Cytotoxic T-cell elimination during anti-CD4-induced rat liver acceptance and rapid replacement of functional graft antigen-presenting cells.

In previous studies, we showed that primed T cells were eliminated in long-term survival Wistar Furth (WF) recipient rats with spontaneously accepted Lewis (LEW) liver graft and that the grafted liver lost the ability to elicit rejection reaction early after liver transplantation. We hypothesized that the same phenomenon may be observed in tolerant animals after immunosuppression in a rejector rat strain combination (WF-->LEW). Furthermore, we proposed the repopulation of liver allograft with host antigen-presenting cells rapidly after transplantation. Recipient LEW rats that underwent anti-CD4 therapy accepted the WF liver allografts after a transient rejection reaction. In tolerant animals, alloreactive CD8 T cell precursors were present, but primed T cells were absent. Intraperitoneal challenge with grafted WF liver homogenates obtained from recipient LEW rats on day 4 after transplantation did not induce transient rejection responses in long-term survival recipient LEW rats, a finding that differed from the results of experiments using normal WF liver homogenates. However, challenge with grafted WF liver homogenates, similar to those of normal LEW liver homogenates, induced rejection responses in long-term survival recipient WF rats with LEW liver allograft. Flow cytometric analysis confirmed that most of nonparenchymal cells in the grafted WF liver were recipient (LEW) genotype. These observations showed that the deletional mechanism of effector T cells also is observed in this setting, and professional donor antigen-presenting cells are replaced by those of recipient genotype within the graft during the early phase of transplantation.     

Circulating alloreactive T cells correlate with graft function in longstanding renal transplant recipients

Monitoring for alloreactive memory T cells after organ transplantation may allow individualization of immunosuppression. Two pathways of T cell allorecognition have been implicated in chronic graft dysfunction: Direct (recipient T cells respond to donor peptides presented by donor antigen-presenting cells) and indirect (donor peptides are processed and presented by recipient antigen-presenting cells). Previous studies have assessed these alloresponses only during the first 2 yr after kidney transplantation,so this study correlated the presence of circulating donor-reactive memory/effector T cells, primed by both pathways, in 34 longstanding living-donor renal transplant recipients using the highly sensitive IFN-gamma Elispot assay. Remarkably, 59% of patients had directly primed donor-reactive T cells, and their presence correlated directly with serum creatinine (P = 0.001) and inversely with estimated GFR (P = 0.042). Multivariate analysis revealed that hyporesponsiveness of direct, donor-specific T cells was the only variable that significantly correlated with graft function and that antidonor indirect alloreactivity was the only variable that significantly correlated with proteinuria. Interestingly, when both allorecognition pathways were considered together, patients with undetectable direct alloreactivity had better longterm graft function, independent of allosensitization by the indirect pathway. In conclusion, circulating donor-specific alloreactive T cells primed by both pathways are detectable long after transplantation and are associated with graft injury. Assessment of alloreactive memory/effector T cells might be helpful to tailor individual immunosuppression regimens for transplant recipients in the future.     

DNA released from rejecting organs is an indicator of the degree of graft cellular damage

Warm and cold ischemia as well as rejection of the transplanted organ or tissue cause destructive changes in the graft parenchyma. Fragments of disintegrated cellular organelles are phagocytized and digested by recipient scavenger cells in lymph nodes, spleen, and liver. Some fragments engulfed by dendritic cells are processed including donor DNA present in the ingested cellular debris. The question arises as to whether the DNA from the disintegrated cells may be used as a measure of graft damage. In this study we provide evidence that both syngeneic and allogeneic organ transplantation followed by "seeding" of donor DNA from graft cells is internalized in recipient macrophages and dendritic cells in lymphoid organs. The kinetics of accumulation of donor DNA in recipient tissues reflected the degree of ischemic and immune graft damage. Immunosuppression with cyclosporine or tacrolimus did not significantly attenuate the DNA release. Measurements of the concentration of donor DNA gives insight into the kinetics of allograft rejection. Real-time polymerase chain reaction for donor DNA in recipient serum and blood leukocytes that have engulfed donor cell debris may be useful for clinical diagnostic application.     

