Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.     

Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival

BACKGROUND: Failure of costimulatory molecule-deficient donor dendritic cells (DCs) to induce indefinite allograft acceptance may be a result of the 'late" up-regulation of these molecules on the DCs after interaction with host T cells. Ligation of CD40 on antigen-presenting cells by its cognate ligand CD40L is thought to induce expression of CD80 (B7-1) and CD86 (B7-2). We examined the influence of anti-CD40L monoclonal antibody (mAb) on the capacity of donor-derived DC progenitors to induce long-term allograft survival. METHODS: High purity DC progenitors were grown from B10 (H2b) mouse bone marrow in granulocyte-macrophage colony-stimulating factor and transforming growth factor beta1 (TGFbeta1). Mature DC were propagated in granulocyte-macrophage colony-stimulating factor and interleukin-4. Their phenotype was characterized by flow cytometric analysis and their function by mixed leukocyte reactivity. Anti-donor cytotoxic T lymphocyte activity in grafts and spleens of vascularized heart allograft recipients was also assessed. RESULTS: The TGFbeta3-cultured cells were (1) DEC 205-positive, MHC class II-positive, CD80dim, CD86dim, and CD40dim, (2) poor stimulators of naive allogeneic T-cell proliferation, and (3) able to prolong significantly B10 cardiac allograft survival in C3H (H2k) recipients when given (2 x 10[6] i.v.) 7 days before organ transplantation (median survival time [MST] 26 days vs. 12 days in controls, and 5 days in interleukin-4 DC-treated animals). Their allostimulatory activity was further diminished by addition of anti-CD40L mAb at the start of the mixed leukocyte cultures. Anti-CD40L mAb alone (250 microg/mouse, i.p.; day -7) did not prolong cardiac graft survival (MST 12 days). In contrast, TGFbeta-cultured DCs + anti-CD40L mAb extended graft survival to a MST of 77 days, and inhibited substantially the anti-donor cytotoxic T lymphocyte activity of graft-infiltrating cells and host spleen cells assessed 8 days after transplant. CONCLUSIONS: The CD40-CD40L pathway appears important in regulation of allogeneic DC-T-cell functional interaction in vivo; its blockade increases markedly the potential of costimulatory molecule-deficient DCs of donor origin to induce long-lasting allograft survival.     

Assessment of donor cell and matrix survival in fresh articular cartilage allografts in a goat model

The long-term survival of allografts of articular cartilage has been proposed to be dependent on the survival of the cells that maintain the unique structural and material properties of the allograft. In this study, we assessed cell survival in 24 fresh articular cartilage allografts of the medial plateau in a Spanish-goat model. A DNA-probe technique was used to distinguish clearly between DNA from donor (allograft) and host cells. The intraarticular survival of viable allograft chondrocytes in the transplanted articular cartilage started to diminish as early as 3 weeks after transplantation; however, there was considerable variation in the amount of donor cell DNA detected in the allografts at 6 and 12 months following transplantation. This contrasts with our experience with fresh allografts of ligament, tendon, and meniscus, in which no donor DNA was detected 4 weeks after transplantation. DNA from host cells was present in all articular cartilage allografts, as evidenced by detectable unique host DNA patterns. Histological and histochemical assays showed that none of the transplants demonstrated normal structure and composition at 1 year after transplantation. The grafts in which large quantities of donor DNA were present appeared grossly superior to those with no or reduced remaining demonstrable donor DNA.     

Flow cytometric analysis of proliferative activity of pleomorphic adenoma of salivary gland

BACKGROUND: In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel graft performed 17 days later in spite of infiltration of the intestinal grafts by mononuclear cells. AIMS: To compare the phenotype of graft infiltrating cells in rejecting and tolerated small bowel grafts in order to elucidate the mechanism(s) which block the graft infiltrating cells from mediating rejection. METHODS: Multiparameter immunofluorescence was used to compare the phenotype and state of activation of donor and recipient cells isolated from intestinal grafts rejected or tolerated after liver transplantation. RESULTS: Three differences were found. Firstly, there was a more rapid replacement of lamina propria (LP) cells by recipient lymphocytes in tolerated than in rejected grafts. Secondly, the proportion of LP recipient CD8alphabeta+ lymphocytes bearing the high affinity receptor for interleukin 2 was significantly less in tolerated grafts (1.1%, range 0-2%) than in rejected grafts (21.3%, range 9-26%). Finally, tolerated grafts contained significantly less NK lymphocytes (NKR-P1+) and macrophages than rejected intestinal allografts. CONCLUSIONS: These observations make it possible to delineate clear cut differences in the phenotype of cells infiltrating rejecting versus tolerated grafts. Furthermore, the data suggest that liver transplantation induces tolerance of intestinal grafts by hampering the activation of recipient TcRalphabeta+ CD8alphabeta+ T cells and subsequently the recruitment of non-specific effector cells.     

