The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4(+) T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4(+) T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4(+) T cells. METHODS: Transgenic RAG2(-/-) mice expressing I-A(b)-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2(k) male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2(k) male allograft and presented by the recipient's own I-A(b) APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-A MHC class II molecules. This observation suggested that graft endothelium replacement by I-A(b)-positive cells of recipient origin could stimulate the rejection of male H-2(k) graft by I-A(b)--restricted H-Y--specific T cells. To investigate further this possibility, hearts from H-2(b)--into--H-2(k) irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-A(b)-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2(k) male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4(+) T cells.     

The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4+ T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4+ T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4+ T cells. METHODS: Transgenic RAG2-/- mice expressing I-Ab-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2k male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2k male allograft and presented by the recipient's own I-Ab APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-Ab MHC class II molecules. This observation suggested that graft endothelium replacement by I-Ab-positive cells of recipient origin could stimulate the rejection of male H-2k graft by I-Ab-restricted H-Y-specific T cells. To investigate further this possibility, hearts from H-2b-into-H-2k irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-Ab-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2k male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4+ T cells.     

A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody

Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1^?/? or B6.RAG1^?/?C3^?/? (H‐2^b) mice received B10.BR (H‐2^k) heart allografts and repeated doses of IgG2a, IgG1 or F(ab’)₂ fragments of IgG2a DSA (anti‐H‐2^k). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti‐NK1.1 reduced significantly DSA‐induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ‐chain knock out) also showed decreased severity of DSA‐induced CAV. Direct NK reactivity to the graft was not necessary. F(ab’)₂ DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho‐extracellular signal‐regulated kinase (pERK), a response not elicited by F(ab’)₂ DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d‐negative chronic antibody‐mediated rejection in humans.     

Islet allograft rejection by contact-dependent CD8+ T cells: perforin and FasL play alternate but obligatory roles.

Though CD8(+) T lymphocytes are important cellular mediators of islet allograft rejection, their molecular mechanism of rejection remains unidentified. Surprisingly, while it is generally assumed that CD8(+) T cells require classic cytotoxic mechanisms to kill grafts in vivo, neither perforin nor FasL (CD95L) are required for acute islet allograft rejection. Thus, it is unclear whether such contact-dependent cytotoxic pathways play an essential role in islet rejection. Moreover, both perforin and CD95L have been implicated in playing roles in peripheral tolerance, further obscuring the role of these effector pathways in rejection. Therefore, we determined whether perforin and/or FasL (CD95L) were required by donor MHC-restricted ('direct') CD8(+) T cells to reject islet allografts in vivo. Islet allograft rejection by primed, alloreactive CD8(+) T cells was examined independently of other lymphocyte subpopulations via adoptive transfer studies. Individual disruption of T-cell-derived perforin or allograft Fas expression had limited impact on graft rejection. However, simultaneous disruption of both pathways prevented allograft rejection in most recipients despite the chronic persistence of transferred T cells at the graft site. Thus, while there are clearly multiple cellular pathways of allograft rejection, perforin and FasL comprise alternate and necessary routes of acute CD8(+) T-cell-mediated islet allograft rejection.     

CD4+ T cells are critical for corneal, but not skin, allograft rejection

BACKGROUND: The relative contribution of CD4+ or CD8+ T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. METHODS: We compared the ability of CD4- knockout (KO), CD8- KO, and normal CD4+/CD8+ mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. RESULTS: Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8- KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 +/- 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 +/- 1 days), although most CD4- KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4- KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4- KO mice that accepted such grafts or that were not grafted. CONCLUSIONS: This study indicates that mice lacking CD4+ T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4+ T cells, the likely mechanism for rejection appears to involve the generation of CD8+ CTLs.     

Mediation of skin allograft rejection in scid mice by CD4+ and CD8+ T cells.

