Alloreactive (CD4-Independent) CD8+ T Cells Jeopardize Long-Term Survival of Intrahepatic Islet Allografts

Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8⁺ T cells that are independent of CD4⁺ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8⁺ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60–90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8⁺ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allo specific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.     

Role of 4-1BB in Allograft Rejection Mediated by CD8+ T Cells

Blockade of traditional costimulatory molecules fails to inhibit rejection in many models where CD8+ T cells are sufficient to mediate rejection. This observation demonstrates that in many settings CD8+ T cells are not dependent upon CD28 or CD154 signals to mediate rejection. 4-1BB (CD137) has been shown to be an important regulatory molecule for CD8+ T cells in a variety of nontransplant models. Here we show that blocking the 4-1BB pathway significantly inhibited rejection of intestinal allografts by CD8+ but not CD4+ T cells. This effect was associated with significantly decreased expression of the genes encoding TNFalpha and secondary lymphoid chemokine (SLC) within the spleens of recipient mice. Disruption of the 4-1BB pathway also impaired the priming of alloantigen-specific CD8+ T cells and the accumulation of recipient dendritic cells within the spleen. These data directly demonstrate an important role for 4-1BB in allograft rejection; particularly rejection mediated by CD8+ T cells. Our data suggest that in addition to providing a direct costimulatory signal to T cells, the 4-1BB pathway may regulate other important steps in the immune response such as the migration of T cells and dendritic cells.     

Activation and Maturation of Alloreactive CD4-Independent, CD8+ Cytolytic T Cells

The goal of this study was to determine the in vivo conditions that promote activation of the (CD4-independent) CD8+ T cell-mediated rejection pathway. We have previously noted that hepatocellular but not islet allografts readily activate this rejection pathway. In the current study, we utilized these two cell transplant models to investigate whether differences in host cell recruitment to the graft site, expression of T-cell activation markers by CD8+ graft infiltrating cells (GICs), and/or development of delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte cell-mediated effector functions could account for the differential transplant outcomes. The collective results demonstrate that recruitment of CD8+ T cells to the site of transplant, CD103 or CD69 expression on CD8+ GICs, and activation of alloreactive DTH responses are insufficient to initiate CD4-independent, CD8-dependent transplant rejection. Instead, rejection by alloreactive (CD4-independent) CD8+ T cells correlated with expression of CD25, CD154 and CD43 by CD8+ GICs, in vitro alloproliferation by recipient CD8+ T cells, and the development of in vivo allospecific cytolytic effector function. These results suggest that tissue-derived factors influence the activation and maturation of (CD4-independent) CD8+ T cells into cytolytic effectors, which correlates with transplant rejection.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.     

EBV-Specific CD8+ T Cell Reactivation in Transplant Patients Results in Expansion of CD8+ Type-1 Regulatory T Cells

Posttransplantation lymphoproliferative disorders (PTLD) are life-threatening complications of solid organ transplantation, triggered by EBV infection in chronically immunosuppressed (IS) patients. Our goal is to establish DC-based protocols for adoptive immunotherapy of refractory PTLD, while understanding how the immunosuppressive drug environment may subvert DC-EBV-specific T cell interactions. Type-1 CD8(+) T cells are critical for efficient immune surveillance and control of EBV infection, whereas type-2 or Treg/type-3 responses may provide an environment conductive to disease progression. We have recently reported that chronic IS inhibits DC function in transplant patients. Here, we have analyzed the comparative ability of mature, type-1 polarized DCs (i.e. DC1) generated from quiescent transplant patients or healthy controls, to boost type-1 EBV-specific CD8(+) T cells in vitro. Our results show that unlike healthy controls, where DC1 loaded with MHC class I EBV peptides preferentially reactivate specific type-1 CD8(+) T cells, DC1 generated from transplant patients reactivate EBV-specific CD8(+) T cells that produce both IFN-gamma and IL-10, up-regulate FOXP3 mRNA, and suppress noncognate CD4(+) T-cell proliferation via cell-cell contact. These data support a novel regulatory pathway for anti-EBV T-cell-mediated responses in IS transplant patients, with implications for the design of adoptive immunotherapies in this setting.     

