Tolerance In Chimeras Research Articles

Murine models of transplantation tolerance through mixed chimerism: advances and roadblocks.

Organ transplantation is the treatment of choice for patients with end-stage organ failure, but chronic immunosuppression is taking its toll in terms of morbidity and poor efficacy in preventing late graft loss. Therefore, a drug-free state would be desirable where the recipient permanently accepts a donor organ while remaining otherwise fully immunologically competent. Mouse studies unveiled mixed chimerism as an effective approach to induce such donor-specific tolerance deliberately and laid the foundation for a series of clinical pilot trials. Nevertheless, its widespread clinical implementation is currently prevented by cytotoxic conditioning and limited efficacy. Therefore, the use of mouse studies remains an indispensable tool for the development of novel concepts with potential for translation and for the delineation of underlying tolerance mechanisms. Recent innovations developed in mice include the use of pro-apoptotic drugs or regulatory T cell (Treg ) transfer for promoting bone marrow engraftment in the absence of myelosuppression and new insight gained in the role of innate immunity and the interplay between deletion and regulation in maintaining tolerance in chimeras. Here, we review these and other recent advances in murine studies inducing transplantation tolerance through mixed chimerism and discuss both the advances and roadblocks of this approach.

Donor Lymphocyte Infusion–Mediated Graft-versus-Host Responses in a Preclinical Swine Model of Haploidentical Hematopoietic Cell Transplantation

We previously described successful hematopoietic stem cell engraftment across MHC barriers in miniature swine without graft-versus-host disease (GVHD) using novel reduced-intensity conditioning regimens consisting of partial transient recipient T cell–depletion, thymic or low-dose total body irradiation, and a short course of cyclosporine A. Here we report that stable chimeric animals generated with these protocols are strongly resistant to donor leukocyte infusion (DLI)–mediated GVH effects. Of 33 total DLIs in tolerant chimeras at clinical doses, 21 failed to induce conversion to full donor hematopoietic chimerism or cause GVHD. We attempted to overcome this resistance to conversion through several mechanisms, including using sensitized donor lymphocytes, increasing the DLI dose, removing chimeric host peripheral blood cells through extensive recipient leukapheresis before DLI, and using fully mismatched lymphocytes. Despite our attempts, the resistance to conversion in our model was robust, and when conversion was achieved, it was associated with GVHD in most animals. Our studies suggest that delivery of unmodified hematopoietic stem cell doses under reduced-intensity conditioning can induce a potent, GVHD-free, immune tolerant state that is strongly resistant to DLI.

Donor Leukocyte Infusion Mediated Graft-Versus-Host Responses in a Pre-Clinical Swine Model of Haploidentical Hematopoietic Cell Transplantation

The donor-anti-host cellular reactivity associated with allogeneic hematopoietic cell transplantation (HCT) is a powerful anti-leukemia treatment. However, graft-versus-host disease (GVHD) continues to limit HCT, especially across major histocompatibility (MHC) barriers. We previously described successful hematopoietic stem cell engraftment in MHC-mismatched swine without GVHD using novel reduced intensity conditioning (RIC) regimens consisting of partial, transient recipient T-cell depletion (with or without thymic or low-dose total body irradiation), and a short course of cyclosporine A (CyA). Here, we report that stable chimeric animals generated with these protocols are strongly resistant to donor leukocyte infusion (DLI) mediated GVH effects (n=17). Delayed DLI in tolerant chimeras at a dose consisting of 10-50X106 T cells /kg (Tc/kg) failed to induce conversion to full donor chimerism or cause GVHD. Sensitizing the donor with recipient skin prior to collecting donor cells for DLI did not have any demonstrable effect on DLI outcome (n=2). This resistance to DLI mediated GVH reactivity could be overcome in some cases (n=9) by either 1) increasing the dose of DLI, 2) removing chimeric host peripheral blood cell populations through extensive leukapheresis prior to DLI or 3) delivering lymphocytes fully mismatched to the host but not to the original HCT donor within the chimera. However, conversion to full donor chimerism in some recipients was associated with GVHD. Our studies suggest that delivery of non-manipulated HCT grafts under reduced intensity conditioning can induce a potent, GVHD free immune tolerant state that is strongly resistant to DLI. DLI induced conversion to full donor chimerism is often associated with GVHD despite full immune reconstitution of the host and lack of known inflammatory stimuli.