Flt-3 ligand increases microchimerism but can prevent the therapeutic effect of donor bone marrow in transiently immunosuppressed cardiac allograft recipients

C3H (H2k) mice received 50 x 10(6) B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 microg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 microg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.     

Influence of aging on rolipram-sensitive phosphodiesterase activity and [3H]rolipram binding in the rat brain

Cytolytic T cells were generated in vitro by culturing purified Balb/c CD4+ T cells with irradiated C57Bl/6 (B6) splenocytes plus anti-IL-4 mAb. Matched, noncytotoxic T cells were similarly generated by culturing purified Balb/c CD4+ T cells with irradiated B6 splenocytes plus recombinant murine IL-4. The latter T cells displayed to cytolytic activity, even in lectin-mediated lysis assays, but produced characteristic cytokines upon contact with specific alloantigens. Transfusion of cytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts resulted in acute allograft rejection within 5 to 10 days. Transfusion of noncytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts also resulted in acute allograft rejection within 7 to 10 days. Limiting dilution analysis (LDA) of infiltrating cells recovered from rejected allografts after collagenase digestion demonstrated that the CD4+ T cells retained their cytolytic or noncytolytic functional phenotypes in vivo throughout the rejection process. These data demonstrate that isolated CD4+ T cell populations can promote rapid acute cardiac allograft rejection, and that cytolytic activity is not necessary for this acute rejection response.     

Cytomegalovirus infection enhances the neointima formation in rat aortic allografts: effect of major histocompatibility complex class I and class II antigen differences

BACKGROUND: The development of chronic rejection has emerged as a major cause of long-term graft failure. Previous studies have demonstrated that cytomegalovirus (CMV) infection is associated with an increased incidence of chronic allograft rejection in renal, cardiac, and aortic allografts. This study was designed to investigate the effects of the major histocompatibility complex (MHC) class I or class II mismatches on CMV-enhanced chronic rejection. METHODS: Aortic transplantation was performed between different inbred rat strain combinations; the Lewis to RP combination was class I-mismatched and Wag/Rij to RP class II-mismatched. At 7, 28, and 90 days after transplantation, the intensity of chronic rejection in mismatched grafts with or without CMV infection was evaluated using histological and immunohistological analysis. RESULTS: The results of this study demonstrated that CMV infection led to an increased influx of monocytes/ macrophages in class I-mismatched grafts at 1 week after transplantation and enhanced infiltration of T lymphocytes in class II-mismatched grafts at 4 weeks. Although more vascular lesions were observed in the class II-mismatched combinations, an intensified neointima formation by CMV infection was observed only in the MHC class I-mismatched allografts. CONCLUSIONS: CMV infection may increase neointima formation of allografts when an MHC class I disparity between donor and recipient is present. This may be associated with the increased perivascular influx of monocytes/macrophages observed in CMV-infected animals early after transplantation.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.     

Distinct mechanisms for the induction and maintenance of allograft tolerance with CTLA4-Fc treatment

A murine CTLA4/Fc gamma2a heavy chain (mCTLA4-Fc) chimeric fusion molecule was used in B6AF1 recipients of BALB/c pancreatic islet allografts to study the induction and maintenance of tolerance following inhibition of the CD28-B7 pathway for T cell activation. Donor-specific tolerance was achieved by administering 100 microg of mCTLA4-Fc on alternate days for 14 days (8 total doses) or a single 500 microg dose of mCTLA4-Fc on day 2 after transplant. Tolerance was mediated by long-lived peripheral lymphocytes and showed features of organ and alloantigen specificity. Whereas tolerance could not be established in allograft recipients receiving simultaneous mCTLA4-Fc and rIL-2, previously tolerant animals did not reject their grafts when given IL-2, suggesting that the induction and maintenance phases of tolerance were distinct and separate. The maintenance of donor-specific tolerance was an active immunologic process that was CD4+ T cell dependent and could be adoptively transferred to naive lymphocytes, but could not be explained by apoptosis or deletion of alloreactive T cells. Although an IL-2-sensitive mechanism such as anergy may contribute toward the induction of tolerance, its maintenance involves active suppression.     