Altered intragraft immune responses and improved renal function in MHC class II-deficient mouse kidney allografts

BACKGROUND: During renal allograft rejection, expression of MHC class II antigens is up-regulated on the parenchymal cells of the kidney. This up-regulation of MHC class II proteins may stimulate the intragraft alloimmune response by promoting their recognition by recipient CD4+ T cells. In previous studies, absence of donor MHC class II antigens did not affect skin graft survival, but resulted in prolonged survival of cardiac allografts. METHODS: To further explore the role of MHC class II antigens in kidney graft rejection, we performed vascularized kidney transplants using donor kidneys from A(beta)b-deficient mice that lack MHC class II expression. RESULTS: At 4 weeks after transplant, GFR was substantially depressed in control allografts (2.18+/-0.46 ml/min/kg) compared to nonrejecting isografts (7.98+/-1.62 ml/min/kg; P<0.01), but significantly higher in class II- allografts (4.38+/-0.60 ml/min/kg; P<0.05). Despite the improvement in renal function, class II- allograft demonstrated histologic features of acute rejection, not unlike control allografts. However, morphometric analysis at 1 week after transplantation demonstrated significantly fewer CD4+ T cells infiltrating class II- allografts (12.8+/-1.2 cells/mm2) compared to controls (25.5+/-2.6 cells/mm2; P=0.0007). Finally, the intragraft profile of cytokines was altered in class II- allografts, with significantly reduced expression of Th2 cytokine mRNA compared to controls. CONCLUSIONS: These results support a role of MHC class II antigens in the kidney regulating immune cells within the graft. Further, effector pathways triggered by class II antigens promote renal injury during rejection.     

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4⁺ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I–mismatched (K^d) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a K^d skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.     

B Cell Depletion With an Anti‐CD20 Antibody Enhances Alloreactive Memory T Cell Responses After Transplantation

Alloreactive memory T cells mediate accelerated allograft rejection and transplant tolerance resistance. Recent studies have shown that B cell deficient–μMT mice fail to mount donor‐specific memory T cell responses after transplantation. At the same time, other studies showed that pretransplant B cell depletion using rituximab (IgG1 anti‐CD20 mAb) combined with cyclosporine A promoted the survival of islet allografts in monkeys. In this study, we investigated the effect of anti‐CD20 antibody‐mediated B cell depletion on the memory T cell alloresponse in mice. Wild‐type and anti‐OVA TCR transgenic mice were treated with an IgG2a anti‐CD20 monoclonal antibody, which depleted nearly all B cells in the peripheral blood and secondary lymphoid organs but spared some B cells in the bone marrow. B cell depletion did not affect the direct alloresponse but resulted in a marked increase of indirect alloresponse after skin transplantation of naïve mice. Furthermore, in allosensitized mice, anti‐CD20 mAb treatment enhanced the reactivation of allospecific memory T cells and accelerated second set rejection of skin allografts. This suggests that the effect of anti‐CD20 antibodies on alloimmunity and allograft rejection might vary upon the nature of the antibodies as well as the circumstances under which they are delivered.     

Impact of both donor and recipient strains on cardiac allograft survival after blockade of the CD40-CD154 costimulatory pathway

BACKGROUND: The effectiveness of anti-CD154 monoclonal antibodies in prolonging the survival of mouse allografts is dependent on the strain combination. In this report, we examined the impact of the donor and the recipient strains on the success of CD40-CD154 blockade. MATERIALS AND METHODS: Cardiac allograft survival was monitored in different donor/recipient strain combinations. Morphometric analyses on the allograft coronary arteries allowed quantification of vessel intimal thickening. RESULTS: Prolonged cardiac allograft survival after the administration of an anti-CD154 monoclonal antibody was found to be dependent on the donor and the recipient strains. The influence of the donor and the recipient strains lay in the ability of CD8 T cells to cause graft rejection despite CD40-CD154 blockade. Elimination of CD8 T cells before transplantation resulted in similar graft prolongation irrespective of the genotype of the donor or the recipient strain. CONCLUSION: These data show that both donor and recipient strains contribute to CD40-CD154-independent CD8 T-cell-mediated rejection.     

