of NP-specific cells from young and aged hosts stained positive for IgG1. Therefore, in this model, we found no age-related defects in the ability of NP-specific cells to undergo Ig class switching.

We also examined the expansion of young donor CD4 T cells in the young and aged hosts. Fig. 9 A shows that there was no difference in the number of the donor cells recovered in either the young or aged hosts at days 7, 14, or 21 after transfer and immunization. In addition, Fig. 9 B shows the CFSE profiles of the donor cells at each time point. There were no differences in these profiles, indicating that the young donor T cells responded similarly in the young and aged hosts, with no age-related differences in donor T cell priming. Taken together the results shown in Figs. 7, 8, and 9 indicate that the age of the host components have no effect on the response to immunization in our NP-PCC model and that age-related differences in CD4 T cell cognate function could account for the reduction in antibody production that we have observed in our model.

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