We analyzed the role of CD4+ and CD8+ T cells in H-2-disparate skin allograft rejection in the mutant mouse strain C.B-17/Icr scid with severe combined immunodeficiency. On the day of skin allografting, scid mice were adoptively transferred with negatively selected CD4+ or CD8+ splenocytes from normal unsensitized C.B-17/Icr mice. These populations were obtained using a double-mAb--plus--complement elimination protocol using anti-CD4 or anti-CD8 mAb that resulted in no detectable CD4+ or CD8+ cells by FACS and negligible numbers of cytolytic T lymphocytes by limiting dilution analysis in anti-CD8 treated populations. Spleen cells were removed from grafted mice at the time of rejection and were tested in vitro for antidonor reactivity in several assays: mixed lymphocyte culture, cell-mediated lympholysis, and LDA for CTL and for IL-2-producing HTL. The presence of Thy 1.2+, CD4+, or CD8+ cells was determined by FACS. All control C.B-17 mice and scid mice adoptively transferred with nondepleted CD4+, and CD8+ cells rejected skin allografts with similar mean survival times (15.6 +/- 1.5, 18.8 +/- 3.4, 18.0 +/- 5.4, respectively), whereas control scid mice retain skin allografts indefinitely (all greater than 100 days). C.B-17 syngeneic grafts survived indefinitely in all groups. At the time of rejection, splenocytes from scid mice receiving CD4+ cells had negligible donor-specific cytotoxicity in CML and negligible numbers of CTL by LDA, but demonstrated a good proliferative response in MLC and IL-2-producing cells by LDA (frequency = 1/1764). There were no detectable CD8+ cells present by FACS analysis. Conversely, splenocytes from scid mice adoptively transferred with CD8+ cells had strong donor-specific cytotoxicity in CML (58.8% +/- 16.1%) and CTL by LDA (frequency = 1/3448), but no significant proliferation was detected in MLC. There were no detectable CD4+ cells by FACS, but there were small numbers of IL-2-producing cells by LDA (frequency = 1/10,204). These data demonstrate that CD4+ cells adoptively transferred into scid mice are capable of mediating skin allograft rejection in the absence of any detectable CD8+ cells or significant functional cytolytic activity. The adoptive transfer of CD8+ cells also results in skin allograft rejection in the absence of detectable CD4+ cells. The detection of small numbers of IL-2 secreting cells in these mice may indicate that CD(8+)-mediated allograft rejection in this model is dependent on IL-2-secreting CD8+ cells.     

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4+ memory T cells in mice

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4⁺ Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5 days to 9 days in recipient mice with CD4⁺ Tm infusion (P < 0.01), which however was not significantly longer than that (8 days) in untreated recipient mice without CD4⁺ Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4⁺ Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4⁺ Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44^highCD62L⁻ effector/memory as well as CD69⁺/CD25⁺ activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4⁺ Tm, for serum level of IFN-γ and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4⁺ Tm cells responding to the alloantigen, indicating that alloreactive CD4⁺ Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4⁺ Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.     

CD4+ T-cell receptor transgenic T cells alone can reject vascularized heart transplants through the indirect pathway of alloantigen recognition

The vast array of epitopes presented by allografts and the diversity of T cells responding to them complicates mechanistic studies of rejection. To minimize these problems, we developed a transgenic (Tg) model system limited to a single T-cell receptor (TCR)/peptide/major histocompatibility complex molecule. Two alloantigen-specific CD4 T-cell clones were used to isolate cDNA encoding the TCRalpha and TCRbeta chains that recognize the Kd54-68/I-Ab epitope. Two different TCR Tg lines were produced in C57BL/6 (B6) mice and crossed onto the B6.Rag1-/- background. B6.Rag1-/- recipients of T cells from TCR Tg Rag1-/-mice promptly rejected B10.D2, but not irrelevant B10.BR, cardiac grafts. Thus, a single allogeneic epitope presented by self-major histocompatibility complex class II is sufficient to activate TCR Tg T cells and serve as a target for rejection.     

Targeting acute allograft rejection by immunotherapy with ex vivo-expanded natural CD4+ CD25+ regulatory T cells

BACKGROUND: Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice. METHODS: Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry. RESULTS: By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection. CONCLUSIONS: Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.     

Suppression mediated by anergic CD4+ T cells requires stimulation by MHC-peptide complexes and can be induced in the presence of costimulation

BACKGROUND: In this study, we have investigated the mechanisms involved in both the induction of suppressive anergy, the stability of the anergy induced, and the possible mechanisms by which the response of immunocompetent CD4+ T cells are suppressed. METHODS: We used immobilized anti-CD3 monoclonal antibody (mAb) to induce anergy in T helper (Th) 1 and Th0 cells reactive with MHC class II molecule H2 I-Ab. RESULTS: We observed that suppressive anergy was induced independently of costimulation in Th0 but not Th1 cells. Although the anergic and suppressive states of Th0 cells were stable in the presence of exogenous interleukin-2, this was not the case for Th1 cells. No evidence for linked epitope suppression was observed for any of the I-Ab reactive cells investigated. Neither anergy nor suppression was observed in Th0 cells upon restimulation with anti-CD3 in the presence of syngeneic antigen-presenting cells (APCs). However, anergy but not suppression was observed in co-cultures restimulated with anti-T-cell antigen receptor (TCR) mAbs/syngeneic APCs and suppression could be restored by the addition of I-Ab+ APCs. CONCLUSIONS: Overall, these data suggested that the MHC-peptide complex recognized by the Th0 cells was required for suppression of the response of immunocompetent cells. We propose that suppression is mediated either by down-modulation of the MHC-peptide complex recognized by the anergic T cells or that a molecule specific to the MHC-peptide/TCR interaction facilitates negative regulation by APC:T or T:T interactions.     

