Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

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.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

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.     

'Chimeric' Grafts Assembled from Multiple Allodisparate Donors Enjoy Enhanced Transplant Survival

Certain components of a graft that provoke alloimmunity may not be vital for graft function or critical as targets of rejection. Corneal transplantation is an example of this, because graft epithelium plays a role in allosensitization, whereas corneal graft endothelium—which shares the same alloantigens—is the critical target in allorejection. In this study, we found that exploiting this biology by replacing donor epithelium of an allograft with an allodisparate third-party epithelium yields a marked enhancement in transplant survival. Such 'chimeric' allografts consisted of a C3H/He (H-2^k) corneal epithelium over a C57BL/6 (H-2^b) epithelial-denuded cornea (or v.v. ) and orthotopically placed on BALB/c (H-2^d) hosts. Conventional corneal allografts (C3H/He or C57BL/6) or isografts (BALB/c) were also transplanted on BALB/c hosts. Alloreactive T-cell frequencies (CD4⁺ interferon [IFN]-γ⁺) primed to the graft endothelium were strongly diminished in chimeric hosts relative to conventionally allografted hosts. This was corroborated by a decreased T-cell infiltration (p = 0.03) and a marked enhancement of allograft survival (p = 0.001). Our results represent the first successful demonstration of chimeric tissue, epithelial-denuded allograft plus third-party allodisparate epithelium, in the promotion of allograft survival. Moreover, chimeric grafting can be readily performed clinically, whereby corneal allograft rejection remains a significant problem particularly in inflamed graft beds.     

Indirect CD4+ TH1 Response, Antidonor Antibodies and Diffuse C4d Graft Deposits in Long-Term Recipients Conditioned by Donor Antigens Priming

Priming of recipients by DST induces long-term survival of mismatched allografts in adult rats. Despite these recipients developing inducible T regulatory cells able to transfer long-term graft survival to a secondary host, a state of chronic rejection is also observed. We revisited the molecular donor MHC targets of the cellular response in acute rejection and analyzed the cellular and humoral responses in recipients with long-term graft survival following transplantation. We found three immunodominant peptides, all derived from LEW.1W RT1.D^u molecules to be involved in acute rejection of grafts from unmodified LEW.1A recipients. Although the direct pathway of allorecognition was reduced in DST-treated recipients, the early CD4⁺ indirect pathway response to dominant peptides was almost unimpaired. We also detected early and sustained antidonor class I and II antibody subtypes with diffuse C4d deposits on graft vessels. Finally, long-term accepted grafts displayed leukocyte infiltration, endarteritis and fibrosis, which evolved toward vascular narrowing at day 100. Altogether, these data suggest that the chronic graft lesions developed in long-term graft recipients are the result of progressive humoral injury associated with a persisting indirect T helper response. These features may represent a useful model for understanding and manipulating chronic active antibody-mediated rejection in human.     

Functionally and phenotypically mature mouse CD8+ T cells develop in porcine thymus grafts in mice

Abstract: Mouse CD4⁺ T cells efficiently develop in fetal pig thymus (FPTHY) grafts and repopulate the periphery of T cell and NK cell-depleted, thymectomized (ATX) mice. However, efficient peripheral repopulation of mouse CD8⁺ T cells does not occur in these mice. We have therefore evaluated the maturation and function of mouse CD8 single positive (SP) thymocytes in fetal pig thymus and liver fragment (FP THY LIV) grafts. Phenotypic maturity, as measured by upregulated expression of TCR, class I MHC, and Qa-2, and downregulated expression of heat stable antigen (HSA) on CD8 SP cells in FP THY grafts, was similar to that in host thymi of euthymic control mice. Cytolytic T lymphocyte (CTL) activity of thymocytes from FP THY grafts was similar to that of thymocytes from host thymi of euthymic mice, indicating that functional maturation of CD8 SP cells had taken place in the grafts. Furthermore, similarly efficient deletion of Vβ5.1/5.2⁺ and Vβ11⁺ CD8 SP cells was observed in FP THY grafts as in host thymi of euthymic control mice. Similar percentages of Vβ6, Vβ7, and Vβ8.1/8.2 expressing cells were also detected among CD8 SP cells in FP THY grafts and host thymi of euthyrnic controls. Together, our results suggest that normal positive and negative selection occurs, and that mouse CD8⁺ cells can undergo normal functional and phenotypic maturation in FPTHY grafts. Thus, other explanations must be sought for the failure of CD8'cells to repopulate the peripheral lymphoid tissues of ATX, T cell-depleted, pig THYLIV-grafted mice.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.     

