Characterization of primary T cell subsets mediating rejection of pancreatic islet grafts

The cellular mechanisms by which pancreatic islet grafts are rejected have not been clearly defined. In order to address the roles of CD4+ and CD8+ T cells in pancreatic islet rejection, we used an adoptive transfer model in which H-2b nude mice were reconstituted with negatively selected H-2b CD4+ or CD8+ T cell subpopulations and engrafted with fully allogeneic pancreatic islet grafts. We found that primary (unprimed) CD4+ T cells mediated the rejection of pancreatic islet grafts, whereas, primary CD8+ T cells failed to do so, even though both T cell subpopulations were competent to reject skin allografts. These data indicate that primary CD4+ T cells are necessary for rejection of allogeneic pancreatic islet grafts, whereas primary CD8+ T lymphocytes are not. Implications concerning the nature of the APC involved in the initiation of the rejection response to islet allografts and the expression of MHC Ag by pancreatic islet cells are discussed.     

Donor lymphocyte infusion induces long-term donor-specific cardiac xenograft survival through activation of recipient double-negative regulatory T cells

Previous studies have shown that pretransplant donor lymphocyte infusion (DLI) can enhance xenograft survival. However, the mechanism by which DLI induces xenograft survival remains obscure. Using T cell subset-deficient mice as recipients we show that CD4+, but not CD8+, T cells are necessary to mediate the rejection of concordant cardiac xenografts. Adoptive transfer of naive CD4+ T cells induces rejection of accepted cardiac xenografts in CD4-/- mice. This rejection can be prevented by pretransplant DLI in the absence of any other treatment. Furthermore, we demonstrate that DLI activates alphabeta-TCR+CD3+CD4-CD8- double-negative (DN) regulatory T (Treg) cells in xenograft recipients, and that DLI-activated DN Treg cells can inhibit the proliferation of donor-specific xenoreactive CD4+ T cells in vitro. More importantly, adoptive transfer of DLI-activated DN Treg cells from xenograft recipients can suppress the proliferation of xenoreactive CD4+ T cells and their ability to produce IL-2 and IFN-gamma in vivo. Adoptive transfer of DLI-activated DN Treg cells also prevents CD4+ T cell-mediated cardiac xenograft rejection in an Ag-specific fashion. These data provide direct evidence that DLI can activate recipient DN Treg cells, which can induce donor-specific long-term cardiac xenograft survival by suppressing the proliferation and function of donor-specific CD4+ T cells in vivo.     

Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte-reconstituted non-obese diabetic severe combined immunodeficient beta2-microglobulin-null mice

Small animal models with the capacity to support engraftment of a functional human immune system are needed to facilitate studies of human alloimmunity. In the present investigation, non-obese diabetic (NOD) severe combined immunodeficient (scid) beta2-microglobulin-null (B2mnull) mice engrafted with human peripheral blood lymphocytes (hu-PBL-NOD-scid B2mnull mice) were used as in vivo models for studying human skin allograft rejection. Hu-PBL-NOD-scid B2mnull mice were established by injection of human spleen cells or PBLs and transplanted with full-thickness allogeneic human skin. Human cell engraftment was enhanced by injection of anti-mouse CD122 antibody. The respective contributions of human CD4+ and CD8+ cells in allograft rejection were determined using depleting antibodies. Human skin grafts on unmanipulated NOD-scid B2mnull mice uniformly survived but on chimeric hu-PBL-NOD-scid B2mnull mice exhibited severe immune-mediated injury that often progressed to complete rejection. The alloaggressive hu-PBLs did not require prior in vitro sensitization to elicit targeted effector cell activity. Extensive mononuclear cell infiltration directed towards human-origin endothelium was associated with thrombosis and fibrin necrosis. No evidence of graft-versus-host disease was detected. Either CD4+ or CD8+ T cells may mediate injury and alloimmune rejection of human skin grafts on hu-PBL-NOD-scid B2mnull mice. It is proposed that Hu-PBL-NOD-scid B2mnull mice engrafted with human skin will provide a useful model for analysis of interventions designed to modulate human allograft rejection.     