Immunobiology of rejection and adaptation

Transplantation is an acceptable therapy for failing organs, however, the balance between prevention of acute rejection and immunosuppressant-induced toxicity remains elusive. Organ transplantation from a genetically disparate donor induces an immune response toward donor antigens in the recipient. An uncontrolled cumulative effect of these responses may jeopardize the recipient's life and destroy the grafted tissue. The donor antigen in the form of passenger leukocytes from the allograft migrating to the organized lymphoid collection is a prerequisite for initiation of acute rejection. In the host lymphoid tissue donor-specific dendritic cells primed with donor peptide activate na?ve CD4 helper T cells which in turn activate effector CD8 T-cell clones through the release of cytokines. Activated effector CD8 cells return to the graft and augment destructive activity with the help of adhesive molecules and perforin. This seems to be the mechanism of adaptive immunity to destroy viral pathogens; the pattern of allograft injury is not much different. Adaptation and tolerance are based on the principle of exhaustion of donor-specific immune responses by an activation-deletion-exhaustion pathway.     

The role of TDTH and Tc populations in organ graft rejection. I. Functional analysis of graft-infiltrating T cells

To analyze the role of T cell subpopulations in the rejection of organ allografts, we developed a new model for obtaining large numbers of graft infiltrating cells (GICs). We isolated W3/25+ Th/DTH and OX8+ Ts/c from vascularized, irradiated rat spleen allografts. W3/25+ GICs obtained from spleen allografts transplanted to normal recipients were highly effective in eliciting cardiac allograft rejection when transferred to sublethally irradiated recipients, however, the OX8+ subset was incapable of eliciting rejection. On the other hand, when OX8+ GICs were obtained from spleen allografts transplanted to previously immunized recipients, they were as efficient as the W3/25+ Th/DTH subset in eliciting cardiac allograft destruction. These results indicate that the W3/25+, OX8- T cell is required for the rejection of primary organ allografts, but that the rejection of a secondary allograft by an immune recipient may be mediated, independently, by both W3/25+ and OX8+ cells.     

Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process.

BACKGROUND: Transient depletion of CD4+ T cells in cardiac allograft recipients prolongs allograft survival; however, grafts exhibit signs of chronic rejection characterized by collagen deposition and neointima development. Although it is believed that Th1 cells promote acute graft rejection, the role of these cells in chronic rejection remains unclear. Hence, our study evaluated whether Th1 cells are associated with the development of chronic cardiac allograft rejection. METHODS: Splenocytes obtained from C57BL/6 recipients bearing BALB/c hearts with signs of chronic rejection were adoptively transferred into C57BL/6 SCID cardiac allograft recipients. As a measure of Th1 function, interferon-y production was determined after restimulation of recipient splenocytes with donor alloantigens. RESULTS: Transfer of splenocytes in SCID allograft recipients resulted in accelerated chronic rejection in the majority of mice. Characterization of these cells before transfer revealed hyporesponsive Th1 function. However, donor-specific proliferative responses and precursor interleukin-2 producing helper and cytotoxic T lymphocyte frequencies were comparable to that of naive splenocytes. Further, splenocytes obtained from SCID recipients with advanced signs of chronic rejection remained deficient in Th1 function, suggesting that Th1 are not involved in this disease process. This possibility was further supported by the development of chronic rejection in IL-12 knockout recipients. Finally, when splenocytes used for adoptive transfer retained Th1 function, transfer of these cells into SCID recipients resulted in acute allograft rejection. CONCLUSIONS: We have established a model in which the mediators of chronic rejection may be further explored. In this system, the absence rather than the presence of donor-reactive Th1 is associated with chronic rejection. These data indicate that Th1-independent effector mechanisms are responsible for chronic rejection in this model.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