Nitric oxide production by mouse sponge matrix allograft-infiltrating cells. Comparison with the rat species

We have recently demonstrated in the rat species that sponge matrix allograft infiltrating cells spontaneously produce nitric oxide (.N = 0) and this .N = 0 production precedes the development of CTL. Compared with our experience in the mouse species, the CTL activity recovered from rat sponge grafts is of shorter duration and less intense. Assessment of the spontaneous .N = 0 production by mouse allograft infiltrating cells reveals a more delayed time course of production, paralleling the recovery of CTL activity from the graft. The in vitro spontaneous .N = 0 production by mouse allograft-infiltrating cells was greater than the production by syngeneic graft-infiltrating cells on all days tested. Exposure of allogeneic but not syngeneic graft infiltrating cells to the sensitizing alloantigen in vitro resulted in enhanced .N = 0 synthesis. In contrast, LPS stimulated .N = 0 production by both syngeneic and allogeneic graft cells on all days postgrafting. Culture of day-14 allograft infiltrating cells with alloantigen in the absence of NG-monomethyl-L-arginine (NMA), the competitive inhibitor of .N = 0 synthesis, resulted in elevated supernatant NO2- levels and decreased 3H-TdR uptake and CTL activity compared with cultures carried out in the presence of NMA. The supernatant NO2- levels, as well as the CTL activity and 3H-TdR incorporation of the cultured cells, was dependent on the concentration of NMA present, and these effects could be reversed by excess L-arginine. Thus, the species difference in .N = 0 synthesis (rat > mouse), observed by others, is evident in the sponge allograft model and may explain why CTL activity recovered from rat allografts is of shorter duration and less intense than that from the mouse allografts.     

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.     

Love, courage, and honor

OBJECTIVES: Despite the increasing success of whole-organ pancreas transplantation, the success of clinical islet allografts has remained limited. One of the factors limiting the success is the difficulty in monitoring islet allografts after transplantation. The aim of these studies is to develop a method of "biopsying" human islet allografts using a forearm islet implantation site. METHODS: A subtherapeutic number of isolated human islets were placed in the forearm under the muscle fascia in three human recipients with Type I insulin dependent diabetes. All of the recipients had undergone successful cadaveric renal transplantation at least one year prior and were maintained on their baseline immunosuppression. Aliquots of the islet grafts were removed 7 and 14 days to assess engraftment and graft infiltrate. To verify that the islets were viable, 400 were handpicked and transplanted into B6-scid mice made diabetic with streptozotocin. RESULTS: The biopsy site was found in all three cases. In one patient, no islets were recovered. In two other patients, viable islet tissue was recovered 7 days after transplantation. Immunohistology at 7 days showed the presence of both insulin and glucagon-staining cells in the islets. At 14 days in these two patients, a mononuclear cell infiltrate was observed in the explanted islet biopsies. Immunohistology showed the relative absence of insulin-staining cells with intact glucagon-staining cells. This finding is consistent with recurrent autoimmunity in the islet grafts. DISCUSSION: This preliminary study shows that the forearm biopsy site is a useful method to retrieve human islet grafts after transplantation. The islets engraft and are easily found in the first weeks after transplantation. These data suggest that recurrent autoimmunity may affect islet allografts. Further studies will be needed to determine if the histology in the forearm will correlate with the fate of intraportal or intraperitoneal islet allografts. Although they were shown to reduce the incidence of early allograft failure, their influence on the long-term graft survival remains to be proven.     

