Eomesodermin^lo CTLA4^hi Alloreactive CD8⁺ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4Ig-Treated Non-Human Primates

Abstract

Background

Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in non-human primates.

Methods

We evaluated Eomes and co-inhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin (Ig), both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion.

Results

In normal monkeys, CD8⁺ T cells expressed significantly more Eomes than CD4⁺T cells. By contrast, CD8⁺T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, Eomes^loCTLA4^hi cells displayed higher levels of CD25 and Foxp3 than Eomes^hiCTLA4^lo CD8⁺ T cells. Following allostimulation, distinct proliferating Eomes^loCTLA4^hi and Eomes^hiCTLA4^lo CD8⁺ T cell populations were identified, with a high proportion of Tcm being Eomes^loCTLA4^hi. CB with CTLA4Ig during allostimulation of CD8⁺T cells reduced CTLA4 but not Eomes expression, significantly reducing Eomes^loCTLA4^hi cells. After transplantation with CB and rapamycin, donor-reactive Eomes^loCTLA4^hi CD8⁺T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly.

Conclusions

Low Eomes and high CTLA4 expression by donor-reactive CD8⁺ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive Eomes^loCTLA4^hi Tcm.

INTRODUCTION

Induction of tolerance to organ allografts can be readily achieved in rodents by a variety of strategies. However, such approaches have proved unsuccessful in non-human primate (NHP) models and in clinical transplantation. Pre-existing alloreactive memory T cells (Tmem) are considered a major barrier to the induction of tolerance (1). In NHP, kidney allograft rejection is associated with the development of costimulation blockade (CB)-resistant Tmem (2–4). Recent clinical testing of cytotoxic T lymphocyte Ag 4 (CTLA4) immunoglobulin (Ig) (belatacept), a chimeric fusion protein that blocks the B7-CD28 pathway, in a calcineurin inhibitor-free regimen, has resulted in an increased incidence of acute cellular rejection in renal transplant recipients (5, 6). There is also recent evidence that CTLA4Ig may prevent regulatory T cell (Treg)-dependent transplant tolerance in rodents (7, 8).

Alloreactive CD8⁺ Tmem are known to be more resistant to CB than CD4⁺ Tmem (9–12). Eomesodermin (Eomes) is a key transcription factor in CD8⁺ Tmem differentiation, fate and function (13, 14). It plays a critical role in the long-term survival of antigen (Ag)-specific central memory T cells (Tcm) (15). Significantly, however, the role of Eomes in the differentiation, regulation and maintenance of donor-specific Tmem in allograft recipients has not been examined.

Using a robust, rhesus monkey model, we have reported recently (16) that a single infusion of donor-derived regulatory dendritic cells (DCreg), one week before transplant, together with CTLA4Ig and tapered rapamycin maintenance monotherapy, can significantly prolong renal allograft survival. This therapeutic effect of DCreg is associated with increased CD4⁺ Treg to CD8⁺ Tmem ratios in peripheral blood and with upregulation of co-inhibitory CTLA4 (CD152) and programmed death-1 (PD1; CD279) by Tmem following their stimulation by donor but not third party Ag. Together, these findings suggest attenuation of donor-specific Tmem responses in DCreg recipients (17).

It has been reported that CTLA4 may reduce Eomes expression by CD8⁺ T cells (18). Here, we examined the expression of Eomes and CTLA4 by normal and allostimulated monkey Tmem and by Tmem in CTLA4Ig-treated renal allograft recipients, without or with DCreg infusion. We found that CD8⁺ T cells express higher levels of Eomes, but lower levels of CTLA4 compared to CD4⁺ T cells, in which population Tcm displayed the highest levels of Eomes. Additionally, Eomes^loCTLA4^hi CD8⁺ T cells expressed higher CD25 and Foxp3 levels than Eomes^hiCTLA4^lo CD8⁺ T cells. CB with CTLA4Ig significantly reduced CTLA4, but not Eomes expression by alloreactive T cells in vitro. This was associated with reduction in the alloreactive Eomes^loCTLA4^hi but not the Eomes^hiCTLA4^lo subpopulation. Our data also reveal that combined CTLA4Ig and pre-transplant DCreg infusion is associated with low Eomes and high CTLA4 expression by donor-reactive CD8⁺ Tcm, consistent with attenuation of donor-specific Tmem and improved graft survival in CB-treated graft recipients.