Successive emergence of two CD 8 subsets in primary CMV infection of allograft recipients

Abstract Allograft recipients with cytomegalovirus (CMV) infection develop increased proportions of circulating CD8 lymphocytes. A longitudinal study of 11 kidney and 5 liver allograft recipients with primary CMV infection but no other aetiological factor to explain graft dysfunction revealed selective imbalances in peripheral blood CD8' T cell subsets. Initially, CMV viraemia was associated with elevated CD8+bright' T cell numbers and T cell activation. Activation markers fell to normal when viral cultures became negative (before the end of the 1st month). During the 2nd-6th months, most (12/16) patients continued to have high CD8⁺ T cell counts (1050–2900 CD8⁺ cells/mm3), comprising an uncommon CD8⁺ T cell subset, as 45–73% of CD8^{+ bright} lymphocytes were CD3⁺ and TCRαβ⁺ but were not stained by anti-CD28, CD11b, CD16, CD56 and CD57 antibody. Unexpectedly, CD 8⁺ CD 57⁺ T cells, a hallmark of CMV infection, did not appear until the 2nd-6th months of primary CMV infection, and their numbers increased progressively thereafter. They became the predominant CD8⁺ T cell subset after about 6 months of infection and their persistence for several (up to 4) years was strongly correlated ( r = 0.87) with expansion of CD8⁺ cells. Persistence of CD 8 lymphocytosis was, thus, directly related to the rate of expansion of an uncommon CD 8⁺CD 57^- subset and its progressive replacement by CD 8⁺CD 57 ⁺ T cells that were chronically elicited by CMV.     

Migration of host and donor T cells in small bowel transplantation

Abstract In this study migration of host and donor CD4⁺ andCD8⁺ T cells in a fully allogeneic model was described and compared with the migration pattern in a graft-versus-host reaction (GVHR) model, where the T-cell traffic in the graft served as a physiological control. Heterotopic small bowel transplantations were performed in a rat model, with animals being sacrificed on postoperative days (POD) 2, 3, 4, 5, and 7. Graft and host mesenteric lymph nodes were harvested, homogenized, and stained with monoclonal antibodies against MHC class I, CD4 ⁺, and CD8 ⁺ antigens. The host and donor T cell migration patterns were studied using a double-staining flow cytometric technique. We found that during the development of rejection, the normal physiological circulation of graft and host T cells was disrupted. In the graft of the allogeneic model, a shift from host cell to graft cell dominance occurred on POD 3–4. This change in migration pattern coincided in the host with a 6 % peak in graft cell infiltration, which disappeared on POD 7. These patterns of T-cell migration may be further explored for diagnostic purposes.     

Development of autoimmunity after skin graft rejection via an indirect alloresponse