Cellular basis of allograft rejection in vivo. V. Examination of the mechanisms responsible for the differing efficacy of monoclonal antibody to CD4+ T cell subsets in low- and high-responder rat strains

MRC OX35, an anti-CD4 mAb, was used to treat high responder Wistar Furth (W/F) (RT1u) and low responder DA (RT1a) rats which had been grafted with directly vascularized hearts from PVG (RT1c) rats across a full MHC plus non-MHC incompatibility. Four doses of mAb at 7 mg/kg given in the first 2 wk postgrafting induced indefinite graft survival (greater than 150 days) in DA hosts, but only delayed rejection to 18 to 42 days in W/F as compared to rejection times of 6 to 8 days in untreated rats. The extension of MRC OX35 treatment to 6 wk in W/F rats induced indefinite graft survival in three of six rats. During treatment MRC OX35 therapy only partially depleted CD4+ cells, and all circulating CD4+ cells were coated with MRC OX35. The capacity of naive CD4+ and CD8+ cells from W/F and DA to be activated to PVG alloantigen was compared both in vitro in an MLC assay and in vivo by an adoptive transfer assay of their capacity to restore rejection of PVG heart grafts in irradiated syngeneic hosts. CD4+ cells from both W/F and DA proliferated in MLC and restored graft rejection. W/F CD8+ cells both proliferated in MLC and restored rejection, but DA CD8+ cells neither proliferated nor reconstituted rejection. Examination of lymphocytes from MRC OX35 treated hosts with long-surviving grafts showed that they were neither depleted of CD4+ T cells nor did they lack the capacity to proliferate to PVG Ag in MLC, this response being similar to that to third-party Ag or by naive lymphocytes. Compared to first-set rejection, PVG skin graft rejection was delayed 2 to 3 days in W/F and 10 to 12 days in DA rats with long-surviving grafts after MRC OX35 therapy, whereas they rejected third-party skin grafts in first-set tempo. These studies show that differences in graft survival in anti-CD4 treated low and high responder strains may be due to the inherent capacity of CD8+ cells to be activated to effect rejection independent of CD4+ cells in W/F but not in DA. In those hosts that accept grafts, there is no evidence of clonal deletion, but there appears to be a form of unresponsiveness akin to that induced in adult rats by other immunosuppressive therapies that protects the graft from rejection.     

Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions

Skin lesions in the allergic form of atopic dermatitis (AD) are induced by allergen-specific T cells that infiltrate the skin at the site of allergen exposure. Although Th2-type CD4+ T cells appear to be crucial in AD pathophysiology, little is known about the contribution of CD8+ T cells in the development of the allergic skin inflammation. In the present study, we have analyzed the respective role of CD8+ and CD4+ T cells in the development of AD skin lesions in a mouse model of allergen-induced AD. In sensitized mice, CD8+ T cells are rapidly and transiently recruited to the allergen-exposed site and initiate the inflammatory process leading to skin infiltration with eosinophils and Th1/Th2-producing cells. CD8+ T cell-depleted mice show no inflammation, demonstrating that these cells are mandatory for the development of AD. In contrast, CD4+ T cell-depleted mice develop a severe form of eczema. Furthermore, adoptive transfer of CD8+ T cells from sensitized mice into naive recipient mice leads to skin inflammation soon after allergen exposure. These data indicate that allergen-primed CD8+ T cells are required for the development of AD-like lesions in mice.     

CD8+ T cells circumvent immune privilege in the eye and mediate intraocular tumor rejection by a TNF-alpha-dependent mechanism