Targeting Cells Causing Split Tolerance Allows Fully Allogeneic Islet Survival With Minimal Conditioning in NOD Mixed Chimeras

Donor-specific tolerance induced by mixed chimerism is one approach that may eliminate the need for long-term immunosuppressive therapy, while preventing chronic rejection of an islet transplant. However, even in the presence of chimerism it is possible for certain donor tissues or cells to be rejected whereas others from the same donor are accepted (split tolerance). We previously developed a nonmyeloablative protocol that generated mixed chimerism across full major histocompatability complex plus minor mismatches in NOD (nonobese diabetic) mice, however, these chimeras demonstrated split tolerance. In this study, we used radiation chimeras and found that the radiosensitive component of NOD has a greater role in the split tolerance NOD mice develop. We then show that split tolerance is mediated primarily by preexisting NOD lymphocytes and have identified T cells, but not NK cells or B cells, as cells that both resist chimerism induction and mediate split tolerance. Finally, after recognizing the barrier that preexisting T cells impose on the generation of fully tolerant chimeras, the chimerism induction protocol was refined to include nonmyeloablative recipient NOD T cell depletion which generated long-term mixed chimerism across fully allogeneic barriers. Furthermore, these chimeric NOD mice are immunocompetent, diabetes free and accept donor islet allografts.

Roles of Deletion and Regulation in Creating Mixed Chimerism and Allograft Tolerance Using a Nonlymphoablative Irradiation-Free Protocol

The induction of mixed chimerism (MC) is a powerful and effective means to achieve transplantation tolerance in rodent models. Host conditioning with irradiation or cytotoxic drugs has been used in many protocols for chimeric induction across allogeneic barriers. The deletion of alloreactive T cell clones has been described as the main mechanism responsible for the induction of a stable MC. In this study, we demonstrate that a stable MC and skin allograft tolerance can be established across MHC barriers by a noncytotoxic, irradiation-free approach using costimulation blockade plus rapamycin treatment. By using an adoptive transfer model of skin allograft and using specific Vbeta TCR probes, we demonstrated that deletion of donor-reactive cytopathic T cell clones is indeed profound in tolerant hosts. Nonetheless, the challenge of tolerant mixed chimeras with 5 million mononuclear leukocytes (MNL) from naive syngeneic mice was neither able to abolish the stable MC nor to trigger skin allograft rejection, a hallmark of peripheral, not central tolerance. Furthermore, in an adoptive transfer model, MNLs harvested from tolerant hosts significantly inhibited the capacity of naive MNLs to reject same donor, but not third-party, skin allografts. Moreover, when we transplanted skin allografts from stable tolerant chimeras onto syngeneic immune-incompetent mice, graft-infiltrating T cells migrated from the graft site, expanded in the new host, and protected allografts from acute rejection by naive syngeneic MNLs. In this model, both deletional and immunoregulatory mechanisms are active during the induction and/or maintenance of allograft tolerance through creation of MC using a potentially clinically applicable regimen.

NK cell tolerance in mixed allogeneic chimeras.

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c-->B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c--> B6 chimeras rejected a low dose of beta(2)-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and beta(2)-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.

Persistent chimerism despite antidonor MHC in vitro responses in miniature swine following allogeneic hematopoietic cell transplantation.

T-cell chimerism predominates in miniature swine receiving hematopoietic-cell transplantation without myelosuppressive conditioning. Several chimeric recipients have become hyporesponsive to donor-major histocompatibility complex (MHC) in vitro and accepted donor-matched renal transplants without immunosuppression. However, some retained antidonor in vitro responses and subsequently rejected donor renal allografts despite the persistence of peripheral blood chimerism. In this study, we characterize the donor cells in both "tolerant" and "nontolerant" chimeric miniature swine. Peripheral blood chimerism was determined by flow cytometry. In vitro antidonor responsiveness was determined by mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Donor cells were separated from chimeras by immunomagnetic bead separation and used as stimulators or targets in CML assays. Phenotypic analysis of donor cells in chimeras was performed using flow cytometry. Peripheral blood chimerism stabilized beyond 100 days and was made up almost entirely of T cells. PBMC from nontolerant chimeras could be stimulated in vitro to kill donor cells isolated from the mixed chimera itself. In contrast, PBMC from tolerant chimeras hyporesponsive to donor-type cells could not be stimulated in vitro to kill their own sorted donor cells. The in vivo persistence of donor T cells in mixed chimeric animals with in vitro antidonor responsiveness is not caused by an inability of these cells to be killed but rather by the poor stimulating capacity of these donor T cells. The nature of donor T cells that persist in the face of in vitro antidonor responses, has important implications for the induction of transplant tolerance by way of the generation of mixed chimerism.