CD4+ T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferon-gamma

BACKGROUND: T cells responding through the indirect pathway can induce allograft rejection, but mechanisms of rejection are not known. Interferon-y (IFN-gamma) may be an important mediator of rejection under these circumstances. METHODS: We transferred CD4+ T cells from IFN-gamma-deficient (IFN-gamma-/-) mice into SCID recipients of MHC II-deficient (MHC II-/-) skin grafts. Under these conditions, rejection can only occur via the indirect pathway and cannot be mediated by T-cell production of IFN-gamma. RESULTS: IFN-gamma-/- CD4+ T cells rejected MHC II -/- skin grafts. Flow cytometry revealed only CD4+ T cells in the recipients. Cytokine enzyme-linked immunosorbent spot assays confirmed only indirect recognition with an associated expansion of an alloreactive population of IL-2-, IL-4-, and IL-5-secreting T cells. CONCLUSION: CD4+ T cells recognizing alloantigens via the indirect pathway can mediate skin graft rejection in the absence of IFN-gamma.     

Alloantigen-specific CD4+ regulatory T cells induced in vivo by ultraviolet irradiation after alloantigen immunization require interleukin-10 for their induction and activation,and flexibly mediate bystander immunosuppression of allograft rejection

Ultraviolet (UV) irradiation prior to antigen immunization is employed to induce antigen-specific regulatory T cells (Tregs). UV-induced Tregs demonstrate unique bystander suppression, although antigen-specific activation is required initially. We previously reported the phenotype of alloantigen-specific transferable Tregs induced by UV-B irradiation after immunization was the same as T regulatory type 1-like CD4⁺ T cells, with antigen-specific interleukin (IL)-10 production. Here, by using semi-allogeneic transplantation models in vivo, we investigated the role of IL-10 in the induction and activation of these Tregs, and the possibility of bystander suppression of third-party allograft rejection. Naïve mice (H-2^b) were immunized with alloantigen (H-2^b/d), and received UV-B irradiation (40 kJ/m²) 1 week later. Four weeks afterwards, splenic CD4⁺ T cells were purified from the UV-irradiated immunized mice, and were transferred into naïve mice (H-2^b). Allografts expressing the same alloantigen as T-cell donors were immunized against (H-2^b/d) or an irrelevant alloantigen (H-2^b/k) were transplanted to CD4⁺ T-cell-transferred mice, and an alloantigen-specific prolongation of allograft survival observed. Experiments where IL-10 was neutralized by monoclonal antibody in the induction or effector phase revealed that IL-10 is critical, not only for induction but also for immunosuppressive function of CD4⁺ Tregs induced by UV irradiation after alloantigen immunization. Third-party allografts (H-2^d/k) were transplanted to CD4⁺ T-cell-transferred mice, and graft survival was also prolonged. Even a graft only partially compatible with immunized alloantigen worked well in vivo to activate CD4⁺ Tregs induced by UV irradiation after alloantigen immunization, which resulted in the bystander suppression of third-party allograft rejection.     

Elimination of self-reactive CD8+, but not CD4+, T cells by a peripheral immune mechanism

Unirradiated (BALB/c X B6)F1 recipients of lymphocytes from either parent or (B6 X DBA/2)F2 recipients of DBA/2 parental lymphocytes specifically remove the function of donor-derived F1-reactive CTL from the spleen, since such cells could not be recovered 1 week after injection. However, donor-derived CTL specific for third-party antigens, as well as donor-derived F1-reactive CD4+ T cells could be recovered. In contrast, CTL in spleens from recipients sublethally irradiated prior to injections consisted predominantly of F1-reactive CTL in all strain combinations tested. Athymic BALB/c nude mice grafted with fetal thymus of B6 develop a T cell compartment tolerant of BALB/c and B6, like (BALB/c X B6)F1 animals. However, unlike the F1 mice, the thymus-grafted nude mice were not able to eliminate B6-reactive lymphocytes after injection of normal BALB/c spleen cells. Our data indicate the existence of a peripheral immune mechanism capable of selectively eliminating self-reactive CD8+ CTL, but not CD4+, T cells. This mechanism requires self antigen expressed on radiosensitive cells. The presence of T cells tolerant to self antigen by thymic negative selection is not sufficient and perhaps not required. Most likely, this mechanism is involved in the relative resistance to lethal GVHR mediated by parental CD8+ T cells in parent-into-F1 situations.     