RESULTS

CD8⁺ Tmem Express High Eomes and Minimal CTLA4 Levels Compared to CD4⁺ Tmem in Normal Rhesus Monkeys

Eomes is a T-box transcription factor that plays a key role in the differentiation of Tmem, particularly Ag-specific Tcm (15). First, we examined the expression of Eomes by normal monkey peripheral blood CD4⁺ and CD8⁺ T cells (Fig. 1A). CD8⁺T cells expressed significantly higher levels (approx. 5-fold) than CD4⁺T cells. Next, we evaluated Eomes expression by naïve and memory subsets of CD4⁺ and CD8⁺ T cells (Fig. 1B), based on their differential expression of CD28 and CD45RA (19). Eomes was expressed more strongly by all CD8⁺ compared to CD4⁺ naïve and memory T cell subsets. In both CD4⁺ and especially CD8⁺ populations, Tcm displayed the highest Eomes expression (Fig. 1B and 1C). In CD4⁺T cells, mean Eomes expression by Tcm (4.1%) was significantly higher than that by effector T cells (Teff; 1%), but not naïve (Tn; 2.3%) or effector memory T cells (Tem; 1.6%). In CD8⁺T cells, mean Eomes expression by Tcm (47.3%) was significantly higher than for all other subsets,- Tem (29.4%), Tn (23.5%) and Teff (18.9%) (Fig. 1C). We then evaluated Eomes versus CTLA4 expression by both CD4⁺ and CD8⁺ naïve and memory T cell subsets (Fig. 1D). In correlation with previous results, low percentages of Eomes^hiCTLA4^lo and high percentages of Eomes^loCTLA4^hi cells were detected in all CD4⁺T cell subsets, compared to CD8⁺T cells. CD4⁺Tcm and CD4⁺Tem showed higher frequencies of Eomes^loCTLA4^hi cells. While CD8⁺T cell subsets exhibited minimal percentages of Eomes^loCTLA4^hi cells (< 4%), CD8⁺Tcm comprised the highest percentage of Eomes^loCTLA4^hi cells.

FIGURE 1.

Expression of Eomes and CTLA4 by normal rhesus monkey naïve and memory CD4⁺ and CD8⁺ T cells. (A) Left, expression of Eomes by total CD4⁺ and CD8⁺ peripheral blood T cells in a representative normal rhesus monkey determined by flow cytometry; right, individual and mean values for 8 normal monkeys. (B) Eomes expression by CD4⁺ and CD8⁺ naive and memory T cell subsets, determined by differential CD45RA and CD28 expression: central memory T cells (Tcm; CD45RA⁻CD28⁺), naïve T cells (Tn; CD45RA⁺CD28⁺), effector memory T cells (Tem; CD45RA⁻CD28⁻) and effector T cells (Teff; CD45RA⁺CD28⁻). (C) Individual and mean incidences of Eomes⁺ CD4⁺ and CD8⁺ T naïve and memory cell subtypes (n=6 normal monkeys). (D) Eomes versus CTLA4 expression by CD4⁺ and CD8⁺ naive and memory T cell subsets in naïve monkeys (data representative of n=4 monkeys). (E) CD122 (IL-2Rβ), CD45RA, CCR7, PD1, CD25 and Foxp3 expression by Eomes^loCTLA4^hi versus Eomes^hiCTLA4^lo CD8⁺ T cells in naïve monkeys. Representative data (above) and means +1SD for n=4 monkeys (below) are shown; ns = not significant.

Eomes^loCTLA4^hi CD8⁺ T Cells Include a Higher Proportion of Putative Regulatory Cells

In humans and rodents, memory-like regulatory CD8⁺T cells suppress both auto- and alloimmune responses (20–22). CD8⁺CD122(IL-2Rβ chain)⁺PD1⁺ T cells suppress T cell proliferation and inflammatory cytokine production (23), inhibit allograft rejection (24, 25) and mediate allograft acceptance (26). We evaluated CD122, CD45RA, CCR7, PD1, CD25 and Foxp3 expression by Eomes^loCTLA4^hi versus Eomes^hiCTLA4^lo CD8⁺ T cells in naïve monkeys (Fig. 1E). The incidence of CD122⁺ cells was higher in the Eomes^hiCTLA4^lo compared to the Eomes^loCTLA4^hi population. Overall, the Eomes^loCTLA4^hi population exhibited a more central memory phenotype, i.e. a significantly higher incidence of CCR7 and a lower incidence of CD45RA⁺ cells than the Eomes^hiCTLA4^lo population. CD25 expression was low on both populations, but higher on Eomes^loCTLA4^hi cells. Furthermore, the Eomes^loCTLA4^hi CD8⁺ population exhibited a higher incidence of Foxp3⁺ cells compared to Eomes^hiCTLA4^lo CD8⁺ T cells. These findings suggest that low Eomes and high CTLA4 expression by rhesus CD8⁺T cells may be associated with a subset of cells with a regulatory phenotype.