The Immunobiology of Inductive Anti-CD40L Therapy in Transplantation: Allograft Acceptance is Not Dependent Upon the Deletion of Graft-Reactive T Cells

CD40–CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti‐CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft‐reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40–CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti‐CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL‐2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti‐CD40L treatment. Interestingly, the spleens of mice bearing long‐term cardiac allografts following inductive anti‐CD40L treatment retained precursor donor alloantigen‐reactive CTL, IL‐2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor‐strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor‐strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long‐term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long‐term graft acceptance. Collectively, these data demonstrate that inductive CD40–CD40L blockade does not result in the deletion of graft‐reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen‐reactive cells are subject to this regulation.     

Dynamic changes in Th1, Th17, and FoxP3+ T cells in patients with acute cellular rejection after cardiac transplantation

Previously, studies suggest that CD4(+) effector T-cell subsets participate in allograft rejection. However, the dynamic changes and relative roles of these CD4(+) effector T-cell subsets, especially Th17 cells, have not been systemically examined in patients with acute rejection after cardiac transplantation. In this study, we have studied and compared these CD4(+) T-cell subsets in peripheral blood and endomyocardial biopsies (EMB) in patients with stable-graft and acute cellular rejection. We observed that the gene expressions including T-bet, IFN-γ, RORγt, IL-17, IL-23, and FoxP3, the functional marker of Th1, Th17, and FoxP3(+) CD4(+) T cells, were elevated in EMB samples from patients with acute graft rejection. Accordingly, the percentages of circulating Th1, Th17, and FoxP3(+) CD4(+) T cells were also significantly increased. The data suggest that Th1, Th17, and FoxP3(+) CD4(+) T cells are associated with acute graft rejection in patients with cardiac transplantation.     

Allopeptide-pulsed dendritic cells and composite tissue allograft survival

Composite tissue allografts (CTAs) contain their own reservoir of vascularized bone marrow, offering novel aspects for the induction of donor-specific tolerance. Additionally, the manipulation of recipient dendritic cells, pulsed with donor allopeptide, has been shown to engender solid organ allograft survival. To exploit these modalities, we have developed a protocol utilizing injection of recipient bone marrow-derived dendritic cells (BMDCs) pulsed with a donor-derived peptide for use in CTA transplantation. Six days prior to orthotopic hind-limb transplantation, Lewis rats received IV injection of donor allopeptide-pulsed, recipient BMDCs, in conjunction with a single dose of anti-lymphocyte serum. Control groups displayed signs of allograft rejection within 5 days postoperatively. Animals within the primary experimental cohort demonstrated prolongation of graft survival to an average of 8 days, and exhibited low numbers of donor T cells. The use of BMDCs in conjunction with transient immunosuppression has potential therapeutic application for induction of donor-antigen-specific tolerance to hind limb allografts.     

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.     

Graft produced interleukin-6 functions as a danger signal and promotes rejection after transplantation

BACKGROUND: Interleukin (IL)-6 is a pleiotropic cytokine that functions in both the innate and adaptive immune responses. However, the role of IL-6 in allograft rejection remains poorly understood. METHODS: In this study, we demonstrate a critical role for graft-produced IL-6 in allograft rejection in a murine model of cardiac allograft transplantation. RESULTS: The results show that IL-6-deficient grafts transplanted into allogeneic wild-type recipients have significantly prolonged survival, approximately three times the survival time of wild-type controls. In contrast, allogeneic cardiac transplants into IL-6-deficient recipients do not have prolonged graft survival, indicating that donor graft cells are the relevant source of IL-6. Our investigation of potential mechanisms shows that graft-produced IL-6 promotes the activation of peripheral CD4 and CD8 T cells. Furthermore, we show that IL-6 deficiency prolongs graft survival only in the presence of CD25+ T cells that have a phenotype consistent with regulatory T cells. Interestingly, IL-6 production by the graft is triggered by antigen-independent innate immune mechanisms. CONCLUSIONS: Thus, our results suggest the paradigm that graft rejection versus tolerance is determined by a balance between the activation of effector T cells versus immune suppression by regulatory T cells, and that after transplantation, IL-6 functions as a systemic danger signal that overcomes constitutive immune suppression mediated by regulatory T cells and promotes the activation of effector T cells.     

