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As increasing numbers of elderly patients require solid organ transplantation, the need to better understand how aging modifies alloimmune responses increases. Here, we examined whether aged mice exhibit augmented, donor-specific memory responses prior to transplantation. We found that elevated donor-specific IL-17, but not IFN-γ, responses were observed in aged mice compared to young mice prior to transplantation. Further characterization of the heightened IL-17 alloimmune response with aging demonstrated that memory CD4+ T cells were required. Reduced IL-2 alloimmune responses with age contributed to the elevated IL-17 phenotype in vitro, and treatment with an anti-IL-17 antibody delayed the onset of acute allograft rejection. In conclusion, aging leads to augmented, donor-specific IL-17 immune responses that are important for the timing of acute allograft rejection in aged recipients. IL-17 targeting therapies may be useful for averting transplant rejection responses in older transplant recipients.
The number of patients over 65 years of age waiting for kidney transplants has increased considerably within the last decade to more than 9% of all patients awaiting donor organs (1, 2). As the elderly subpopulation is increasing in Western countries and many endstage organ disorders are associated with increased age, it is likely that a growing number of older patients will be considered as transplant recipients in coming years (2, 3).
Older patients suffer from increased morbidity and mortality resulting from infections and malignancy, but also exhibit an impaired ability to reject organ allografts (4-11). Some reports have indicated that reduced alloimmune responses are due to declining cellular immunity (12, 13). An experimental study has shown that the reduced ability of aged recipients to reject skin allografts is associated with declining CD4+ T cell responses (14). While several clinical studies have indicated that aging is associated with lower frequencies of acute allograft rejection, other reports have suggested that when rejection occurs in older recipients the episodes are more severe, reviewed in (8). Furthermore, there is evidence indicating that aging is associated with increased chronic rejection (15).
Studies using non-allogeneic models have indicated that aging impairs adaptive T cell function. Specifically, TCR activated aged T cells exhibit impairments in CD28 signaling, IL-2 receptor activation, and downstream TCR signaling pathways (16-18). Additionally, immune senescence may be influenced by the accumulation of memory T cells (4, 19). This is potentially significant because cross-reactive effector memory T cells participate in increased rates of acute allograft rejection and can impair the induction of transplantation tolerance (20). Cross-reactive memory T cells can be detected through rapid production of IFN-γ. However, it is not clear whether aged hosts exhibit augmented donor-specific memory responses prior to transplantation, and if this is the case, the characteristics of the memory response are not well elucidated. Finally, the impact of any aged-altered, pre—transplant, donor specific memory responses on transplantation outcomes has not been clearly defined.
We examined whether aged mice exhibit increased donor specific IFN-γ responses prior to transplantation. We found that aged hosts showed increased pre-transplant, donor-specific IL-17 responses, but not IFNγ responses, compared to young hosts. CD4+ memory T cells were responsible for the heightened IL-17 alloimmune response with aging. Aged mice exhibited a slightly delay to time of allograft rejection as compared to young mice, and inhibiting IL-17 in aged mice significantly extended allograft survival. Our results indicate that augmented IL-17 alloimmune responses influence the tempo of allograft rejection in aged transplant recipients.
Aged (18-23 months) and young (2-4 months) CBA (H2k) and C57BL/6 (H2b) mice were purchased from the NIA rodent facility, and animal use was approved by theYale University IACUC. All mice were housed under pathogen free conditions, and animals were not used if they had skin lesions, or exhibited weight loss or lymphadenopathy. Additionally, sentinel mice were regularly tested for common murine pathogens: mouse hepatitis virus, parvovirus, mycoplasma pneumonia, sendai virus, lymphocytic choriomengitis virus, ectromelia virus, pneumonia virus, and epizootic diarrhea.
Full-thickness trunk skin was removed from donor mice and transplanted (stapled) onto recipients as previously described (21). Rejection was defined as graft necrosis for >90% of the graft area.
The generation of BMDCs has been described previously (22). To perform the MLR, 1×105 purified T cells/well were cultured with 1×105 irradiated (28Gy) allogeneic BMDCs and incubated in 96-well plates at 37°C in a 5% CO2 incubator for 4 days.