Role of CD4+ and CD8+ T cells in early and late acute rejection of small bowel allograft

BACKGROUND/PURPOSE: Results of small bowel transplantation remain unsatisfactory because of severe immune rejection. The current study aims to elucidate the role of activation of CD4+ and CD8+ T cells in early and late acute rejection of small bowel allograft and, hence, provide the immunologic basis for developing new therapeutic strategies. METHODS: We used an MHC fully mismatched (DA to Lewis) heterotopic rat small bowel transplant model and a unique FK506-based immunosuppressive regimen, which suppresses early acute rejection but does not prevent late acute rejection. Flow cytometric analysis was used to quantitate the number of activated CD4+ and CD8+ T cells in graft and host mesenteric lymph nodes. RESULTS: The survival (mean +/- SD) of intestinal allograft was significantly prolonged, from 6.6 +/- 0.84 days for the untreated group to 40.7 +/- 14.1 days for the FK506-treated group. Activation of CD4+ cells was suppressed significantly in the FK506-treated group on postoperative day 7 compared with the untreated group (29.4% +/- 3.55% v 52.83% +/- 11.9%; P <.01). Activation of CD8+ cells was similarly suppressed (31.5 +/- 10.34% v 48.53 +/- 14.34%; P <.05). Interestingly, at late acute rejection, activated CD4+ and CD8+ T cells remained at almost the same low levels as those on postoperative day 7 in the FK506-treated group. The spleen to body weight ratio was significantly increased in the untreated group (0.53 +/- 0.07), and slightly increased in the FK treated group (0.27 +/- 0.07, on postoperative day 7; 0.24 +/- 0.07 at late acute rejection) compared with the syngeneic group (0.18 +/- 0.02). CONCLUSION: The activation of CD4+ and CD8+ T cells was suppressed effectively by early potent immunosuppressive treatment resulting in prolonged survival of intestinal allograft. At late acute rejection, the CD4+ and CD8+ T cells remained at low-level activation status, in contrast to the surge of CD4+ and CD8+ activation during early acute rejection. This suggests that persistent T cell activation even at low level is sufficient to cause the late acute rejection eventually. A therapeutic strategy targeting these cells is needed for long-term engraftment.     

The role of CD8+ and CD4+ cells in islet allograft rejection

The requirements of CD8+ and CD4+ cells for islet graft rejection in combinations with different histoincompatibilities were investigated by in vivo administration of anti-Lyt-2.2 (CD8) mAb, anti-L3T4 (CD4) mAb, or both to recipient mice. In B10.AQR----B10.A (H-2K-incompatible) and B10.A(5R)----B10.A (H-2K- and IA-incompatible) combinations, administration of either anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb completely blocked islet graft rejection, indicating that neither CD8+ cells nor CD4+ cells alone were capable of mediating rejection, and that collaboration of CD8+ cells and CD4+ cells was necessary. On the other hand, in the BALB/c----B6 (H-2- and non-H-2-incompatible) combination, administration of anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb resulted in rejection of most of the grafts, although survival was prolonged significantly, and administration of both anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb together completely blocked rejection. These results suggested that either CD8+ or CD4+ cells were capable of mediating rejection, but that rejection was maximal in the presence of both T cell subsets. Immunohistochemical analyses showed marked depletion of CD8+ cells and CD4+ cells in grafted islets as well as spleens when anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb, respectively, were injected.     

Renal allograft rejection in CD4+ T cell-reconstituted athymic nude rats. The origin of CD4+ and CD8+ graft-infiltrating cells

PVG-rnu/rnu nude rats reject a fully allogeneic DA renal allograft after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many CD8+ leukocytes within the graft. In order to clearly establish the provenance of these CD8+ cells infiltrating rejecting kidney allografts, nude recipients (PVG-RT7a) were injected with CD4+ T cells from the PVG-RT7b congenic strain bearing an allotypic variant of the leukocyte-common antigen. Dual fluorescence and immunohistochemistry demonstrated that approximately 75% of the total infiltrate was host-derived; the donor-derived RT7b population was almost entirely (92-99%) CD4+, CD5+, CD3+, and alpha beta TCR+. At least 97% of the CD8+ cells were of nude origin. There was no evidence of donor-derived CD8+ cells or of a CD4+8+ double-staining population. Unexpectedly, nearly half of the alpha beta TCR+ cells from the grafts were of nude origin.     

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.