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.     

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.     

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.     

Role of Integrin CD103 in Promoting Destruction of Renal Allografts by CD8+ T Cells

Infiltration of CD8(+)TCRalphabeta(+) T-effector populations (CD8 effectors) into graft epithelial compartments has long been recognized as a key lesion in progression of clinical renal allograft rejection. While the afferent phase of allograft immunity is increasingly well-defined, the efferent pathways by which donor-reactive CD8-effector populations access and ultimately destroy the graft renal tubules (rejection per se) have received remarkably little attention. This is an important gap in our knowledge of transplantation immunology, because epithelial compartments comprise the functional elements of most commonly transplanted organs including not only kidney, but also liver, lung, pancreas, and intestine. Furthermore, there is increasing evidence that attack of graft epithelial elements by CD8-effector populations not only causes short-term graft dysfunction but is also a major contributor to development of chronic allograft nephropathy and late graft loss, which now represent the salient clinical problems. Recent studies of the T-cell integrin, alpha(E)beta(7) (CD103), have provided insight into the mechanisms that promote interaction of CD8 effectors with graft epithelial compartments. The purpose of this communication is to review the known properties of the CD103 molecule and its postulated role in the efferent phase of renal allograft rejection.     

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.     

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.     

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.     

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.     

Striking Dichotomy of PD-L1 and PD-L2 Pathways in Regulating Alloreactive CD4+ and CD8+ T Cells In Vivo

Programmed death-1 (PD-1) is a recently identified coinhibitory molecule that belongs to the CD28 superfamily. PD-1 has two ligands PD-L1 and PD-L2. There is some evidence that PD-L1 and PD-L2 serve distinct functions, but their exact function in alloimmunity remains unclear. In the present study, we used a GVHD-like model that allows detailed analyses of T-cell activation at a single cell level in vivo to examine the role of PD-1/PD-L1 and PD-1/PD-L2 interactions in regulating proliferation of CD4(+) and CD8(+) T cells in response to alloantigen stimulation. We found that both CD4(+) and CD8(+) T cells proliferated vigorously in vivo and that PD-L1 and PD-L2 exhibit strikingly different effect on T-cell proliferation. While blocking PD-L1 did not affect the in vivo proliferation of CD4(+) and CD8(+) T cells regardless of CD28 costimulation, blocking PD-L2 resulted in a marked increase in the responder frequency of CD8(+) T-cells in vivo. The effect of PD-L2 on the CD8(+) T-cell proliferation is regulated by CD28 costimulation and by the CD4(+) T cells. We conclude that PD-L1 and PD-L2 function differently in regulating alloreactive T-cell activation in vivo, and PD-L2 is predominant in this model in limiting alloreactive CD8(+) T-cell proliferation.     

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.     

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.     

Differential Requirement of CD27 Costimulatory Signaling for Naïve Versus Alloantigen‐Primed Effector/Memory CD8+ T Cells

CD8⁺ memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T‐cell costimulatory pathway in alloreactive CD8⁺/CD4⁺ T‐cell activation. CD27‐deficient (CD27^?/?) and wild‐type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4⁺ or CD8⁺ T cells, suggesting that CD27 is not essential during primary T‐cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27^?/? or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor‐type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40‐ but not day 10‐sensitized CD27^?/? recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8⁺ T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27^?/? recipients of class I‐ but not class II‐mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8⁺ memory T cells in acute and chronic allograft rejection.     

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.     

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4⁺ regulatory T cells play a critical role in tolerance induction in transplantation. CD8⁺ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8⁺ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8⁺Foxp3⁺ T cells can be induced and significantly expanded by stimulating naïve CD8⁺ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8⁺Foxp3⁺ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8⁺Foxp3⁺ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8⁺Foxp3⁺ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4⁺Foxp3⁺ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8⁺Foxp3⁺ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.     

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.     

Allopeptide-Specific CD4+ T Cells Facilitate the Differentiation of Directly Alloreactive Graft-Infiltrating CD8+ T Cells

To investigate the mechanism of CD4(+) T-cell help during the activation and differentiation of directly alloreactive CD8(+) T cells, we examined the development of obliterative airways disease (OAD) following transplantation of airways into fully mismatched recipient mice deficient in CD4(+) T cells. BALB/c trachea allografts became fibrosed significantly less frequently in B6 CD4(-/-) recipients as compared to wildtype controls. Furthermore, class I-directed cytotoxicity failed to develop in the absence of CD4(+) T cells. The infiltration of graft tissue by primed L(d)-specific directly alloreactive 2C CD8(+) T cells was not found to depend on the presence of CD4(+) T cells. Nevertheless, graft-infiltrating 2C CD8(+) T cells failed to express CD69 and granzyme B when CD4(+) T-cell help was unavailable. Importantly, reconstitution of B6 CD4(-/-) recipient mice with graft peptide-specific TCR-Tg CD4(+) T cells (OT-II or TEa) capable of recognizing antigen only on recipient APC allowed for full expression of CD69 and granzyme B by the directly alloreactive CD8(+) T cells and restored the capacity of recipients to reject their allografts. These results demonstrate that indirectly alloreactive CD4(+) T cells ensure the optimal activation and differentiation of graft-infiltrating directly alloreactive CD8(+) T cells independent of donor APC recognition.     

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.     

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.