Reciprocal Relationship Between Eomes and CTLA4 Expression by Alloreactive T Cells

The association between high CTLA4 and low Eomes expression relates to recent evidence (18) that co-inhibitory CTLA4 may selectively inhibit Eomes expression by rodent CD8⁺T cells. We therefore hypothesized that an inverse correlation might exist between CTLA4 and Eomes expression by alloactivated rhesus T cells. All proliferating CD4⁺T cells displayed low Eomes expression (Fig. 2A, left), whereas proliferating CD8⁺T cells displayed two distinct populations, with either low or high Eomes expression (Fig. 2A, right). Furthermore, proliferation of CD8⁺T cells with low Eomes expression was significantly greater than that of those with high Eomes expression (Fig. 2B, left). We also examined CTLA4 expression by proliferating CD4⁺ and CD8⁺ T cells. Proliferating CD4⁺T cells (Eomes^lo) displayed higher levels of CTLA4 than proliferating Eomes^lo and Eomes^hi CD8⁺T cells Interestingly, CD8⁺Eomes^lo T cells expressed significantly higher levels of CTLA4 than CD8⁺Eomes^hi T cells (Fig. 2 A, right panel and Fig. 2B, right panel).

FIGURE 2.

Inverse correlation between Eomes and CTLA4 expression by alloreactive CD4⁺ and CD8⁺ T cells in normal monkeys. Two distinct populations of Eomes^hi and Eomes^lo proliferating CD8⁺ T cells are observed following allostimulation of normal monkey T cells. (A) Proliferation of Eomes^hi and Eomes^lo CD4⁺ and CD8⁺ T cells following allostimulation in CFSE-MLR. Histograms show CTLA4 expression by proliferating cells (values in parentheses indicate MFI). Grey histograms indicate isotype controls. (B) Left, proliferation of Eomes^hi compared with Eomes^lo CD8⁺ T cells; right, CTLA4 expression (MFI) by proliferating Eomes^lo and Eomes^hi CD8⁺ T cells. Responder PBMC were co-cultured with allogeneic T cell-depleted PBMC for 5 days in CFSE-MLR. Proliferation of Eomes^hi and Eomes^lo T cell populations were determined after gating on CD4⁺ or CD8⁺ T cells (n=10 normal monkeys).

In addition, we evaluated expression of CTLA4 by non-proliferating CD4⁺ and CD8⁺ T cells in relation to that of Eomes. In non-proliferating CD8⁺T cells, no significant differences were observed in CTLA4 expression by Eomes negative, lo or hi subsets. In non-proliferating CD4⁺T cells, Eomes⁺ cells showed slightly higher CTLA4 expression than Eomes⁻ cells (Supplementary Fig. 1). These data indicate that proliferation of alloreactive CD8⁺ T cells is associated with increased frequency of a subset of Eomes^loCTLA4^hi cells.

Alloreactive Eomes^loCTLA4^hi CD8⁺ T Cells Exhibit an Increased Incidence of Tcm

As Eomes is critical for maintenance of Tcm, we next evaluated the memory phenotype of Eomes^loCTLA4^hi and Eomes^hiCTLA4^lo CD8⁺ T cells following allostimulation. When cultured without stimulation, CD8⁺ T cells comprised a much higher proportion of Eomes^hiCTLA4^lo than Eomes^loCTLA4^hi T cells (Fig. 3A, left). As in previous experiments, no significant changes were observed in the percentages of Eomes^hiCTLA4^lo or Eomes^loCTLA4^lo T cells following allostimulation, however, Eomes^loCTLA4^hi T cells increased significantly (Fig. 3A left and Fig. 3B left). Next, we examined naïve and memory T cell subsets among Eomes^loCTLA4^hi CD8⁺ T cells following allostimulation (Fig. 3A, right). Compared with Eomes^hiCTLA4^lo or the Eomes^loCTLA4^lo T cells, Tcm in the Eomes^loCTLA4^hi CD8⁺ T cell population were markedly enriched (Fig. 3B, right).

FIGURE 3.

Increased frequency of central memory T cells (Tcm) in the Eomes^loCTLA4^hi CD8⁺ T cell population following allostimulation. (A) Frequencies of Eomes^hiCTLA4^lo and Eomes^loCTLA4^hi CD8⁺ T cell subpopulations in MLR. Data are from a representative experiment. (B) left, combined data from 8 allogeneic monkey stimulator-responder combinations; right, frequencies of naïve and memory T cell subsets in the Eomes^loCTLA4^hi CD8⁺ T subpopulation after allo-stimulation. The combined data are from 8 different allogeneic monkey responder and stimulator pairs.