Cell survival following bone-anterior cruciate ligament-bone allograft transplantation: DNA fingerprints, segregation, and collagen morphological analysis of multiple markers in the canine model

Bone-anterior cruciate ligament-bone allograft transplantation has become recognized as a potential solution to reconstruction of the anterior cruciate ligament (ACL). The purpose of this study was to determine the time-dependent fibrocyte donor cell survival rate after cryopreserved bone-ACL-bone allograft transplantation. Additionally, bony incorporation of the pediculated bone plugs was examined. The ability to successfully transplant allogenous ACL fibrocytes and have them survive has not previously been documented. In this study, DNA fingerprints identified and documented the survival rate of the cellular DNA in transplanted ACL allografts for ACL re-construction in the knee joints of 10 skeletally mature dogs. At 4, 8, 26 and 52 weeks after ACL allograft transplantation, DNA probes, H & E, Giemsa, Goldner, PAS and polarized light staining was done to demonstrate the time-dependent changes in the allografts after transplantation. At 4 weeks host fibrocytes began to grow into the graft; however, histologically the cells could not be distinguished as to host or donor origin. After 4 weeks the DNA pattern reflected only the band pattern of the host. This reveals the early cellular infiltration activity of the host into the ACL allograft, also demonstrated in the light microscopy stainings. The survival rate of transplanted allogenous ACL fibrocytes had not been documented before this study. There is no evidence that ACL allograft cells survive in the intra-articular environment of the host's knee. Within 4 weeks ACL allografts became completely repopulated with host cells. The cells that migrate early into the ACL allografts are probably of synovial origin because they are present before revascularization and collagen reorganization occur. We conclude from this study that viable cells in transplanted ACL allografts did not survive longer than 4 weeks after intra-articular transplantation. Advances in molecular biology may offer new approaches to alter or stimulate fibrocyte population and function in the transplanted ACL allograft used for ACL reconstruction. New methods to maintain the viability of donor cells may be necessary to improve the biomechanical and histological properties of autografts or allografts for ACL reconstruction.     

Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation

OBJECTIVE: To delineate the mechanisms by which topical interleukin 1 receptor antagonist (IL-1RA) treatment promotes orthotopic corneal allograft survival. METHODS: Corneal buttons were prepared from eyes of C57BL/6 mice and placed orthotopically in normal or neovascularized (high-risk) eyes of BALB/c mouse recipients. Topical IL-1RA (or vehicle alone) was applied to grafts 3 times daily until the grafted eyes were enucleated. Corneal specimens were evaluated for content of Langerhans cells. A week after enucleation, 1 group of recipients was tested for allospecific delayed-type hypersensitivity elicited by intrapinnae injections of donor splenocytes. In companion experiments, a second group of mice that underwent transplantation, IL-1RA treatment, and enucleation was challenged with orthotopic skin grafts from B10.D2 donor mice (sharing minor H antigens with C57BL/6 mice) to determine whether the second group of mice could reject grafts bearing corneal donor minor H alloantigens in an accelerated fashion. RESULTS: Mice whose orthotopic corneal allografts were treated topically with IL-1RA acquired neither donor-specific delayed-type hypersensitivity (P<.001) nor the capacity to reject orthotopic donor-type skin allografts in an accelerated manner (P<.05), whereas controls treated with vehicle alone developed delayed-type hypersensitivity and rejected B10.D2 grafts in an accelerated manner. Moreover, IL-1RA-treated grafts placed in both high-risk (P = .01) and normal-risk (P = .004) eyes displayed significantly reduced levels of infiltrating Langerhans cells compared with vehicle-treated controls. CONCLUSIONS: Topical IL-1RA promotes corneal allograft survival in large part by preventing activity of recipient Langerhans cells, and thereby preventing these cells from inducing systemic allosensitization. These data suggest that IL-1 plays a key role in promoting allosensitization when corneal allografts are placed orthotopically. ClINICAL RELEVANCE: Suppression of allosensitization by topical IL-1RA may prove a clinically useful method for enhancing corneal transplant survival.     

Estradiol inhibits allograft-inducible major histocompatibility complex class II antigen expression and transplant arteriosclerosis in the absence of immunosuppression