Although intraocular tumors reside in an immune-privileged environment, T cells can circumvent immune privilege and mediate tumor rejection without inducing damage to normal ocular tissue. In this study, we used a well-characterized tumor, Ad5E1 (adenovirus type 5 early region 1), to analyze the role of CD8+ T cells in the pristine rejection of intraocular tumors. It has been previously documented that Ad5E1 tumor rejection can occur in the absence of CD8+ T cells. However, here we find that CD8+ T cells infiltrated intraocular Ad5E1 tumors in C57BL/6 mice. Surprisingly, CD8+ T cells from tumor-rejector mice could mediate intraocular tumor rejection following adoptive transfer to SCID mice. In determining the mechanisms behind CD8+ T cell-mediated tumor rejection, we discovered that antitumor CTL activity was neither observed nor necessary for rejection of the intraocular tumors. CD8+ T cells from rejector mice did not produce IFN-gamma in response to Ad5E1 tumor Ags or use FasL to mediate intraocular tumor rejection. Also, CD8+ T cells did not use perforin or TRAIL, as CD8+ T cells from perforin knockout (KO) and TRAIL KO mice conferred protection to SCID recipient mice following adoptive transfer. We discovered that CD8+ T cells used TNF-alpha to mediate tumor rejection, because Ad5E1 tumor cells were highly sensitive to TNF-alpha-induced apoptosis and CD8+ T cells from TNF-alpha KO mice did not protect SCID mice from progressive Ad5E1 tumor growth. The results indicate that CD8+ T cells circumvent immune privilege and mediate intraocular tumor rejection by a TNF-alpha-dependent manner while leaving the eye intact and vision preserved.     

Cellular interactions and the role of interleukin 2 in the expression and induction of immunity against a syngeneic murine sarcoma

We have previously demonstrated that following the adoptive transfer of immune cells, the regression of established pulmonary metastases from a weakly immunogenic sarcoma, MCA 105, required the collaboration of two T cell subsets. In this study, we found that the critical role played by L3T4+ immune cells was to provide a helper function since tumor regression proceeded in the absence of L3T4+ immune cells if exogenous interleukin 2 (IL-2) was administered. To extend these observations, we analyzed the events leading to the induction and generation of L3T4+ and Lyt-2+ immune T cells after immunization of mice with viable tumor cells admixed with Corynebacterium parvum. The basic protocol involved immunization, surgical excision of the immunization site on day 7, and challenge with viable tumor cells on day 21. The ability of mice to reject tumor challenge provided a means to evaluate the occurrence of a systemic antitumor immunity. With the use of this experimental protocol, we have found that depletion of T cell subsets in vivo with either L3T4 or Lyt-2 monoclonal antibodies after active immunization abrogated the development of antitumor immunity. Mice immunized and depleted of L3T4+ but not Lyt-2+ T cells were able to reject tumor challenge if exogenous IL-2 was given for 7 days. However, the rejection of tumor challenge required 3 days of additional exogenous IL-2 administration. These results indicate that the induction of Lyt-2+ immune T cells depended on the helper function of L3T4+ T cells via the secretion of IL-2. In the absence of L3T4+ immune lymphocytes, the expression of antitumor immunity by Lyt-2+ immune cells could be facilitated by in vivo administration of exogenous IL-2. The induction of L3T4+ immune T cells, on the other hand, occurred independently of the Lyt-2+ T cell response because the transfer of spleen cells from Lyt-2+ cell-depleted, immunized animals was able to restore antitumor reactivity in L3T4+ cell-depleted, immunized mice. These results demonstrate the intricate cellular interactions leading to the induction as well as the expression of antitumor immunity.     

Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing.

Although CD4 cells are major mediators in cellular rejection of fetal pig pancreas (FPP) in the mouse, rejection still occurs in the absence of CD4 cells, albeit with delayed kinetics. CD4 cell-independent mechanisms of cellular rejection are poorly understood. To investigate the involvement of CD8 T cells in FPP rejection and their activation requirements, we used mice transgenic for anti-CD4 Ab; this is the most complete model of CD4 cell deficiency. We showed that in such mice FPP was infiltrated with CD8 cells starting from 2 wk posttransplantation and FPP was eventually rejected 8 wk posttransplantation. Ab depletion of CD8 cells greatly improved the survival of FPP and reduced cell infiltration at the graft site. This suggests that CD8 cells can mediate the rejection of porcine xenografts in the absence of CD4 cells. This CD8-mediated rejection of FPP is independent of their perforin-mediated lytic function, as graft survival was not affected in mice deficient in perforin. The production of IFN-gamma and IL-5 by the graft infiltrates indicates that CD8 cells may act through cytokine-mediated mechanisms. Remarkably, in the absence of CD4 cells, lymphocyte infiltration at the graft site was absent in mice transgenic for CTLA4Ig such that the islet grafts flourished beyond 24 wk. In contrast, rejection was little affected by CD40 ligand deficiency. Therefore, we show that CD8 cells are activated to mediate FPP rejection independent of perforin and that this CD4-independent activation of CD8 cells critically depends on B7/CD28 costimulation.     