Role of in situ IL-2r and TGF-β expression in tolerant vascularized bone marrow (limb) transplant chimeras

We hypothesized that an increase in IL-2 activated T cells in situ within the marrow component of a transplanted limb may adversely affect development of tolerance, while increased TGF-β expression locally would facilitate tolerance induction and/or maintenance. Digital image analysis of cellular expression of IL-2r in the bone marrow was significantly increased in the CON and TXP limbs for both GVHD and tolerant recipients as compared to normal limb marrow ( P < .02). The amount of cellular expression of TGF-β was significantly increased in the GVHD animals, both CON and TXP, as compared to the tolerant animals (43.2 ± 3.1 vs 10.6 ± 2.6; P < .000001). Our results show that increased IL-2r and TGF-β expression in situ within the bone marrow is an important effect common to both alloimmune tolerance and GVHD induction with VBMT chimeras. The dramatic increase in the expression of TGF-β in the GVHD transplanted limbs may explain the profound immunosuppression that results. Additionally, moderate expression of TGF-β in situ in tolerant chimeras may represent a mechanism for the induction and maintenance of tolerance.

Cellular mechanisms of alloimmune non-responsiveness in tolerant mixed lymphocyte chimeras induced by vascularized bone marrow transplants.

It has been demonstrated that the development of stable mixed lymphocyte chimerism is associated with alloimmune tolerance induction in vascularized bone marrow transplant (VBMT) recipients. The underlying mechanisms of immune non-responsiveness in tolerant VBMT chimeras remains unclear. Our VBMT model involves the transplantation of a parental donor limb (Lewis rats) onto a hybrid (Lewis x Brown Norway) F1 recipient. Tolerogenic mechanisms and cellular immune regulation to self and host allodeterminants were investigated during the early post-transplant phase of tolerance induction. Flow cytometric analysis of sIg+-depleted experimental peripheral blood lymphocytes from tolerant VBMT recipients demonstrated low level stable mixed immune chimerism. Chimeric cells tested for responsiveness against self-LEW determinants showed activated proliferation and immune dysregulation 30 days post-transplantation. However, direct immunocytolytic activity against LEW determinants was not found. Tolerant chimeras also demonstrated elevated cellular proliferation and cytolytic responses against host-specific BN allodeterminants at 30 days. Consistent with these in vitro findings, limited clinical signs compatible with GVH reactivity were evident in vivo at this time. Following this initial period, the tolerant VBMT animals returned to normal clinical condition and remained otherwise healthy throughout the study. Consistent with these results, VBMT chimeras then showed declining proliferative responses from the elevated values seen at 30 days against self-LEW determinants. Proliferative and immunocytolytic responses also decreased against host-specific BN allodeterminants from peak levels at 30 days. In conclusion, these results provide evidence that the initial phases of tolerance induction in VBMT chimeras consist of self- and alloimmune regulation that follow an early period of immune dysregulation. Sequential phases of immune dysregulation and re-regulation elucidated in VBMT stable mixed chimeras within the first 100-day period may represent important mechanisms of tolerance induction.

Effective graft-versus-leukemia effects independent of graft-versus-host disease after T cell-depleted allogeneic bone marrow transplantation in a murine model of B cell leukemia/lymphoma. Role of cell therapy and recombinant IL-2.