CD28 CB with CTLA4Ig Reduces CTLA4 Expression by CD8⁺ T Cells After Allostimulation

The co-inhibitory molecules CTLA4 and PD1 are considered markers of T cell exhaustion and regulation, and they are also expressed by activated T cells (27, 28). In our previous study (16), DCreg infusion in transplanted monkeys was associated with upregulation of CTLA4 and PD1 double positive donor-reactive Tmem. We tested the influence of CTLA4Ig on CTLA4 and PD1 expression in relation to Eomes expression by alloreactive CD8⁺ T cells following allostimulation in MLR. As seen in Fig. 4A, proliferation of CD8⁺ T cells was significantly reduced, as expected. In the absence of CTLA4Ig, mean CTLA4 expression by stimulated CD8⁺ T cells was upregulated 4-5-fold. Meanwhile, expression of PD1 was increased modestly, although not significantly. Reduced CD8⁺ T cell proliferation in the presence in CTLA4Ig was accompanied by a significant reduction in proliferating CTLA4⁺ cells, in a CTLA4Ig concentration-dependent manner. Concurrently, the total percentage of CTLA4⁺CD8⁺ T cells was also reduced significantly by CTLA4Ig. While proliferating PD1⁺ cells were also reduced by CTLA4Ig, no similar reduction was observed for total PD1⁺ cells (Figures 4A and 4B). Notably, we found no evidence that, under these conditions, anti-CTLA4 mAb used to stain CTLA4 bound to any residual cell-bound fusion protein with consequent ‘artificial’ reduction in CTLA4 staining (data not shown).

FIGURE 4.

Effect of CTLA4Ig on PD1 and CTLA4 expression by allostimulated normal monkey CD8⁺ T cells. (A) CTLA4Ig inhibits total CD8⁺T cell, CTLA4⁺CD8⁺T cell and PD1⁺CD8⁺T cell proliferation in a concentration-dependent manner. CFSE dilution and percentages of CTLA4⁺ and PD1⁺ populations were determined after gating on CD8⁺ T cells. (B) Percentages of CD8⁺PD1⁺ and CD8⁺CTLA4⁺ T cells following allostimulation in the absence or presence of CTLA4Ig. Responder PBMC were co-cultured with allogeneic T cell-depleted stimulator PBMC in CFSE-MLR for 5 days, in the absence or presence of CTLA4Ig (1 or 100 μg/ml). Bars represent means +1 SD (n=5 independent experiments); ns=not significant.

CD28 CB with CTLA4Ig Does Not Affect Eomes Expression by CD8⁺ T Cells After Allostimulation

As Eomes is critical for Tmem maintenance and differentiation, preservation of high Eomes expression associated with low CTLA4 expression by T cells, may play a role in the development CB-resistant Tmem. Having shown that CD28 CB reduces proliferating alloreactive CD8⁺CTLA4⁺ T cells (Figures 4A and 4B), we next examined the influence of CTLA4Ig on Eomes expression by CD8⁺ T cells following allostimulation. Proliferation of Eomes^hi and Eomes^lo cells was increased following allostimulation and reduced in the presence of CTLA4Ig (Fig. 5A). However, the total percentage of Eomes⁺ cells was not reduced by CTLA4Ig. In parallel, expression of Eomes by CD8⁺ T cells was increased significantly following allostimulation, but was not reduced by addition of CTLA4Ig at the start of MLR cultures. At the same time, however, CTLA4 expression was reduced significantly (Fig. 5A, lower left). In correlation, no marked changes in the incidence of Eomes^hiCTLA4^loCD8⁺T cells were observed after allostimulation in the absence or presence of CTLA4Ig (Fig. 5B, upper panel), while the incidence of Eomes^loCTLA4^hi CD8⁺T cells was reduced markedly by CTLA4Ig, in a concentration-dependent manner (Fig. 5B, lower panel).

FIGURE 5.

CTLA4Ig does not reduce Eomes expression compared to CTLA4 expression by allostimulated normal monkey CD8⁺ T cells. (A) Proliferation of Eomes^lo and Eomes^hi normal monkey CD8⁺ T cells following allo-stimulation in MLR, in the presence or absence of CTLA4Ig. Representative data from one experiment are shown (top). CTLA4 and Eomes expression by CD8⁺ T cells following allostimulation is also shown (bottom panels). Representative and mean values +1 SD of 4 independent experiments are shown; ns=not significant. (B) Frequencies of Eomes^loCTLA4^hi and Eomes^hiCTLA4^lo CD8⁺ T cells following allostimulation in the absence or presence of CTLA4Ig. Representative data (top) and means +1SD of 4 independent experiments are shown (bottom).