BACKGROUND: T cell allorecognition occurs through direct contact with donor peptide: MHC complexes on graft cells and through indirect recognition of donor-derived determinants expressed by recipient MHC molecules. As both indirect allorecognition and autoantigen recognition are self-restricted, we hypothesized that chronic activation of indirectly primed T cells might result in determinant spreading to involve autoantigens, analogous to that which occurs during chronic autoimmune diseases. METHODS: We placed C57BL/6 MHC II knockout (B6 II-/-) skin grafts onto BALB/c SCID mice reconstituted with wild-type (WT) CD4+ T cells. Under these conditions the CD4+ cells could not recognize any antigen on the graft, but could respond through the indirect pathway. CD4+ cell-mediated rejection of WT B6 skin was studied to determine if autoreactivity was induced after direct allorecognition. Recall immune responses against donor- and self-stimulator cells were determined by ELISPOT and animals were tested for their ability to reject second isografts. RESULTS: WT allografts were rejected by day 14 although B6 II-/- grafts underwent delayed rejection over 4-5 weeks. CD4+ cells reisolated from the recipients of the MHC II-/- grafts, but not from the recipients of WT grafts, vigorously produced interferon-gamma and interleukin-2 in response to self, BALB/c stimulators. These autoreactive CD4+ T cells mediated rejection of a second isogenic BALB/c skin graft, demonstrating that the autoimmune response was pathogenic. CONCLUSION: Autoreactivity can develop after transplant rejection via the indirect pathway. Although the direct alloresponse is likely to be the driving force in acute graft rejection, posttransplantation induced autoimmune responses may be important elements of delayed or chronic rejection.     

Infusion of Mesenchymal Stem Cells and Rapamycin Synergize to Attenuate Alloimmune Responses and Promote Cardiac Allograft Tolerance

The inherent immunosuppressive properties and low immunogenicity of mesenchymal stems cells (MSCs) suggested their therapeutic potential in transplantation. We investigated whether MSCs could prolong allograft survival. Treatment involving infusion of MSCs into BALB/c recipients 24 hours after receiving a heart allograft from a C57BL/6 donor significantly abated rejection and doubled graft mean survival time compared to untreated recipients. Furthermore, combination therapy of MSCs and low-dose Rapamycin (Rapa) achieved long-term heart graft survival (>100 days) with normal histology. The treated recipients readily accepted donor skin grafts but rejected third-party skin grafts, indicating the establishment of tolerance. Tolerant recipients exhibited neither intragraft nor circulating antidonor antibodies, but demonstrated significantly high frequencies of both tolerogenic dendritic cells (Tol-DCs) and CD4⁺CD25⁺Foxp3⁺T cells in the spleens. Infusion of GFP⁺C57BL/6-MSCs in combination with Rapa revealed that the GFP-MSCs accumulated in the lymphoid organs and grafts of tolerant recipients. Thus, engraftment of infused MSCs within the recipient's lymphoid organs and allograft appeared to be instrumental in the induction of allograft-specific tolerance when administered in combination with a subtherapeutic dose of Rapamycin. This study supports the clinical applicability of MSCs in transplantation.     

Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.     

Primed CD8+ T-Cell Responses to Allogeneic Endothelial Cells Are Controlled by Local Complement Activation

CD8 T cells primed by transplantation recognize allogeneic class I MHC molecules expressed on graft vascular endothelium and contribute to allograft injury. We previously showed that immune cell-derived complement activation fragments are integral to T cell activation/expansion. Herein we tested the impact of local complement production/activation on T cell/endothelial cell (EC) interactions. We found that proinflammatory cytokines upregulated alternative pathway complement production by ECs, yielding C5a. We further found that ECs deficient in the cell surface C3/C5 convertase regulator decay accelerating factor (DAF, CD55) induced greater CD8 T-cell proliferation and more IFNγ⁺ and perforin⁺ effector cells than wild-type (WT) ECs. Allogeneic C3^−/− EC induced little or no CD8 responses. Abrogation of responses following C5a receptor (C5aR) blockade, or augmentation following addition of recombinant C5a demonstrated that the effects were mediated through T-cell-expressed-C5aR interactions. Analyses of in vivo CD8 cell responses to transplanted heart grafts deficient in EC DAF showed similar augmentation. The findings reveal that EC-derived complement triggers secondary CD8 T-cell differentiation and expansion and argue that targeting complement and/or C5aR could limit T-cell-mediated graft injury.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

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.