After allogeneic bone marrow transplantation (BMT) for leukemia, beneficial graft-vs-leukemia (GVL) effects are usually accompanied by potentially serious graft-vs-host disease (GVHD). Because T cell depletion is the only effective way to prevent GVHD it seems important to understand whether effective GVL can develop after BMT with T cell depletion in GVHD-free recipients. Well-established C57BL/6-->BALB/c chimeras that were free of GVHD, reconstituted with T cell-depleted allogeneic bone marrow cells, and inoculated 3 mo after BMT with a high inoculation of murine B cell leukemia (BCL1) showed no evidence of disease, whereas all control mice developed leukemia and died within 58 days. Results from adoptive transfer experiments in secondary naive BALB/c recipients indicated that all BCL1 cells were eliminated in the chimeras within 14 days. Hence, complete resistance to BCL1 developed in the chimeras despite complete tolerance to host alloantigens. The GVL effects observed in tolerant chimeras were further amplified by administration of immunocompetent allogeneic C57BL/6 spleen cells, low dose rIL-2, or both for 5 days. Our data suggest that GVL effects can develop even after T cell depletion in the absence of clinically overt GVHD and that GVL can be further amplified by rIL-2, either with or without use of additional immunocompetent donor T cells. Our data may provide the basis for new approaches to induce effective GVL after allogeneic BMT with cell therapy and rIL-2 at the stage of minimal residual disease, while avoiding early GVHD induced by the BMT procedure.

Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination

A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

Attenuation of Graft-Versus-Host Disease and Graft Rejection by Ex Vivo Immunotoxin Elimination of Alloreactive T Cells in an H-2 Haplotype Disparate Mouse Combination

AbstractA mouse anti—interleukin-2 receptor A-chain—specific PC61 -immunotoxin (PC61 -IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61 -IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodeple-tion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61 -IT— treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allodepletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 X 106 T-cell-depleted BM cells and 0.5 X 106 PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

Do donor cells function as veto cells in the induction and maintenance of tolerance across an MHC disparity in mixed lymphoid radiation chimeras?

Mixed lymphoid chimeras can be established across H-2b-->H-2d by injection of C57BL/6J (B-6) donor bone marrow cells into BALB/c hosts conditioned by sublethal irradiation, 235 cGy x3. These chimeras are specifically tolerant to both donor and host alloantigens. Tolerance cannot be broken even by injection of 4 x 10(8) normal BALB/c spleen cells (SC), suggesting a suppressor mechanism. In contrast to conventional suppression, however, in which suppressors are syngeneic to the cells they suppress, tolerance can be transferred only by cells of the allogeneic donor allotype (1,2). Thy 1+ cell depletion eliminates the capacity of the donor population to transfer tolerance and markedly reduces the capacity of B-6 BMC to induce tolerance (3). In the present studies spleen cells from tolerant chimeras (CSC), when coinjected in a 1:1 ratio, reduced the high GVHD mortality induced in irradiated H-2d/b F1 hybrids by injection of 2 x 10(7) normal BALB/c SC alone (P < .01). When coinjected at a 5:1 ratio CSC eliminated the GVHD mortality and weight loss induced by 5 x 10(6) BALB/c SC (P < .01). Depletion of B-6 cells from CSC removed their capacity to inhibit the GVHD induced by normal BALB/c SC. In contrast to conventional suppression, which requires the continued presence of suppressor cells, the BALB/c cells isolated from CSC, although unable to inhibit the GVHD of normal BALB/c SC, remained nonreactive against the H-2b allotype for a prolonged period following depletion of the B-6 cells. This prolonged response reduction dependent upon some interaction with allogeneic donor cells--which, in turn, are specifically nonreactive to host antigens--is compatible with a veto mechanism.

INDUCTION OF STABLE CHIMERISM AND TRANSPLANTATION TOLERANCE TO RAT ISLET AND HEART ALLOGRAFTS BY ULTRAVIOLET-B MODULATION OF BONE MARROW CELLS