To determine whether reduction in CTLA4 expression by alloreactive CD8⁺T cells was unique to CD28 pathway blockade (by CTLA4Ig), we evaluated the influence of CD40/CD40L blockade using anti-CD40 monoclonal antibody. Neither proliferation of CD8⁺T cells nor the Eomes^loCTLA4^hi CD8⁺T cell population was reduced in the presence of anti-CD40 compared to CTLA4Ig (Supplementary Fig. 2).

CTLA4 and PD1 Expression by CD8⁺ T Cells in Renal Allograft Recipients

Recently we reported (16) prolonged graft survival in rhesus renal allograft recipients given CTLA4Ig and DCreg infusion compared with those that did not receive DCreg. This was associated with upregulation of co-inhibitory CTLA4 and PD1 by circulating CD8⁺Tmem in response to ex-vivo donor but not 3^rd party stimulation. We hypothesized that a similar profile of donor-reactive T cells might be observed in the grafts of these DCreg recipients. Thus, we examined and quantified CTLA4 and PD1 expression by graft-infiltrating CD8⁺ T cells, one month post-transplant in two recipients from each of the control and DCreg groups. In the control group, graft-infiltrating CD8⁺ T cells showed minimal CTLA4 and PD1 expression, whereas strong expression of CTLA4 and PD1 by CD8⁺ T cells was observed in renal allografts of the DCreg group (Fig. 6). This was consistent with the corresponding upregulation of these inhibitory molecules on host CD8⁺ T cells following ex-vivo stimulation with donor Ag at the same time post-transplant (16).

FIGURE 6.

CTLA4 and PD1 expression by graft-infiltrating CD8⁺T cells in rhesus monkey kidney transplant recipients. (A) CTLA4 (CD152) and PD1 (CD279) expression by CD8⁺ T cells was examined by immunofluorescence staining. Graft tissue from a representative monkey (M143) in the control (CTRL) group (treated with CTLA4Ig and rapamycin) at the time of graft rejection (postoperative day [POD]28) is shown. Tissue from a graft recipient in the group that also received DCreg (M148) was obtained by open kidney graft biopsy, also on POD 28. Co-localization of CTLA4 (green) and PD1 (blue) with CD8⁺ T cells (in red) is shown (white arrows) in the middle and bottom panels, respectively. Nuclei were stained with DAPI (blue; upper panels). Slides were examined with a Nikon Eclipse E800 microscope equipped with a CCD camera (Nikon). Leukocyte infiltrates were quantified at 200x on at least 3 sections per allograft, with MetaMorph Offline 7.7.50n software. (B) Numbers of CTLA4⁺ and PD1⁺ CD8⁺T cells per high power field (HPF) in the allograft tissue of control (CTRL) and DCreg-treated monkeys.

DCreg Infusion Promotes Donor-Specific Eomes^loCTLA4^hi CD8⁺ Tcm in CTLA4g-Treated Renal Allograft Recipients

Next, we tested the in vivo relevance of differential Eomes and CTLA4 expression by alloreactive CD8⁺ Tmem in our transplant model (Fig. 7), in which DCreg infusion in CTLA4Ig-treated recipients is associated with selective attenuation of anti-donor Tmem responses (16). We examined the percentages (Fig. 7A and 7B, top panels) and absolute numbers (Fig. 7B; bottom panels) of peripheral blood alloreactive Eomes^loCTLA4^hi CD8⁺ Tcm following donor or 3^rd party stimulation, before and 1 month post-transplant, with or without DCreg infusion. In control monkeys (no DCreg infusion), donor-reactive Eomes^loCTLA4^hi CD8⁺ Tcm were reduced significantly post- compared to pre-transplant (Fig. 7B). By contrast, in graft recipients given DCreg infusion, the frequency of Eomes^loCTLA4^hi CD8⁺ Tcm following donor stimulation was increased modestly post- compared to pre-transplant (Fig. 7B, upper panels). More importantly, the mean percentage and absolute numbers of Eomes^loCTLA4^hi CD8⁺ Tcm in the DCreg group were significantly higher than in the control group post-transplant. No similar differences were observed in response to 3^rd party stimulation.

FIGURE 7.

DCreg infusion spares reductions in donor-specific Eomes^loCTLA4^hi CD8⁺Tcm in CTLA4Ig-treated renal allograft recipients. (A) Incidences of Eomes^loCTLA4^hi and Eomes^hiCTLA4^lo CD8⁺T cells in response to donor or 3^rd party stimulation, before transplant and on day 28 post-transplant in representative control (CTRL) and DCreg-treated monkeys. (B) Percentages (top) and absolute numbers ±1SD (bottom) from 4 allograft recipients in each group. Responder PBMC were co-cultured with donor or third party T cell-depleted PBMC for 5 days. Percentages and absolute numbers of Eomes^loCTLA4^hi were determined after gating on CD8⁺T cells. ns = not significant.