T cell-activated macrophages are capable of both recognition and rejection of pancreatic islet xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4(+) T cells from BALB/c mice. Twelve days after CD4(+) T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4(+) T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4(+) T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4(+) T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4(+) T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.     

Effect of localized cytokine dysregulation: accelerated rejection of IL-2-expressing skin grafts

Transgenic mice were created in which a sheep keratin promoter directed the expression of IL-2 into the dermis. These KIL-2 transgenic mice were used to investigate the effects of localized IL-2 dysregulation on immune responses. Peripheral tolerance to skin antigens was not broken by in situ IL-2 expression because syngeneic KIL-2 skin grafts were not rejected. However, MHC Class I-disparate skin grafts from KIL-2 donors were rejected faster (median survival time (MST) 12 days) than grafts of non-transgenic littermate skin (MST 18 days). In contrast, the kinetics of KIL-2 H-Y-disparate skin graft rejection (MST 14 days) did not differ significantly from controls (MST 16 days), suggesting that upregulation of IL-2 at the effector site could affect CD4+ T cell- independent, but not CD4+ T cell-dependent, responses. No effect on rejection kinetics was observed when wild type allogeneic skin was grafted onto transgenic mice that expressed bcl2 constitutively in their lymphocytes (MST of 14 days, both sets), indicating that this was not simply due to increased longevity of T cells within the IL-2 expressing graft. We therefore suggest that aberrant expression of IL-2 can accelerate helper-independent CD8+ T cell responses by increasing proliferation and/or differentiation of cytolytic T cells at the effector site.     

Maturation and function of mouse T-cells with a transgenic TCR positively selected by highly disparate xenogeneic porcine MHC.

Remarkably normal cellular immune function, along with specific T-cell tolerance to highly disparate xenogeneic donors, can be achieved by grafting fetal pig thymus (FP THY) tissue to T and NK cell-depleted, thymectomized (ATX) mice. Porcine MHC can mediate positive selection of mouse CD4+ T-cells with a mouse MHC-restricted TCR in FP THY-grafted, T- and NK cell-depleted, ATX TCR-transgenic "AND" mice. However, functional studies were not performed on transgenic mouse T-cells selected in a FP THY graft. We have now performed further studies to confirm the ability of porcine MHC to mediate the positive selection of mouse T-cells with a mouse MHC-restricted TCR, and to exclude the possibility that the maturation of mouse T-cells with a mouse MHC-restricted TCR in FP THY grafts in ATX "AND" mice is a special case. For this purpose, TCR-transgenic mice with an unrelated transgenic TCR ["3A9", specific for hen egg lysozyme (HEL) peptide 46-61 presented by I-Ak] were employed. Similar to FP THY-grafted ATX "AND" mice, large numbers of mouse CD4 single positive thymocytes expressing the transgenic TCR (Vbeta8.2) and expressing a mature phenotype (Qa-2high and heat stable antigen, HSAlow) were detected in FP THY grafts. Porcine thymus grafting led to a high level of peripheral repopulation with mouse naive-type (CD44low CD45RBhigh CD62Lhigh) CD4+ cells expressing the transgenic TCR in T and NK cell-depleted ATX "3A9" mice, regardless of whether the recipients had a positive selecting or a non-selecting, class II deficient MHC background. The mouse CD4+ T-cells expressing the "3A9" TCR showed efficient primary proliferative responses to the protein antigen (HEL) when it was presented by mouse class II+ antigen presenting cells (APC) in vitro. These results, collectively, support the general conclusion that discordant xenogeneic porcine MHC can mediate positive selection of mouse T-cells with mouse MHC-restricted TCR. This study has implications for the potential clinical use of xenogeneic thymus transplantation to reconstitute cellular immunity in the setting of thymic insufficiency or thymectomy, and hence for its applicability to the induction of xenograft tolerance and in the treatment of immunodeficiency diseases.     