Ultraviolet-B irradiation (UV-B) (700 J/m2) of BM cells prior to transplantation into lethally gamma-irradiated (1050 rads) allogeneic rats prevents the development of GVHD and results in stable chimerism. This study was developed to determine if UV-B modulation of BMT is useful for preconditioning recipients for the induction of tolerance to donor islets and heart allografts. Lethally irradiated Lewis rats that received UV-B irradiated (700 J/m2) WF BMT (10(8) BM cells) demonstrated stable chimerism without any evidence of GVHD. The stable Lewis chimeras were made diabetic with streptozotocin (STZ) at 28-35 days after BMT and subdivided into 3 experimental groups that received 1000-1200 islets from WF, Lewis, or BN (third-party), respectively. The results showed that group I diabetic Lewis chimeras accepted permanently (greater than 300 days) BM donor WF islets and became normoglycemic. When 3 of 6 Lewis chimeras transplanted with WF islets were rechallenged with WF hearts 60 days after islet grafts, they accepted both islets and cardiac allografts permanently (greater than 240 days). Similarly, the remaining 3 animals accepted Lewis cardiac allografts permanently, thus indicating tolerance to both donor and recipient alloantigens. Group II diabetic chimeras accepted permanently (greater than 300 days) recipient (Lewis) islets. In contrast, group III chimeras rejected acutely (7-8 days) third-party (BN) islets. However, when these animals that rejected BN islets and again became diabetic were retransplanted with BM donor-type (WF) islets, they became permanently normoglycemic (greater than 200 days). This finding emphasizes the specificity of the induction of tolerance in this model and the apparent lack of organ-specific sensitization. To define the underlying mechanism of tolerance, in vivo adoptive transfer of 10(8) spleen cells to naive Lewis or WF recipients, obtained from tolerant Lewis chimeras carrying donor islets and heart allografts, showed no prolongation of cardiac allografts in the unmodified syngeneic hosts, thus questioning the role of suppressor mechanisms in the tolerant rats. Furthermore, cells from the tolerant chimeras that showed no mixed lymphocyte reaction (MLR) response to Lewis or; WF alloantigens failed to suppress anti-Lewis and anti-WF MLR-response in coculture MLR. These results suggest that tolerance to donor alloantigens in the UV-B BMT model is most likely due to selective elimination of anti-BM donor helper or effector cell precursors (clonal deletion) rather than induction of suppressor cell activity. This study demonstrates that this relatively simple and effective approach to modulation of T cells in BM treatment may be potentially useful in the induction of tolerance to donor organs.

Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance.

The ability of normal recipient-type lymphocytes to break tolerance in long-term allogenic radiation chimeras has been investigated. Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic T cell-depleted (TCD) bone marrow (BM) has previously been shown to lead to mixed chimerism and permanent, specific tolerance to donor and host alloantigen (3-5). If allogeneic T cells are not depleted from the reconstituting inoculum, complete allogeneic chimerism results; however, no clinical evidence for GVHD is observed, presumably due to the protective effect provided by syngeneic TCD BM. This model has now been used to study the effects of allogenic T cells administered in reconstituting BM inocula on stability of long-term tolerance. We have attempted to break tolerance in long-term chimeras originally reconstituted with TCD or non-TCD BM by challenging them with inocula containing normal, nontolerant recipient strain lymphocytes. tolerance was broken with remarkable ease in recipients of mixed marrow inocula in which both original BM components were TCD. In contrast, tolerance in chimeras originally reconstituted with non-TCD allogeneic BM was not affected by such inocula. Susceptibility to loss of chimerism and tolerance was not related to initial levels of chimerism per se, but rather to T cell depletion of allogeneic BM, since chimeras reconstituted with TCD allogeneic BM alone (mean level of allogeneic chimerism 98%) were as susceptible as mixed chimeras to the tolerance-breaking effects of such inocula. The possible contribution of GVH reactivity to this resistance was investigated using an F1 into parent strain combination. In these animals, the use of non-TCD F1 BM inocula for reconstitution did not lead to resistance to the tolerance-breaking effects of recipient strain splenocytes. These results suggest that the ability of T cells in allogeneic BM inocula to confer resistance to late graft failure may be related to their graft-vs.-host reactivity, even in situations in which they do not cause clinical GVHD. These findings may have relevance to the mechanism whereby T cell depletion of allogeneic BM leads to an increased incidence of late graft failure in clinical BM transplantation situations.

Alloantigen persistence in induction and maintenance of transplantation tolerance.

Infusion of parental bone marrow cells into F1 hybrids conditioned by total lymphoid irradiation (TLI) results in chimeras with a high percentage of donor-type cells, and without clinical signs of graft-vs.-host reaction. In these chimeras, a state of tolerance has been shown to be associated with paucity of cytotoxic T lymphocyte percursors (pCTL) reactive with host-type alloantigens. To determine whether the presence of tolerizing alloantigens is essential for maintenance of unresponsiveness, lymphohematopoietic cells obtained from such tolerant chimeras were transferred into supralethally irradiated recipients of two different genotypes: in one case the adoptive recipients were syngeneic with host-type cells, and in the other they were syngeneic with donor-type cells of the original chimeras, thus providing the chimeric cells with a tolerogen-free environment. After "parking" for 4 d in syngeneic donor-type mice, the transferred cells displayed a marked increase in the frequency of pCTL directed against tolerizing alloantigens, whereas a low pCTL frequency directed against the same H-2 target cells was maintained in allogeneic tolerizing-type adoptive recipients. Multiple injections of adoptive donor-type mice with tolerizing-type cells of the original chimera reestablished a low level of cytotoxic precursors. Cytotoxic activity against unrelated alloantigens was independent of the presence of tolerogen-presenting cells in the adoptively transferred mice. Our experimental model suggests that persistence of cells bearing tolerizing alloantigens is an essential requirement for maintenance of previously established tolerance.