DISCUSSION

Pre-existing alloreactive Tmem are considered a major barrier to induction of allograft tolerance (1, 29–31). As Tmem require less co-stimulation compared to naïve T cells (32–34), alloreactive Tmem are thought to play a fundamental role in CB-resistant rejection (10, 35, 36) and to preclude CB-induced tolerance (36, 37). The T-box transcription factors T-box expressed in T cells (T-bet and Eomes) are considered master regulators of CD8⁺ effector Tmem differentiation and function (13, 14, 38, 39). Both T-bet and Eomes have cooperative and redundant roles in CD8⁺ T cell function, but they also have distinct roles in CD8⁺ Tmem development. Eomes plays a critical role in long-term survival of Ag-specific Tcm (15). In rodents, Eomes is upregulated in early Teff, where its expression increases as T cells progress from an effector to a memory phenotype (15, 40, 41). Additionally, Eomes knockouts are deficient in long-term formation and homeostatic renewal of Tmem (14, 15, 42).

The role of Eomes in the differentiation, regulation and maintenance of donor-specific Tmem in allograft recipients has hitherto not been examined. Furthermore, the expression of Eomes by Tn and Tmem in NHP has not previously been reported. We examined the expression of Eomes by alloreactive CD4⁺ and CD8⁺ T cells in normal monkeys and found that CD8⁺ T cells expressed significantly higher levels than CD4⁺ T cells. Similarly to humans (43), Tcm expressed the highest levels of Eomes compared to Tn, Tem and Teff. These observations suggest that Eomes may play a role in the development of donor-reactive CD8⁺ Tmem after transplantation.

It has been reported that CTLA4 may reduce Eomes expression by CD8⁺ T cells (18). In the current study, we evaluated Eomes and CTLA4 expression by rhesus alloreactive CD8⁺ T cells. Following allostimulation, CD8⁺ T cells upregulated both CTLA4 and Eomes. Following allostimulation, all proliferating CD4⁺T cells exhibited low Eomes but high CTLA4 expression. In contrast, proliferating CD8⁺T cells comprised two distinct populations, one with high and one with low Eomes expression. Interestingly, CTLA4 expression by Eomes^hi CD8⁺T cells was lower than that by Eomes^lo CD8⁺ T cells, suggesting an inverse relationship between CTLA4 and Eomes expression. In correlation, we observed a significant increase in Eomes^loCTLA4^hi CD8⁺ T cells following allo-stimulation, compared to Eomes^hiCTLA4^lo or Eomes^loCTLA4^lo T cells. Furthermore, ~ 70% of these Eomes^loCTLA4^hi CD8⁺ T cells were Tcm. CD28 CB with CTLA4Ig during allostimulation did not reduce Eomes expression on CD8⁺ T cells, despite efficient inhibition of T cell proliferation and significant reduction in CTLA4 expression. In the presence of CTLA4Ig, there was a reduction in the Eomes^loCTLA4^hi population, with minimal effect on Eomes^hiCTLA4^lo T cells. These observations indicate that blocking CD28 costimulation maintains alloreactive T cells with high Eomes levels, while reducing those with high CTLA4 expression.

Regulatory CD8⁺T cells with a memory phenotype suppress both auto- and allo-immune responses (20–22). In rodents, CD122⁺PD1⁺ regulatory CD8⁺T cells suppress T cell responses in vitro and in vivo in an IL-10-dependent manner (23). Additionally, bystander CD8⁺Tcm (and not Tem) prevent islet allograft rejection (24) and mediate lung allograft acceptance (26). Furthermore, naturally-occurring CD8⁺CD122⁺ T cells appear more suppressive than CD4⁺CD25⁺ Treg (25) and are usually considered Ag-specific memory T cells with a central memory phenotype (44–46). In this study, we evaluated CD122, CD45RA, CCR7, PD1, CD25 and Foxp3 expression by Eomes^loCTLA4^hi CD8⁺ T cells in vitro and found that these cells expressed lower CD122 than Eomes^hiCTLA4^lo CD8⁺ T cells. In humans, CD8⁺CD122⁺ Treg are not well-characterized. Moreover, while mouse CD8⁺CD122⁺ Treg are CXCR3⁺, human CD8⁺CXCR3⁺ Treg are mostly CD122⁻ (47).

