Transplantation Tolerance In Mice Research Articles

A Review of Cyclophosphamide-Induced Transplantation Tolerance in Mice and Its Relationship With the HLA-Haploidentical Bone Marrow Transplantation/Post-Transplantation Cyclophosphamide Platform.

The bone marrow transplantation (BMT) between haplo-identical combinations (haploBMT) could cause unacceptable bone marrow graft rejection and graft-versus-host disease (GVHD). To cross such barriers, Johns Hopkins platform consisting of haploBMT followed by post-transplantation (PT) cyclophosphamide (Cy) has been used. Although the central mechanism of the Johns Hopkins regimen is Cy-induced tolerance with bone marrow cells (BMC) followed by Cy on days 3 and 4, the mechanisms of Cy-induced tolerance may not be well understood. Here, I review our studies in pursuing skin-tolerance from minor histocompatibility (H) antigen disparity to xenogeneic antigen disparity through fully allogeneic antigen disparity. To overcome fully allogeneic antigen barriers or xenogeneic barriers for skin grafting, pretreatment of the recipients with monoclonal antibodies (mAb) against T cells before cell injection was required. In the cells-followed-by-Cy system providing successful skin tolerance, five mechanisms were identified using the correlation between super-antigens and T-cell receptor (TCR) Vβ segments mainly in the H-2-identical murine combinations. Those consist of: 1) clonal destruction of antigen-stimulated-thus-proliferating mature T cells with Cy; 2) peripheral clonal deletion associated with immediate peripheral chimerism; 3) intrathymic clonal deletion associated with intrathymic chimerism; 4) delayed generation of suppressor T (Ts) cells; and 5) delayed generation of clonal anergy. These five mechanisms are insufficient to induce tolerance when the donor-recipient combinations are disparate in MHC antigens plus minor H antigens as is seen in haploBMT. Clonal destruction is incomplete when the antigenic disparity is too strong to establish intrathymic mixed chimerism. Although this incomplete clonal destruction leaves the less-proliferative, antigen-stimulated T cells behind, these cells may confer graft-versus-leukemia (GVL) effects after haploBMT/PTCy.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

Overexpression of Fibrinogen-Like Protein 2 Promotes Tolerance in a Fully Mismatched Murine Model of Heart Transplantation.

Fibrinogen-like protein 2 (FGL2) is an immunomodulatory protein that is expressed by regulatory T cells (Tregs). The objective of this study was to determine if recombinant FGL2 (rFGL2) treatment or constitutive FGL2 overexpression could promote transplant tolerance in mice. Although rFGL2 treatment prevented rejection of fully mismatched cardiac allografts, all grafts were rejected after stopping treatment. Next, we generated FGL2 transgenic mice (fgl2(Tg) ) that ubiquitously overexpressed FGL2. These mice developed normally and had no evidence of the autoimmune glomerulonephritis seen in fgl2(-/-) mice. Immune characterization showed fgl2(Tg) T cells were hypoproliferative to stimulation with alloantigens or anti-CD3 and anti-CD28 stimulation, and fgl2(Tg) Tregs had increased immunosuppressive activity compared with fgl2(+/+) Tregs. To determine if FGL2 overexpression can promote tolerance, we transplanted fully mismatched cardiac allografts into fgl2(Tg) recipients. Fifty percent of cardiac grafts were accepted indefinitely in fgl2(Tg) recipients without any immunosuppression. Tolerant fgl2(Tg) grafts had increased numbers and proportions of Tregs and tolerant fgl2(Tg) mice had reduced proliferation to donor but not third party antigens. These data show that tolerance in fgl2(Tg) recipients involves changes in Treg and T cell activity that contribute to a higher intragraft Treg-to-T cell ratio and acceptance of fully mismatched allografts.

Induction of tolerance through mixed chimerism.

"Mixed chimerism" refers to a state in which the lymphohematopoietic system of the recipient of allogeneic hematopoietic stem cells comprises a mixture of host and donor cells. This state is usually attained through either bone marrow or mobilized peripheral blood stem cell transplantation. Although numerous treatment regimens have led to transplantation tolerance in mice, the induction of mixed chimerism is currently the only treatment modality that has been successfully extended to large animals and to the clinic. Here we describe and compare the use of mixed chimerism to establish transplantation tolerance in mice, pigs, monkeys, and in the clinic. We also attempt to correlate the mechanisms involved in achieving tolerance with the nature of the tolerance that has resulted in each case.