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-gamma is essential for tumor rejection, we isolated live T cells based on their IFN-gamma production. IFN-gamma secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-gamma+ but not IFN-gamma- T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-gamma capture assay.     

The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.     

The potential of human regulatory T cells generated ex vivo as a treatment for lupus and other chronic inflammatory diseases

Regulatory T cells prevent autoimmunity by suppressing the reactivity of potentially aggressive self-reactive T cells. Contact-dependent CD4+ CD25+ 'professional' suppressor cells and other cytokine-producing CD4+ and CD8+ T-cell subsets mediate this protective function. Evidence will be reviewed that T cells primed with transforming growth factor (TGF)-beta expand rapidly following restimulation. Certain CD4+ T cells become contact-dependent suppressor cells and other CD4+ and CD8+ cells become cytokine-producing regulatory cells. This effect is dependent upon a sufficient amount of IL-2 in the microenvironment to overcome the suppressive effects of TGF-beta. The adoptive transfer of these suppressor cells generated ex vivo can protect mice from developing chronic graft-versus-host disease with a lupus-like syndrome and alter the course of established disease. These data suggest that autologous T cells primed and expanded with TGF-beta have the potential to be used as a therapy for patients with systemic lupus erythematosus and other chronic inflammatory diseases. This novel adoptive immunotherapy also has the potential to prevent the rejection of allogeneic transplants.     

Human TNF can induce nonspecific inflammatory and human immune-mediated microvascular injury of pig skin xenografts in immunodeficient mouse hosts

TNF activates endothelial cells to express cell surface molecules that are necessary to recruit a local infiltrate of leukocytes. Because the actions of this proinflammatory cytokine are not species restricted, we investigated whether human TNF can up-regulate porcine endothelial adhesion molecules to elicit human T cell infiltration and damage of pig skin xenografts in a chimeric immunodeficient mouse model. We have previously demonstrated the vigorous rejection of human skin allografts and the absence of injury to porcine skin xenografts in human PBMC-SCID/beige mice. Intradermal administration of human TNF at high doses (600 or 2000 ng) caused nonspecific inflammatory damage of pig skin grafts, whereas low concentrations of TNF (60 or 200 ng) resulted in human PBMC-dependent injury of porcine endothelial cells. There was a strong correlation among pig skin xenograft damage, human T cell infiltration, and the TNF-induced up-regulation of swine MHC class I and class II molecules, VCAM-1, and, in particular, the de novo expression of porcine E-selectin. The microvascular damage and leukocytic infiltration elicited by TNF were enhanced by porcine IFN-gamma, suggesting that xenografts may be less prone to cytokine-mediated injury due to the species-restricted effects of recipient IFN-gamma. Our results indicate that maintenance of a quiescent endothelium, which does not express E-selectin or other activation-dependent adhesion molecules, is important in preventing human anti-porcine T cell xenoresponses in vivo and that TNF signaling molecules and TNF-responsive gene products are appropriate therapeutic targets to protect against human T cell-mediated rejection of pig xenografts.     

Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells

In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.     

Human effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury in vivo.     

Both infiltrating regulatory T cells and insufficient antigen presentation are involved in long-term cardiac xenograft survival