The Eomes^loCTLA4^hi population exhibited a more central memory phenotype than the Eomes^hiCTLA4^lo population. Interestingly, despite low CD25 expression on both populations, Eomes^loCTLA4^hi cells displayed higher CD25 compared to Eomes^hiCTLA4^lo CD8⁺ T cells. Of note, the Eomes^loCTLA4^hi CD8⁺ population also exhibited a higher frequency of Foxp3^hi cells compared to Eomes^hiCTLA4^lo CD8⁺ T cells. Collectively, these findings suggest a more regulatory component within the Eomes^loCTLA4^hi CD8⁺ T cell population. To our knowledge, regulatory CD8⁺ T cells have not been characterized in NHP and our future studies will address the regulatory function of these cells in our monkey transplant model.

Regulatory immune cell therapy offers considerable potential for promotion of transplant tolerance (48–53). DCreg can control Tmem responses (54–56) and are considered promising agents to promote clinical transplant tolerance (49, 57–61). We have reported (16) that a single infusion of donor-derived DCreg, one week before transplantation, combined with CTLA4Ig, prolongs renal allograft survival associated with enhanced co-inhibitory CTLA4 and PD1 expression by circulating Tmem following stimulation with donor, but not third party alloAg. While PD1 and CTLA4 are considered markers of T cell exhaustion, they have distinct inhibitory effects on T cell activation (62). Tumors evade adaptive immune responses through upregulation of CTLA4 and PD1, while co-expression of CTLA4 and PD1 is associated with significant dysfunction of Ag-specific T cells (63). CTLA4 expression, with or without PD1, is associated with reduced CD8⁺ T cell proliferation and cytokine production. Furthermore, blocking of CTLA4 and PD1 interaction in vivo upregulates T-bet and Eomes in CD8⁺ T cells (63) required for in anti-tumor responses (64).

Our observations suggest that, when Tmem encounter donor Ag following infiltration of the graft, they may upregulate PD1 and CTLA4 that, in turn, may inhibit donor-reactive Tmem activation. To address this hypothesis, we examined PD-1 and CTLA4 expression by CD8⁺ T cells in kidney allografts 28 days post-transplant. We found that graft-infiltrating CD8⁺ T cells displayed more CTLA4 and PD1 in the DCreg group than in the control group (no regulatory cell infusion). Since this enhanced expression of CTLA4 and PD1 by donor-reactive Tmem may underlie improved graft survival in the DCreg group, we explored whether upregulation of these co-inhibitory molecules might be associated with regulation of donor-specific Tmem responses in the monkeys given DCreg infusion. Thus we ascertained the frequency of Eomes^loCTLA4^hi CD8⁺ Tcm in allograft recipient monkeys in response to donor stimulation before and after transplant. In correlation with our in vitro observations, the frequency of Eomes^loCTLA4^hi CD8⁺ Tcm in response to donor stimulation was reduced significantly in the CTLA4Ig-treated monkeys (control group) post-transplant. By contrast, the frequency of Eomes^loCTLA4^hi CD8⁺ Tcm was increased modestly after transplantation in the CTLA4Ig-treated monkeys given DCreg. More importantly, the mean percentages and absolute numbers of Eomes^loCTLA4^hi CD8⁺ Tcm,- a phenotype that suggests low persistence and exhaustion, was significantly higher in the DCreg group than in the control group post-transplant.

Our observations provide further insight to the limitations of CD28 CB in organ transplantation. Significant reduction of the co-inhibitory receptor CTLA4 by alloreactive Tmem in the presence of CTLA4Ig in vitro and in CTLA4Ig-treated graft recipients post-transplant, together with no reduction in Eomes expression by donor-reactive Tmem, may be a key factor in CB resistance. While CTLA4Ig reduces effector T cell responses against donor Ag efficiently, this may be achieved at the expense of regulatory mechanisms that favor donor-specific Treg and attenuate donor-specific Tmem, resulting in increased rates of acute cellular rejection, as observed in belatacept-treated renal allograft recipients (5, 6).

These findings in rhesus monkeys identify CTLA4 as a marker of regulation of donor-specific, alloreactive Tmem, associated with improved transplant outcome. Upregulation of CTLA4 expression may play a role in attenuation of CB-resistant Tmem after transplantation. Whether pre-transplant DCreg infusion promotes the development of Eomes^loCTLA4^hi donor-reactive CD8⁺T cells, that are maintained after transplantation in response to the allograft, requires further investigation. The role of Eomes in development of alloreactive Tmem after transplantation has until now, not been ascertained. As Eomes plays a critical role in development and maintenance of Tcm (15), its reduced expression may attenuate donor-reactive Tcm after transplantation. Together with upregulation of CTLA4 by Tmem that we have shown in graft recipients given DCreg, this might further mitigate anti-donor T cell responses. Furthermore, our observations suggest that DCreg infusion before renal transplantation may help preserve donor-specific Tmem regulation that is compromised with use of CD28 CB.