An anti-IL-2 monoclonal antibody (5 μg/ml, clone S4B6), an isotype control, and recombinant IL-2 (2000 pg/ml) were obtained from BD Biosciences (San Diego, CA). An anti-IL-17 monoclonal antibody (clone M210, IgG2a) was generously provided by Amgen (Seattle, Washington) (23). For the in vivo experiments, anti-IL-17 was administered at a dose of 100 μg on days -1, +1, +3, +5, and +7 relative to transplantation. Isotype control antibody was obtained from BioXcell (New Lebannon, NH).
ELISPOT analysis was performed as described previously (22).
Mice were immunized with 1×107 allogeneic spleen cells via i.p. injection.
T cells were purified via negative magnetic selection using EasySep reagents (StemCell Technologies, Vancouver, Canada). CD4, CD8, CD44, CD62L, IL-2, and IL-17 fluorescently labeled monoclonal antibodies and isotype controls were purchased from eBiosciences (San Diego, CA). Memory (CD44hi/CD62Llo) / naïve (CD44lo/CD62Lhi) cells were further sorted by FACS on a FACsAria cell sorter (BD Bioscience, San Diego, CA). Intracellular cytokine staining was achieved by harvesting spleen cells post-transplantation and stimulating the cells ex-vivo with PMA/ionomycin in the presence of cell permeability agents and golgi stop (eBiosciences). All analysis was performed on a FACS CALIBUR flow cytometer (BD Bioscience) and analyzed with Flow Jo software (Treestar, San Carlos, CA).
Survival analysis between groups was calculated using the Logrank method. Comparison of means was performed using a two-tailed t-test, and repeated measures using analysis of variance (ANOVA). All results were generated using GraphPad prism software (San Diego, CA). Statistical significance was considered at a p value < 0.05.
Cross-reactive effector memory T cells increased the rates of acute allograft rejection, impaired the induction of transplantation tolerance (20), and could be detected based on rapid production of IFNγ (i.e., within 20 h of stimulation) (24). Accordingly, we examined whether aged or young non-transplanted CBA mice produced, rapid anti-donor (i.e. C57BL/6) IFNγ responses upon exposure to donor spleen cells. We found that aged (20-22 months of age) and young spleen cells (2-4 months of age) obtained from non-transplanted mice exhibited comparable numbers of IFNγ producing cells upon ex vivo exposure to irradiated donor spleen cells (Figure 1A). Young CBA mice that were immunized with C57BL/6 spleen cells for one month demonstrated increased IFNγ producing cells compared to other groups, and this effect was donor-specific (Figure 1A). Based on these results, aged non-transplanted mice do not exhibit significant anti-donor memory T cell responses. However, spleen cells harvested from non-transplanted aged CBA mice exhibited significantly increased numbers of anti-donor IL-17 producing spleen cells as compared to young spleen cells (Figure 1B). IL-17 is another cytokine produced by memory T cells (25). Similar results were observed in non-transplanted C57BL/6 recipients stimulated ex vivo with irradiated BALB/c spleen cells (data not shown). In summary, these results demonstrate that aged hosts exhibit increased anti-donor IL-17 responses, but not anti-donor IFNγ responses, prior to transplantation.
To further examine the altered IL-17 alloimmune response with aging, we cultured CBA T cells with irradiated, young C57BL/6 BMDCs. The results demonstrated that aged T cells manifested increased IL-17 responses both over time and in dose response, compared to young T cells (Figure 2A). Similar results were obtained when using a different stimulator and responder strain combination (Supplemental Figure 1A). Furthermore, we found that increased IL-17 alloimmune response occurred with advanced aged (17 months of age), but not in intermediate aged T cells (8 months of age, Supplemental Figure 1B). Further analysis demonstrated that the augmented IL-17 T cell alloimmune response with aging was a consequence associated with CD4+ T cells rather than CD8+ T cells (Supplemental Figure 2). In contrast to the IL-17 response, aged T cells exhibited an impaired ability to produce IL-2 over time and in response to a dose range of stimulators (Figure 2B), which was in agreement with a prior report (13). Our results indicate that increased age augments IL-17 in vitro T cell alloimmune response, but impairs IL-2 alloimmunity.
IL-2 is a negative regulator of the Th17 pathway (26), and results from in vitro assays showed that aged T cells produce less IL-2 than young T cells. It is possible that the reduced IL-2 environment contributed to the augmented IL-17 response with aging. We examined whether IL-2 influences the IL-17 alloimmune response in aged CD4+ T cells. Inhibiting IL-2 in young CD4+ T cells resulted in a greater than double proportion of IL-17 producing cells (Figure 2C). Furthermore, the addition of recombinant IL-2 reduced the IL-17 response of aged CD4+ T cells (Figure 2C). These data provide evidence that reduced IL-2 production may facilitate increased IL-17 response by aged CD4+ T cells during allostimulation and that heightened IL-2 response in young T cells may suppress the IL-17 response.
