Characterization of eomesodermin and T-bet expression by allostimulated CD8+ T cells of healthy volunteers and kidney transplant patients in relation to graft outcome.

Memory T cell (Tmem) responses play a critical role in the outcome of allo-transplantation. While the role of the T-box transcription factor Eomesodermin (Eomes) in the maintenance of antigen-specific Tmem is well studied, little is known about Eomes+ CD8+ T cell responses after transplantation. We evaluated the phenotype and function of allo-reactive Eomes+ CD8+ T cells in healthy volunteers and kidney transplant patients and their relation to transplant outcome. High Eomes expression by steady-state CD8+ T cells correlated with effector and memory phenotype. Following allo-stimulation, the expression of both the T-box proteins Eomes and T-bet by proliferating cells increased significantly, where high expression of Eomes and T-bet correlated with higher incidence of allo-stimulated IFNγ+ TNFα+ CD8+ T cells. In patients with no subsequent rejection, Eomes but not T-bet expression by donor-stimulated CD8+ T cells, increased significantly after transplantation. This was characterized by increased Eomeshi T-bet-/lo and decreased Eomes-/lo T-bethi CD8+ T cell subsets, with no significant changes in the Eomeshi T-bethi CD8+ T cell subset. No upregulation of exhaustion markers programmed-death-1 (PD-1) and cytotoxic-T-lymphocyte-associated-antigen-4 (CTLA4) by donor-stimulated Eomes+ CD8+ T cells was observed. Before transplantation, in patients without rejection, there were higher incidences of Eomeshi T-bet-/lo , and lower incidences of Eomeshi T-bethi and Eomes-/lo T-bethi donor-stimulated CD8+ T cell subsets, compared to those with subsequent rejection. Overall, our findings indicate that high Eomes expression by allo-stimulated T-bet+ CD8+ T cells is associated with enhanced effector function, and that an elevated incidence of donor-stimulated CD8+ T cells co-expressing high levels of Eomes and T-bet before transplantation, may correlate with an increased incidence of acute cellular rejection.

Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

Impact of Human Mutant TGFß1/Fc Protein on Memory and Regulatory T Cell Homeostasis Following Lymphodepletion in Nonhuman Primates.

Transforming growth factor ß1 (TGFß1) plays a key role in T cell homeostasis and peripheral tolerance. We evaluated the influence of a novel human mutant TGFß1/Fc (human IgG4 Fc) fusion protein on memory CD4+ and CD8+ T cell (Tmem) responses in vitro and their recovery following antithymocyte globulin (ATG)-mediated lymphodepletion in monkeys. TGFß1/Fc induced Smad2/3 protein phosphorylation in rhesus and human peripheral blood mononuclear cells and augmented the suppressive effect of rapamycin on rhesus Tmem proliferation after either alloactivation or anti-CD3/CD28 stimulation. In combination with IL-2, the incidence of CD4+ CD25hi Foxp3hi regulatory T cells (Treg) and Treg:Th17 ratios were increased. In lymphodepleted monkeys, whole blood trough levels of infused TGFß1/Fc were maintained between 2 and 7 µg/mL for 35 days. Following ATG administration, total T cell numbers were reduced markedly. In those given TGFß1/Fc infusion, CD8+ T cell recovery to predepletion levels was delayed compared to controls. Additionally, numbers of CD4+ CD25hi CD127lo Treg increased at 4-6 weeks after depletion but subsequently declined to predepletion levels by 12 weeks. In all monkeys, CD4+ CD25hi Foxp3hi Treg/CD4+ IL-17+ cell ratios were reduced, particularly after stopping TGFß1/Fc infusion. Thus, human TGFß1/Fc infusion may delay Tmem recovery following lymphodepletion in nonhuman primates. Combined (low-dose) IL-2 infusion may be required to improve the Treg:Th17 ratio following lymphodepletion.

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients.

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4(+) CD25(+) CD127(-) Foxp3(+) (Treg) in clinically relevant large animal models. We infused ex vivo-expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin-lymphodepleted, MHC-mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti-interleukin-6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon-γ production by host CD8(+) T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed "Treg-friendly" agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.

In Vivo Mobilization and Functional Characterization of Nonhuman Primate Monocytic Myeloid-Derived Suppressor Cells.

Increasing evidence from small animal models shows that myeloid-derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3(-)CD20(-)HLA-DR(-)CD14(+)CD33(+)CD11b(+) cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin(-)HLA-DR(-) peripheral blood mononuclear cells. Administration of granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10(6) (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti-CD3- and anti-CD28-stimulated autologous T cells markedly suppressed CD4(+) and CD8(+) T cell proliferation and cytokine secretion (interferon γ, IL-17A). Moreover, these MDSCs enhanced CD4(+)CD25(hi)Foxp3(+) regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase-1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8(+) T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

Sequential monitoring and stability of ex vivo-expanded autologous and nonautologous regulatory T cells following infusion in nonhuman primates.

Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs.

BACKGROUND: In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy. METHODS: As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept + anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered. RESULTS: When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response. CONCLUSION: Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.

Post-transplant repopulation of naïve and memory T cells in blood and lymphoid tissue after alemtuzumab-mediated depletion in heart-transplanted cynomolgus monkeys.

Repopulation of memory T cells (Tmem) in allograft recipients after lymphodepletion is a major barrier to transplant tolerance induction. Ineffective depletion of naïve T cells (Tn) and Tmem may predispose to repopulation of Tmem after transplantation. Cynomolgus macaque monkeys given heart allografts were lymphodepleted using Alemtuzumab (Campath-1H; anti-CD52). Peripheral blood (PB) and lymph nodes (LN) were analyzed for CD95(-) (Tn) and CD95(+) cells (Tmem), one day, one month and up to three months after Alemtuzumab infusion. CD52 expression, susceptibility to Alemtuzumab cytotoxicity and pro-apoptotic caspase-3 were evaluated in Tn and Tmem. In vivo, Alemtuzumab induction profoundly depleted lymphocytes in PB (99% reduction) but exerted a lesser effect in LN (70% reduction), with similar depletion of Tn and Tmem subsets. After transplantation, Tmem comprised the majority of lymphocytes in PB and LN. In vitro, LN T cells were more resistant to Alemtuzumab-mediated cytotoxicity than PB lymphocytes. CD4(+) Tn and Tmem were equally susceptible to Alemtuzumab-mediated cytotoxicity, whereas CD8(+) Tn were more resistant than CD8(+) Tmem. However, no significant differences in CD52 expression between lymphocyte subsets in PB and LN were observed. Caspase-3 expression was higher in PB than LN T cells. CD4(+) and CD8(+) Tn expressed lower levels of Caspase-3 than Tmem, in both PB and LN. Thus, after Alemtuzumab infusion, residual Tn in secondary lymphoid tissue may predispose to rapid recovery of Tmem in allograft recipients.

Regulatory dendritic cell infusion prolongs kidney allograft survival in nonhuman primates.

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.