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo -expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT_reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo -expanded nT_reg may be a practical means of preventing acute allograft rejection.     

T cell receptor beta chain (TCR-Vβ) repertoire of circulating CD4+ CD25−, CD4+ CD25low and CD4+ CD25high T cells in patients with long-term renal allograft survival

The mechanisms underlying maintenance of renal allografts in humans under minimal or conventional immunosuppression are poorly understood. There is evidence that CD4⁺ CD25⁺ regulatory T cells and clonal deletion, among other mechanisms of tolerance, could play a key role in clinical allograft survival. Twenty‐four TCR‐Vβ families were assessed in CD4⁺ CD25^?, CD4⁺ CD25^low and CD4⁺ CD25^high T cells from patients with long‐term renal allograft survival (LTS), patients exhibiting chronic rejection (ChrRx), patients on dialysis (Dial) and healthy controls (HC) by flow cytometry. LTS patients presented a higher variability in their TCR‐Vβ repertoire, such decreased percentage of Vβ2⁺, Vβ8a⁺ and Vβ13⁺ in CD4⁺ CD25^low and ^high compared with CD4⁺ CD25^? subset and increased Vβ4 and Vβ7 families in CD4⁺ CD25^high T cells exclusively. Additionally, LTS patients, particularly those that were not receiving calcineurin inhibitors (CNI), had increased percentages of CD4⁺ CD25^high T cells when compared with Dial (P < 0.05) and ChrRx (P < 0.05) patients. Our results suggest that a differential expression of particular TCR‐Vβ families and high levels of circulating CD4⁺ CD25^high T cells in long‐term surviving renal transplant patients could contribute to an active and specific state of immunologic suppression. However, the increase in this T cell subset with regulatory phenotype can be affected by CNI.     

CD4+ CD25bright+ Regulatory T Cells Can Mediate Donor Nonreactivity in Long-Term Immunosuppressed Kidney Allograft Patients

CD4+ CD25bright+ FoxP3+ T cells are potent regulators of T-cell reactivity, but their possible involvement in donor-specific nonresponsiveness after clinical kidney transplantation remains to be elucidated. We assessed the proliferative donor-reactivity in 33 kidney allograft recipients who were maintained on a combination of proliferation inhibitors (mycophenolate mofetil (MMF) or Azathioprine (Aza)) and prednisone, long (> 5 years) after transplantation. Of the 33 patients, 8 still exhibited donor-reactivity, whereas 25 were classified as donor nonreactive patients. Within these 25 donor nonreactive patients, we assessed the involvement of CD4+ CD25bright+ regulatory T cells both by depleting them from the responder population as well as by reconstituting them to the CD25(-/dim) effector population. The absence of proliferation in these 25 patients, was abolished in 7 (28%) recipients upon depletion of the CD4+ CD25bright+ T cells. Reconstitution of these cells suppressed the donor-reactivity in a dose-dependent manner. Adding-back CD4+ CD25bright+ T cells inhibited the anti-third party response in all recipients, indicating that functional CD4+ CD25bright+ T cells circulate despite more then 5 years of immunosuppressive treatment. Altogether, we conclude that in long-term immunosuppressed kidney allograft patients functional regulatory CD4+ CD25bright+ T cells circulate but that these cells mediate donor non reactivity only in a subset of patients.     

CD4+CD25+ Regulatory T Cells in Transplantation: Progress, Challenges and Prospects

The involvement of CD4(+)CD25(+) regulatory T cells (Treg) in general immune homeostasis and protection from autoimmune syndromes is now well established. Similarly, there has been increasing evidence for Treg involvement in allograft rejection and current immunotherapies. However, despite significant advances in understanding the development, function, and therapeutic efficacy of Treg in certain well-defined rodent models, the relevance of Treg to clinical transplantation remains unclear. In this review, we summarize our current understanding of the role of Treg in immunity and organ transplantation in experimental and clinical settings. In addition, we review advances in using Treg as a form of immune therapy. The goal is to highlight the complexities and opportunities in the field and to provide evidence to support the use of antigen-specific Tregs in the context of transplantation to facilitate a robust and selective state of immune tolerance.     