Donor and recipient HLA/KIR genotypes do not predict liver transplantation outcome

Whether or not Natural Killer (NK) cells affect the immune response to solid organ allografts is still controversial. Main determinants of NK‐cell activation are specific HLA/killer‐cell immunoglobulin‐like receptors (KIR) interactions that, in transplantation, may induce NK‐cell alloreactivity. So far, in liver transplantation (LTX) donor‐versus‐recipient alloreactivity has not been investigated; in addition, studies of predicted recipient‐versus‐donor NK‐cell alloreactivity have led to contradicting results. We typed a cohort of LTX donors and recipients for HLA‐C/Bw4 and KIRs. We estimated the effect of NK‐cell alloreactivity, as predicted by classically used models, in the donor‐versus‐recipient direction. The results indicate that HLA/KIR mismatches in the donor‐versus‐recipient direction do not predict graft rejection nor graft or patient survival, suggesting that donor‐derived NK cells do not play a major role in LTX outcome. In addition, when considering predicted NK‐cell alloreactivity in the reverse direction (recipient‐versus‐donor), we first confirmed that donor HLA‐C genotype was not associated with acute rejection, graft or patient survival and secondly we found that none of the models describing NK‐cell alloreactivity could predict LTX outcome. Overall our observations suggest that, in contrast to what is shown in haematopoietic stem cell transplantation, donor‐derived NK cells may not contribute in preventing liver graft rejection, and that recipient‐versus‐donor NK‐cell alloreactivity does not predict LTX outcome.     

Donor apoptotic cell–based therapy for effective inhibition of donor‐specific memory T and B cells to promote long‐term allograft survival in allosensitized recipients

Allosensitization constitutes a major barrier in transplantation. Preexisting donor‐reactive memory T and B cells and preformed donor‐specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long‐term immunosuppression‐free allograft survival. We demonstrate that donor‐specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti‐CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen‐presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti‐CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long‐term islet allograft survival in such recipients.     

Acceptance of allogeneic fibroblasts in skin equivalent transplants

Living skin equivalents (SE) were prepared by combining cultured fibroblasts with a collagen matrix and overlaying this lattice with keratinocytes. SEs prepared using allogeneic female rat fibroblasts or xenogeneic rabbit or human fibroblasts and keratinocytes isogeneic to the graft recipient were transplanted to recipient male rats. Biopsies of some of these SE grafts were examined histologically at intervals ranging from 5 days to 2 months. Biopsies of other grafts were done, and fibroblasts grown from them were karyotyped to determine the percentage of donor fibroblasts remaining in the graft. SEs containing xenogeneic fibroblasts were rejected. Allografted fibroblasts in SEs were accepted by recipient rats after a transient mononuclear cell response. A second SE allograft from the same donor strain did not provoke rejection either in the original allograft or in the challenge allograft. A secondary graft of allogeneic skin did not provoke rejection in the original SE graft, although the skin graft was rejected. Grafting the recipient first with allogeneic skin and then with the SE allograft led to rejection of the skin but not of the SE graft, ruling out the possibility that suppressor T cells were responsible for SE allograft acceptance. Allografted fibroblasts in SEs do not provoke a rejection response, even in presensitized animals, do not render the recipient tolerant to allogeneic skin, and do not act as targets when active rejection is taking place. We propose that cells bearing class I antigens may be acceptable graft constitutents if incorporated in a tissue equivalent excluding cells with class II antigens.