Cellular basis of graft versus host tolerance in chimeras prepared with total lymphoid irradiation.

BALB/c mice given allogeneic (C57BL/Ka) bone marrow cells after toal lymphoid irradiation become stable chimeras approximately 80% donor-type and 20% host-type cells in the spleen. The chimeras doe not develop graft vs. host disease (GVHD). Purified cells of C57BL/Ka origin from the chimeras mediated GVHD in lightly irradiated C3H (third party), but not in BALB/c (host-strain) mice. Thus graft vs. host tolerance in the chimeras could not be explained by complete immunodeficiency of donor-type cells, serum blocking factors, or suppressor cells of host (BALB/c) origin. Clonal deletion or suppression of lymphocytes reactive with host tissues remain possible explanations. The transfer of donor-type chimeric spleen cells to BALB/c recipients given 500-550 rad whole-body irradiation WBI led to stable mixed chimerism in approximately 50% of recipients. The cells were presumably acting as tolerogens because similarly irradiated BALB/c mice given (BALB/c X C57BL/Ka)F1 spleen or bone marrow cells also became stable mixed chimeras.

Nonspecific suppressor elements in murine allogeneic radiation chimeras

Spleen cells from long-term mouse allogeneic radiation chimeras were tested for their ability to modulate the graft-versus-host (GVH) or plaque-forming cell (PFC) response of normal lymphocytes transplanted in lethally X-irradiated recipients. In vivo GVH proliferation of normal lymphocytes (syngeneic to donor cells of the chimera) against antigens of host-type in which the chimeric state had been established was reduced by chimera cells. Inhibition varied, some chimeras suppressing GVH more than others and a few not suppressing at all. The suppressive effect was abrogated if the chimera cells were treated with anti-θ; treatment with anti-IgM did not eliminate this activity. When mixtures of normal donor lymphocytes and chimera cells were given to irradiated recipients genetically different from host or donor, reduction of donor cell GVH also occurred. Further, chimera cells reduced the GVH activity of normal host cells in irradiated recipients differing from the host at one H-2 locus and from the donor at minor histocompatibility loci. The modulating effect of spleen cells from chimeras on the PFC response by normal lymphocytes also varied. Six chimeras induced a 25 to 90% suppression, two enhanced the response, and one showed no effect. Where suppression occurred, treatment of chimera cells with anti-θ most often, but not always, restored PFC production. Our results show that the suppressive action of splenic lymphoid cells by chimeras is highly nonspecific and variable in expression. We suggest that tolerance in chimeras may be mediated by nonspecific suppressor elements leading to unresponsiveness to a variety of antigens including SRBC.

Induction de la tolérance aux allogreffes de peau dans les chimères de l’amphibien modèle, Pleurodeles waltlii Michah.

ABSTRACT Induction of skin allograft tolerance in chimaeras of urodele amphibian, Pleurodeles waltlii Michah Allogenic chimaeras were produced in Pleurodeles waltlii (Amphibian, Urodele) by associating the anterior and the posterior parts of two different embryos. A reciprocal exchange between two homologue embryos gave rise to reciprocal allogenic chimaeras. Skin grafts performed in the adult stage between two reciprocal chimaeras are always tolerated. On the other hand, when two skin grafts coming from the anterior and the posterior part of one chimaera are grafted on a common host, one of them may be tolerated and the other rejected : each part of a chimaera keeps its own antigenicity. In allogenic chimaeras, the average natural tolerance for skin allografts is 30 % higher than in sibling controls.

Cellular control of lymphocytes initiating graft vs. host reactions

Spleen and lymph nodes of rats, in which a state of transplantation tolerance has been deliberately abolished by adoptive transfer, contain cells which can inhibit syngeneic lymphocytes from normal donors in the initiation of a graft versus host reaction. The suppressive effect shows immunologic specificity. Suppressor cells were not detected in tolerant chimeras.