Human CD4+CD25+ Regulatory T Cells Suppress Anti-Porcine Xenogeneic Responses

Due to the shortage of human organs, xenotransplantation is being explored as an alternative to allotransplantation, but immune rejection remains a major hurdle to its implementation. We tested the ability of human CD4+CD25+ T cells (Treg cells) to suppress CD4+ T cell-mediated anti-porcine xenoresponses usingin vitroassays. Human Treg cells were hyporesponsive to porcine cell stimulation and suppressed the proliferative response of CD4+CD25- T cells in a dose-dependent manner, and comparison of the allo- and xenoresponses indicated that more Treg cells might be required to suppress the xenogeneic response than the allogeneic response. Stimulation of CD4+CD25- T cells with porcine cells resulted in secretion of IFN-gamma, TNF-alpha, IL-10, IL-6 and IL-2, and Treg cells suppressed the secretion of these cytokines, as well as the CD4+CD25- T-cell cytolytic response against porcine cells. These results suggest a potential role for Treg cells in promoting xenograft survival.     

Donor-specific Immune Regulation by CD8+ Lymphocytes Expanded from Rejecting Human Cardiac Allografts

To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8⁺ GL subset of these cultures that inhibited the antidonor response (65–91% inhibition of the proportion of proliferating cells); the CD4⁺ GLs of the expanded GL cultures were not suppressive. In conclusion, CD8⁺ GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro . We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8⁺ type with the potential to specifically inhibit antidonor immune reactivity.     

Essential Role of Sphingosine-1-Phosphate Receptor 1-Bearing CD8+CD44+CCR7+ T Cells in Acute Skin Allograft Rejection

A subset of naturally formed sphingosine‐1‐phosphate receptor 1 (S1P1)‐bearing CD8⁺CD44⁺CCR7⁺ memory T cells has been identified in transplant recipient BALB/c (H‐2^d) mice. The frequency of this subset of memory T cells is significantly increased in the spleen, lymph nodes and skin grafts in the recipient BALB/c mice during acute skin allograft rejections. The immune‐reconstitution with CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells facilitates acute skin allograft rejection in SCID mice. Being Th1‐polarized and cytotoxic, CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells proliferate and differentiate immediately into effectors upon encountering allo‐antigens. A siRNA against S1P1 inhibits CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cell‐mediated acute skin allograft rejection in SCID mice by means of knocking‐down S1P1‐expression. CCL21 mutant (CCL21‐ΔCT) has been used to compete with wild‐type CCL21 in the course of binding to CCR7. Combined administration of siRNA S1P1 and CCL21‐ΔCT significantly prolongs the survival of skin allograft in the recipient BALB/c mice by means of inhibiting accumulation of CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells in the spleen and the skin grafts. Our data provide direct evidence that S1P1 and CCR7 are involved in the proliferation and trafficking of CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells. S1P1 may serve as a functional marker for CD8⁺CD44⁺CCR7⁺ memory T cells. Targeting CD8⁺CD44⁺CCR7⁺S1P1⁺ T cells may be a useful strategy to prolong the survival of allograft transplant.     

Monitoring of Human Cytomegalovirus-Specific CD4+ and CD8+ T-Cell Immunity in Patients Receiving Solid Organ Transplantation

Absolute and human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T-cell counts were monitored in 38 solid organ (20 heart, 9 lung and 9 kidney) transplant recipients during the first year after transplantation by a novel assay based on T-cell stimulation with HCMV-infected autologous dendritic cells. According to the pattern of T-cell restoration occurring either within the first month after transplantation or later, patients were classified as either early (n = 21) or late responders (n = 17). HCMV-specific CD4+ and CD8+ T-cell counts were consistently lower in late compared to early responders from baseline through 6 months after transplantation. In addition, in late responders, while HCMV infection preceded immune restoration, HCMV-specific CD4+ restoration was significantly delayed with respect to CD8+ T-cell restoration. The number of HCMV-specific CD4+ and CD8+ T-cells detected prior to transplantation significantly correlated with time to T-cell immunity restoration, in that higher HCMV-specific T-cell counts predicted earlier immune restoration. Clinically, the great majority of early responders (18/21, 85.7%) underwent self-resolving HCMV infections (p = 0.004), whereas the great majority of late responders (13/17, 76.5%) were affected by HCMV infections requiring antiviral treatment (p = <0.0001). Simultaneous monitoring of HCMV infection and HCMV-specific T-cell immunity predicts T-cell-mediated control of HCMV infection.     