We have previously shown that pretransplant donor lymphocyte infusion (DLI) together with transient depletion of CD4(+) T cells could induce permanent rat-to-mouse heart graft survival, whereas depleting CD4(+) T cells alone failed to do so. In this study, we investigated the mechanism leading to long-term xenograft survival. We found that peripheral CD4(+) T cells from DLI/anti-CD4-treated mice could mount rat heart graft rejection after adoptive transfer into B6 CD4(-/-) mice. Infusing donor-Ag-loaded mature dendritic cells (DCs) could break long-term cardiac xenograft survival in DLI/anti-CD4-treated mice. Interestingly, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, we observed a significant increase in both the number and suppressive activity of alphabeta-TCR(+)CD3(+)CD4(-)CD8(-) double negative regulatory T cells and decrease in the numbers of CD4(+) and CD8(+) T cells in the xenografts of DLI/anti-CD4-treated mice. Moreover, there was a significant reduction in MHC class II-high DCs within the xenografts of DLI/anti-CD4-treated recipients. DCs isolated from the xenografts of anti-CD4- but not DLI/anti-CD4-treated recipients could stimulate CD4(+) T cell proliferation. Our data indicate that functional anti-donor T cells are present in the secondary lymphoid organs of the mice that permanently accepted cardiac xenografts. Their failure to reject xenografts is associated with an increase in double negative regulatory T cells as well as a reduction in Ag stimulation by DCs found within grafts. These findings suggest that local regulatory mechanisms need to be taken into account to control anti-xenograft T cell responses.     

CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses.

Specific and selective immunological unresponsiveness to donor alloantigens can be induced in vivo. We have shown previously that CD25+CD4+ T cells from mice exhibiting long-term operational tolerance to donor alloantigens can regulate rejection of allogeneic skin grafts mediated by CD45RB(high)CD4+ T cells. In this study, we wished to determine whether donor-specific regulatory cells can be generated during the induction phase of unresponsiveness, i.e., before transplantation. We provide evidence that pretreatment with anti-CD4 Ab plus a donor-specific transfusion generates donor-specific regulatory CD25+CD4+ T cells that can suppress rejection of skin grafts mediated by naive CD45RB(high)CD4+ T cells. Regulatory cells were contained only in the CD25+ fraction, as equivalent numbers of CD25-CD4+ T cells were unable to regulate rejection. This pretreatment strategy led to increased expression of CD122 by the CD25+CD4+ T cells. Blockade of both the IL-10 and CTLA-4 pathways abrogated immunoregulation mediated by CD25+ T cells, suggesting that IL-10 and CTLA-4 are required for the functional activity of this population of immunoregulatory T cells. In clinical transplantation, the generation of regulatory T cells that could provide dynamic control of rejection responses is a possible route to permanent graft survival without the need for long-term immunosuppression.     

Rejection of skin allografts by CD4+ T cells is antigen-specific and requires expression of target alloantigen on Ia- epidermal cells

The effector mechanism of skin allograft rejection has been characterized as Ag specific, rejecting cells that express the target alloantigen but sparing those that do not. However, the rejection of MHC class II disparate skin grafts, in which very few cells (Langerhans cells) actually express the target Ia Ag could conceivably proceed by either one of two distinct rejection mechanisms. One possibility is that Ia- cells are destroyed by a sequence of events in which CD4+ T cells, activated by Ia+ LC, elaborate soluble factors that are either directly cytolytic or that recruit and activate non-specific effector cells. The alternative possibility is that activated CD4+ T cells elaborate soluble factors which induce Ia expression on Ia- cell populations, and that these Ia+ cells are subsequently destroyed by effector cells specific for the induced Ia alloantigens. We found that rejection of Ia+ LC was not of itself sufficient to cause rejection of skin grafts, indicating that skin allograft rejection is contingent on the destruction not only of LC but of other graft cell populations as well. We then investigated whether CD4+ T cells rejected allogeneic skin grafts in an antigen specific fashion. To do so, we engrafted immunoincompetent H-2b nude mice with trunk skin grafts from B6----A/J allophenic mice because such skin is composed of mutually exclusive cell populations expressing either H-2a or H-2b histocompatibility Ag, but not both. The engrafted mice were subsequently reconstituted with H-2b CD4+ T cells. The CD4+ T cells destroyed keratinocytes of A/J origin but spared keratinocytes of B6 origin, even though neither cell population constitutively expresses target IAk alloantigen. The targeted rejection of A/J keratinocytes but not of B6 keratinocytes indicates that the target Ia alloantigen must have been induced on Ia- A/J keratinocytes, rendering them susceptible to destruction by anti-Iak-specific CD4+ effector cells. These data demonstrate that CD4+ T cell rejection of skin allografts is mediated by Ag-specific CD4+ cytolytic T cells and hence, requires the induction of target Ia alloantigens on epidermal cells within the graft.