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.     

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.     

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor-Specific CD8+ Memory T Cells

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8⁺ T-cell responses in mice that lack the IFN-I receptor (IFN-IR^−/−). We found that IFN-IR^−/− mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR^−/− male skin was transplanted to syngeneic IFN-IR^−/− female mice. Quantitation of the male (H-Y)-specific CD8⁺ T-cell response in these mice revealed normal generation of donor-specific CD8⁺ effector T cells but fourfold reduction in CD8⁺ memory T cells. Memory CD8⁺ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR^−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8⁺ T cells into long-lived, functional memory T cells.     

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.     

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.     

Epidermal Langerhans Cells Promote Skin Allograft Rejection in Mice With NF-κB-impaired T Cells

T cells play a major role in the acute rejection of transplanted organs. Using mice transgenic for a T-cell-restricted NF-κB super-repressor (IκBαΔN-Tg mice), we have previously shown that T-cell-NF-κB is essential for the acute rejection of cardiac but not skin allografts. In this study, we investigated the mechanism by which skin grafts activate IκBαΔN-Tg T cells. Rejection was not due to residual T-cell-NF-κB activity as mice with p50/p52^−/− T cells successfully rejected skin grafts. Rather, skin but not cardiac allografts effectively induced proliferation of graft-specific IκBαΔN-Tg T cells. Rejection of skin grafts by IκBαΔN-Tg mice was in part dependent on the presence of donor Langerhans cells (LC), a type of epidermal dendritic cells (DC), as lack of LC in donor skin grafts resulted in prolongation of skin allograft survival and injection of LC at the time of cardiac transplantation was sufficient to promote cardiac allograft rejection by IκBαΔN-Tg mice. Our results suggest that LC allow NF-κB-impaired T cells to reach an activation threshold sufficient for transplant rejection. The combined blockade of T-cell-NF-κB with that of alternative pathways allowing activation of NF-κB-impaired T cells may be an effective strategy for tolerance induction to highly immunogenic 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.     

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.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with 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.     

CD4+CD25+FOXP3+ Regulatory T Cells Increase De Novo in Kidney Transplant Patients After Immunodepletion with Campath-1H

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3⁺ regulatory T (Treg) cells. Flow cytometry demonstrated that CD4⁺CD25⁺FOXP3⁺ lymphocytes increased significantly within the CD4⁺ T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4⁺CD25⁺FOXP3⁺ cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo .     

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.     

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.     

Isolated CD39 Expression on CD4+ T Cells Denotes both Regulatory and Memory Populations

Foxp3⁺ regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4⁺CD39⁺ T‐cell pool contains two roughly equal size Foxp3⁺ and Foxp3^? populations. While Foxp3⁺CD39⁺ cells are CD73^bright and are the bone fide Tregs, Foxp3^?CD39⁺ cells do not have suppressive activity and are CD44⁺CD62L^?CD25^?CD73^dim/?, exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP‐induced apoptosis. Compared with Foxp3^?CD39^? naïve T cells, Foxp3^?CD39⁺ cells freshly isolated from non‐immunized mice express at rest significantly higher levels of mRNA for T‐helper lineage‐specific cytokines IFN‐γ (Th1), IL‐4/IL‐10 (Th2), IL‐17A/F (Th17), as well as pro‐inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3^?CD39⁺ cells inhibits TGF‐β induction of Foxp3 in Foxp3^?CD39^? cells. Furthermore, when transferred in vivo, Foxp3^?CD39⁺ cells rejected MHC‐mismatched skin allografts in a much faster tempo than Foxp3^?CD39^? cells. Thus, besides Tregs, CD39 is also expressed on pre‐existing memory T cells of Th1‐, Th2‐ and Th17‐types with heightened alloreactivity.     

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.     

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.