In agreement with prior work (6), we found that aging led to an increased proportion of memory CD4+ T cells and a reduced proportion of naïve CD4+ T cells (Figure 3A). We next FACs purified memory (i.e., CD44hi, CD62Llo) or naïve (i.e., CD44lo, CD62Lhi) populations from CD4+ T cells to determine if aged memory or naïve CD4+ T cells produced elevated IL-17 responses on a per cell basis compared to young T cells during allostimulation. In the MLR, naïve CD4+ T cells did not produce IL-17, regardless of age (Figure 3B). However, we found that aged memory CD4+ T cells exhibited a higher proportion of IL-17 secreting cells than young memory CD4+ T cells (Figure 3C). Furthermore, we noted that aged memory CD4+ T cells that secreted IL-17 did not co-secrete IL-2, but we observed a small proportion of double cytokine-producing memory T cells in this assay (Figure 3C). Aged memory CD4+ T cells produce augmented IL-17 alloimmune responses on a per cell basis, but aged mice also generate higher numbers of memory CD4+ T cells that could contribute to the elevated IL-17 alloimmune response of the greater T cell pool with age.
It is possible that the augmented IL-17 phenotype with aging is due to the accumulation of memory T cells, which characteristically produce IL-17 (25). If this is the case, it might be possible to replicate the aged phenotype in young hosts by immunizing them and allowing them to generate memory T cells. To test this hypothesis, we immunized young CBA mice with C57BL/6 spleen cells to generate memory T cells reactive to alloantigens. One-month post immunization, T cells were purified and stimulated with allogeneic C57BL/6 BMDCs, and IL-17 production was measured. Our results indicate that T cells from young, immunized mice produced a similar amount of IL-17 to T cells from young, non-immunized mice. Both of these groups demonstrated an inferior IL-17 response compared to that of T cells from aged non-immunized mice (Figure 4A). However, T cells from young, immunized mice produced more IL-2 than the other groups (Figure 4B).
We then transplanted young CBA mice with C57BL/6 skin allografts, representing a more chronic exposure to alloantigens than injection with allogeneic spleen cells. One month after transplantation, T cells from young mice were compared by MLR to T cells from non-transplanted aged and young mice, respectively. However, T cells from the young transplanted mice exhibited an inferior IL-17 response during the MLR as compared to T cells from aged, non-transplanted mice (Figure 4C). (Although, at 72h of culture, T cells from the transplanted young mice exhibited slightly increased IL-17 levels compared to T cells from young non-transplanted mice). These results indicate that the generation of donor specific memory T cells in a young host with these immunization protocols does not replicate the aged T cell phenotype.
We investigated the impact of recipient age on alloimmunity using in vivo T cell polyclonal systems. Aged or young CBA recipients received a skin transplant from young C57BL/6 donors. ELISPOT examination of harvested recipient splenocytes at days seven and fourteen post transplantation demonstrated that aged recipients exhibit a reduced ability to produce IL-2 and IFN-g after ex vivo stimulation by young, irradiated donor spleen cells (Figure 5A-B). Additionally, aged transplant recipients expressed lower serum Th1 (IgG2a) alloantibodies than young transplant recipients (data not shown). However, Th2 immune responses, quantified by the number of IL-4 producing spleen cells, were preserved in aged recipients at 14 days post transplantation (Figure 5B). In contrast to the above data, at 14 days post transplantation, aged recipients exhibited augmented IL-17 alloimmune responses (Figure 5C). These data indicate that age impairs recipient Th1 T cell responses, but elevates IL-17 responses after skin allotransplantation.
We repeated the experiment where aged or young CBA recipients received a young C57BL/6 skin allograft, and this time we measured the tempo of allograft rejection. We observed a slightly delayed time to allograft rejection in the aged recipients in this model (Figure 6A), although a majority of aged mice were able to reject their allografts at a similar rate to that observed for young transplant recipients.