Different mechanisms of Campath-1H-mediated depletion for CD4+ and CD8+ T cells in peripheral blood

It is assumed that complement and noncomplement-mediated mechanisms are similarly responsible for Campath-1H-mediated killing of all T-cell subtypes in vivo. However, the differing surface expression of CD52 on T-cell subtypes suggests that may not be the case. The purpose of this study is to determine the extent and mechanism of Campath-1H-mediated elimination of different T-cell subtypes in peripheral blood. Whole blood or lymphocytes isolated from peripheral blood of healthy volunteers by Ficoll density centrifugation were incubated with Campath-1H, with or without complement and/or serum, and the resultant T-cell elimination mechanisms studied. For CD4(+) T lymphocytes, 60% and 40% cell death and for CD8(+) T lymphocytes 23% and 77% cell death, in peripheral blood, was mediated by complement and noncomplement mediated mechanisms, respectively. CD4(+) T cells demonstrated approximately twice the amount of surface CD52 compared with CD8(+) T cells, consistent with primarily complement-mediated killing for CD4(+) T cells. Thus, peripheral blood supports differential and partial elimination of T-cell subtypes, suggesting that the complete T-cell elimination seen in transplant recipients is most likely due to contribution from other lymphoid organs.     

Contrasting Alloreactive CD4+ and CD8+ T Cells: There''s More to It Than MHC Restriction

Surface expression of CD4 or CD8 is commonly used to identify T-cell subsets that recognize antigen presented by class II MHC or class I MHC, respectively. This holds true for T cells that respond to allogeneic MHC molecules that are directly recognized as foreign, as well as peptides from allogeneic MHC molecules that are indirectly presented by self MHC molecules. CD4 or CD8 expression was initially believed to define cytokine secreting helper T cells or cytotoxic cells, respectively. However, this association of phenotype and function is not absolute, in that CD4+ cells may possess lytic activity and CD8+ cells secrete cytokines, notably IFNgamma. Recently, additional fundamental differences in the immunobiology of these T-cell subsets have been identified. These include differences in costimulatory requirements, cytokine responsiveness, cytokine production, cell survival, and the maintenance of memory. This review will survey these differences, emphasizing alloreactive T-cell responses as well as relevant observations that have been made in other systems.     

Role of IFNγ in Allograft Tolerance Mediated by CD4+CD25+ Regulatory T Cells by Induction of IDO in Endothelial Cells

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.     

Everolimus and Basiliximab Permit Suppression by Human CD4+CD25+ Cells in vitro

Immunosuppressive drugs are essential for the prevention of acute transplant rejection but some may not promote long-term tolerance. Tolerance is dependent on the presence and regulatory function of CD4(+)CD25(+) T cells in a number of animal models. The direct effects of immunosuppressive drugs on CD4(+)CD25(+) cells, particularly those that interfere with IL-2 signaling are uncertain. We studied the effects of the rapamycin derivative everolimus and the anti-CD25 monoclonal antibody basiliximab on the regulatory capacity of human CD4(+)CD25(+) cells in vitro. Both drugs permitted the suppression of proliferation and IFN-gamma secretion by CD4(+)CD25(-) cells responding to allogeneic and other polyclonal stimuli; CTLA-4 expression was abolished on CD4(+)CD25(+) cells without compromising their suppressive ability. Everolimus reduced IFN-gamma secretion by CD4(+)CD25(-) cells before the anti-proliferative effect: this is a novel finding. Exogenous IL-2 and IL-15 could prevent the suppression of proliferation by CD4(+)CD25(+) cells and the drugs could not restore suppression. By contrast, suppression of IFN-gamma secretion was only slightly impeded with the exogenous cytokines. Finally, CD4(+)CD25(+) cells were more resistant than CD4(+)CD25(-) cells to the pro-apoptotic action of the drugs. Together these data suggest that CD4(+)CD25(+) cells may still exert their effects in transplant patients taking immunosuppression that interferes with IL-2 signaling.