MATERIALS AND METHODS

Experimental Animals

Indian male juvenile rhesus macaques (Macacca mulatta; 5–7 kg), obtained from the NIAID-sponsored colony (Yemasse, S.C.) were maintained in the NHP research facility of the Department of Laboratory Animal Resources at the University of Pittsburgh School of Medicine. All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Experiments were conducted according to the guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Specific environment enrichment was provided.

Renal Transplantation, DCreg Infusion and Immunosuppression

Leukapheresis, generation of donor-derived DCreg and renal transplantation were performed as described (16, 65). Recipient pairs, i.e. control (no DCreg infusion) and experimental animals (DCreg infusion) received kidney grafts from the same MHC mis-matched donor. In the experimental group, DCreg (3.5–10×10⁶/kg) were infused intravenously, 7 days before transplantation. All recipients in the control and DCreg groups were given CTLA4Ig (abatacept; Bristol-Myers Squibb; Princeton, NJ)-based immunosuppression and maintenance rapamycin (16).

Mixed Leukocyte Reactions (MLR)

MLR were performed as described (16). In some MLRs, CTLA4Ig (1 μg or 100 μg/ml) or primatized anti-CD40 antibody (Clone 2C10R4; 100 μg/ml) was added at the start of culture. Samples were also obtained from normal monkeys or kidney allograft recipients as described previously (16). Thus, PBMC were isolated before and after transplantation (post-operative day [POD] 28) and co-cultured in MLR with either donor or third party stimulator cells. Data were acquired using an LSR II flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star, San Carlos, CA).

Phenotypic Analysis of Alloreactive T Cells

The following fluorochrome-labeled monoclonal antibodies (mAbs) were used for cell surface or intra-cellular flow staining of rhesus T cells: CD3 (clone: SP34-2) PerCP-Cy5.5, CD4 (clone: L200) APC-H7, CD28 (clone: CD28.2) APC-H7, CD45RA (clone: 5H9) PE-Cy7 or FITC, CTLA4 (CD152; clone: BNI3) APC or VB450, CD122 (clone: Mik-β2) PE (all from BD Biosciences; San Jose, CA), CD8 (clone: RPA-T8) AF700, and CD95 (clone: DX2) PE-Cy7, Foxp3 (clone: 206D) AF488, CD25 (clone: BC96) AF700 (all from Biolegend; San Diego, CA), PD-1 (CD279; clone: eBioJ105) PE and Eomesodermin (clone: WD1928) eFluor 660, CCR7 (clone: 3D12) PE (all from eBioscience; San Diego, CA). Data were acquired and analyzed as described above. For renal allograft recipients’ samples, percentages obtained for specific populations were used to determine absolute numbers based on WBC in the peripheral blood.

Immunofluorescence Staining of Kidney Allografts

Tissues were collected from graft recipients in the control group on the day of euthanasia following clinical evidence of rejection and from those in the DCreg group on POD 28 by open biopsy of the kidney graft. Tissues were embedded in O.C.T. (Miles), snap-frozen and stored at −80°C. Cryostat sections (8–10μm) were mounted on slides pre-coated with Vectabond (Vector) then fixed in 96% ethanol and allowed to dry. Sections were blocked successively with 5% goat serum and an avidin/biotin blocking kit (Vector). Next, sections were incubated with anti-human CD8 Ab (clone LT8, Abcam, 1:100, overnight, 10°C), followed by Alexa Fluor 555-goat anti-mouse IgG (Molecular Probes, 1:100, 1h, RT). The slides were then blocked with mouse irrelevant IgG1 (BD Biosystems, 1:100, 1h, RT) and incubated successively with (i) biotin anti-human CTLA4 (CD152) (clone BNI3, BD Biosystems, 1:100, 1h, RT), (ii) DyLight 488-streptavidin (Jackson ImmunoResearch Laboratories, 1:400, 30 min, RT), and (iii) Alexa Fluor 647-conjugated anti-human PD1 (CD279) Ab (clone EH12.2H7, Biolegend, 1:100, 1h, RT). Cell nuclei were stained with DAPI (Molecular Probes).

Statistical Analyses

The significance of differences between groups was determined using Kruskal–Wallis one-way analysis of variance or Mann–Whitney U test, as appropriate. Significance was defined as p < 0.05.

Abbreviations

CB

costimulation blockade

CFSE

carboxyfluorescein succinimidyl ester

CTLA4 (-Ig)

cytotoxic T lymphocyte antigen 4 (-immunoglobulin)

DC

dendritic cells

DCreg

regulatory DC

Eomes

eomesodermin

PD1

programed death 1

Tcm

central memory T cells

Teff

effector T cells

Tem

effector memory T cells

Tmem

T memory cells

Tn

naïve T cells