BACKGROUND: The etiology of transplant arteriosclerosis is unknown, but current data point to the alloimmune response. Previously, we found that estradiol-17beta (E2) with immunosuppressant cyclosporine abolishes major histocompatibility complex (MHC) class II expression in the allograft. This study determines the effect of E2 on MHC class II antigen expression in the allograft, in the absence of immunosuppression. METHODS: Lewis male rats received orthotopic abdominal aorta allografts from male Brown-Norway rats. The recipients were treated continuously subcutaneously with either 20 microg x kg(-1) x day1 of E2 (n=20) or placebo (n=20), from 2 days before transplantation until death on posttransplant days 1, 3, 7, and 14. The allografts were harvested and processed for morphometry and for immunohistochemical staining of MHC class II antigens, macrophages, CD4 and CD8 T lymphocytes, interferon-gamma (IFN-gamma), and IFN-gamma receptor. RESULTS: With E2 treatment, we observed that inducible MHC class II antigen expression is abolished in the media of the vascular allograft; the expression of IFN-gamma and IFN-gamma receptor is unaffected; and macrophage infiltration of the vascular allograft is inhibited significantly (P<0.01), whereas the CD4 and CD8 T lymphocytes are not significantly (P=0.07) suppressed. The myointimal hyperplasia in the allografts from E2-treated-recipients was 3-4-fold less than that from the placebo-treated recipients. CONCLUSIONS: Without immunosuppression, E2 inhibition of transplant arteriosclerosis is still associated with inhibition of inducible MHC class II antigen expression in the allografts. The estradiol-17beta abolition of inducible MHC class II antigen expression in the aorta allograft occurs in spite of up-regulation of IFN-gamma ligand and receptor protein.     

Rejection of cardiac allografts by T cells expressing a restricted repertoire of T-cell receptor V beta genes

We have recently shown that T cells infiltrating cardiac allografts early in graft rejection use a limited T-cell receptor (TCR) V beta repertoire. In this study we tested whether this limited repertoire of V beta genes is important for graft rejection. A cell line, AL2-L3, was established from LEW lymphocytes infiltrating ACI heart allografts 2 days after transplantation. This cell line is composed of CD4+ T cells that primarily recognize the class II RTI.B major histocompatibility complex (MHC) molecule expressed by the donor graft. This cell line precipitated acute rejection of donor hearts with a median survival time (MST) of 10.5 days following adoptive transfer to sublethally irradiated LEW recipients. This rate of graft rejection was significantly (P < 0.0007) accelerated when compared with a MST of 60 days for allografts in irradiated control recipients. The AL2-L3-mediated acceleration of graft rejection was donor specific as WF third-party heart allografts were rejected with a delayed tempo (MST = 28.5 days). The V beta repertoire of this cell line was primarily restricted to the expression of V beta 4, 15 and 19 genes. The nucleotide sequence analysis of the beta-chain cDNAs from this cell line demonstrated that the restricted use of the V gene repertoire was not shared with the N, D and J regions. A wide variety of CDR3 loops and J beta genes were used in association with selected V beta genes. These data provide evidence for the role a restricted repertoire of V beta genes plays in cardiac allograft rejection in this model. The restricted usage of the V beta repertoire in an early T-cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T-cell populations for elimination at the time of organ transplantation.     

Tolerogenic behavior of skin allografts from neonatal mice

Neonatal skin allografts can be tolerogenic when transplanted to appropriately immunosuppressed hosts. Single grafts of neonatal skin survive longer than adult skin grafts when recipients are treated with antilymphocyte serum (ALS) and donor bone marrow cells (BMC). Neonatal skin grafts can also prolong the survival of adult grafts of the same donor strain simultaneously cotransplanted with the neonatal grafts. To probe the mechanisms involved in this cotransplantation phenomenon, we delayed placement of the neonatal cotransplants relative to grafting with adult skin. Neonatal allografts placed either 7-9 days or 14 days after grafting with adult skin significantly prolonged adult graft survival in mice treated with ALS and BMC. However, day 0-placed neonatal cotransplants must remain on the recipient for > 2 weeks to prolong adult graft survival. Removal of cotransplants from ALS- and BMC-treated recipients after 7 or 14 days abrogated the cotransplantation effect. If left in place until day 21, neonatal cotransplants could significantly prolong adult graft survival, but did not induce the long-term graft survival observed in approximately 50% of the recipients whose cotransplants were not removed. Cotransplant removal after 1 year did not affect subsequent adult graft survival. Additionally, cotransplants were removed from recipients either on day 14 or from long-term graft-bearing mice and retransplanted to other ALS/BMC-treated recipients. These retransplanted grafts were unable to prolong survival of adult grafts on the new recipients. After transplant, but not before transplant, cyclophosphamide treatment of recipients prevented expression of the cotransplant effect in ALS-treated mice. However, recipient splenectomy > or = 1 week before grafting did not interfere with the effect. These results reflect on the contributions of the donor tissue, and the recipients' response, to the tolerogenic signals that permit a neonatal cotransplant to prolong adult graft survival.