To examine the role of heightened IL-17 alloimmune responses with age, we transplanted CBA aged recipients with C57BL/6 skin allografts and treated the recipients with an anti-IL-17 mAb or isotype control. Previously, anti-IL-17 was observed to impair IL-17-dependent collagen induced arthritis (23). Both aged and young recipients that were treated with an anti-IL-17 antibody exhibited significantly increased time to acute allograft survival as compared to isotype control treated recipients. The median survival time to acute allograft rejection in aged recipients (20 days) treated with anti-IL-17 was significantly longer than that for young recipients treated with the identical therapy (17 days, p = 0.03). These data indicate that heightened IL-17 alloimmune responses that are associated with aging are an important pathway for acute allograft rejection.
Aging has a complex effect on immune responses (6). One consistent effect is that aging leads to an accumulation of memory T cells (5, 6), even though a prior study indicated that the function of memory T cells declines with aging (27). Based on these studies, we predicted that aged hosts would exhibit increased numbers of donor-specific effector memory responses, characterized by rapid IFN-γ secretion (24), as compared to young hosts, prior to transplantation. However, our study found that that aged hosts exhibited augmented donor-specific, effector memory IL-17, but not IFN-γ, responses prior to transplantation compared to young mice. We showed that CD4+ memory T cells are responsible for the elevated alloimmune response with aging, a phenotype that may be exacerbated by weaker IL-2 alloimmune responses. Importantly, we demonstrated that the heightened IL-17 allommune responses contributed to acute allograft rejection in aged transplant recipients. Our results indicate that aging elevates memory IL-17 alloimmune responses.
IFN-γ producing donor-specific memory T cells, predominantly of the effector memory phenotype, may be an indicator of an increased tendency toward acute allograft rejection (28). Based on our results, IFNγ measurement may not be sufficient for indicating the presence of donor-specific memory responses with aging. Our work suggests that IL-17 may be a more reliable indicator of the presence of cross-reactive effector memory T cells in aged recipients. This is particularly relevant to solid organ transplantation, for which the fastest growing cohort of patients awaiting transplantation is greater than 65 years of age. Our results await validation using human cells.
Prior studies have associated increased systemic IL-6 levels, a Th17 promoting factor, with aging (29). Furthermore, aging is associated with an increased number of regulatory T cells (19), which can act as a source of TGF-β, another Th17 promoting factor (30). In contrast, it is well known that aging leads to reduced IL-2 responses, a negative regulator of the Th17 pathway (4, 31). Apparently, with aging there are increases in Th17 promoting factors and a reduction in Th17 regulatory factors. Thus, it is plausible that, with aging, CD4+ T cells may transition to a Th17 phenotype upon exposure to environmental antigens. Some of these CD4+ T cell effectors may then differentiate into IL-17 producing memory T cells, which may cross-react with alloantigens. This may explain why the IL-17 T cell alloimmune response in young mice was lower than that in aged mice after immunization with alloantigens. Young hosts may not be prone to IL-17 immune responses. Future studies will be required to mechanistically examine how aging leads to accumulated numbers of IL-17 producing memory CD4+ T cells.
Our study shows that aged hosts exhibit increased numbers of memory CD4+ T cells that produce IL-17 when stimulated with donor alloantigens. These memory cells could present a barrier to transplantation. However, since aged hosts have increased numbers regulatory T cells (19), which may aid in immune tolerance induction, it is not clear how age will affect the application of protocols that induce transplantation tolerance. This will clearly be an important topic for future investigation.
In contrast, a previous report found that aged recipients exhibit increased IFNγ and IL-2 responses after transplantation (32). However, a major difference between that study and ours is that acute rejection in the prior study was abrogated by the use of cyclosporine. Clinically, this distinction may be relevant, because several clinical studies have documented an association of increasing recipient age with reduced rates of acute allograft rejection (9-11). In contrast, aging reportedly leads to increased chronic rejection (33). Given the recent findings that IL-17 may contribute to chronic lung rejection (34), it will be important for future studies to determine whether heightened IL-17 alloimmune responses with aging impact the development of chronic rejection.
Our study provides evidence that aging leads to elevated IL-17 alloimmune responses that can influence the rate of acute allograft rejection in individuals. When translated to the clinic, this information may be important for tailoring therapies to treat acute allograft rejection in older transplant recipients.
This Work was supported by the Paul B. Beeson Award in Aging Research (NIA [AG026772], AFAR, Hartford Foundation and Starr Foundation) and NIH grant AI064660 to DRG