Inhibiting cardiac allograft rejection with interleukin-35 therapy combined with decitabine treatment in mice

BackgroundRegulatory T (Treg) cells play a pivotal role in the maintenance of transplantation tolerance. It is of great interest to induce allograft tolerance mediated by regulatory CD4+CD25+ T cells. MethodsHere we investigated the effect of hydrodynamic IL-35-expressing plasmid injection in combination with a methyltransferase inhibitor (decitabine) on immune function and transplantation tolerance in mice. ResultsWe showed that IL-35 and decitabine stimulated the proliferation of CD4+CD25+ Tregs and suppressed CD8+ T cell proliferation in an allogenic mixed lymphocyte culture in vitro. IL-35 gene therapy and decitabine administration prolonged the survival of the transplanted heart in the heterotopic abdominal heart transplantation model in mice. ConclusionsThe possible mechanism through which IL-35 and decitabine treatment increased the survival of graft tissues is to enhance the proliferation of CD4+CD25+ Treg cells and suppress the generation and function of effector T cells. Thus, IL-35 gene therapy combined with decitabine provides a novel approach to induce transplantation tolerance.

IL-6 Induced by Staphylococcus aureus Infection Prevents the Induction of Skin Allograft Acceptance in Mice

Clinical correlations between bacterial infections and rejection suggest a hypothesis that innate immune stimulation by bacterial infections results in the production of inflammatory cytokine that facilitate bystander T-cell activation, increased alloreactivity and inhibition of tolerance induction. Previous studies demonstrated that IFNβ produced during an infection with a model bacterium, Listeria monocytogenes, prevented the induction of transplantation tolerance in mice with anti-CD154 and donor-specific transfusion (DST) (1). We investigated the impact of two clinically relevant bacterial infections at the time of transplantation on the ability of anti-CD154 and DST to induce skin allograft acceptance in mice. Staphylococcus aureus (SA) infection prevented skin allograft acceptance whereas maximally tolerated doses of Pseudomonas aeruginosa infection had no effect. SA induced an acute production of IL-6, which was necessary and sufficient for the prevention of skin allograft acceptance. Furthermore, a single pulse of methylprednisolone modulated IL-6 production during SA infection and facilitated skin allograft acceptance in SA-infected recipients. Taken together, our results suggest that bacterial infections elicit specific proinflammatory cytokines signatures that can serve as barriers to tolerance induction, and that inhibiting the production of or neutralizing these inflammatory cytokines can synergize with costimulatory blockade-based therapies to facilitate the development of transplantation tolerance.

Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice.

One of the main unresolved questions in solid organ transplantation is how to establish indefinite graft survival that is free from long-term treatment with immunosuppressive drugs and chronic rejection (i.e., the establishment of tolerance). The failure to achieve this goal may be related to the difficulty in identifying the phenotype and function of the cell subsets that participate in the induction of tolerance. To address this issue, we investigated the suppressive roles of recipient myeloid cells that may be manipulated to induce tolerance to transplanted hearts in mice. Using depleting mAbs, clodronate-loaded liposomes, and transgenic mice specific for depletion of CD11c+, CD11b+, or CD115+ cells, we identified a tolerogenic role for CD11b+CD115+Gr1+ monocytes during the induction of tolerance by costimulatory blockade with CD40L-specific mAb. Early after transplantation, Gr1+ monocytes migrated from the bone marrow into the transplanted organ, where they prevented the initiation of adaptive immune responses that lead to allograft rejection and participated in the development of Tregs. Our results suggest that mobilization of bone marrow CD11b+CD115+Gr1+ monocytes under sterile inflammatory conditions mediates the induction of indefinite allograft survival. We propose that manipulating the common bone marrow monocyte progenitor could be a useful clinical therapeutic approach for inducing transplantation tolerance.

Rapamycin Promotes the Enrichment of CD4 +CD25 hiFoxP3 + T Regulatory Cells From Naïve CD4 + T Cells of Baboon That Suppress Antiporcine Xenogenic Response In Vitro

The CD4 +CD25 +FoxP3 + regulatory T (Treg) cells play an important role in regulating the immune response. These Treg cells are present in peripheral blood and lymphoid organs and have a high potential for immunotherapy in clinics. Adoptive cell transfer therapy using CD4 +CD25 + cells has been shown to prevent autoimmune diseases and has also induced transplant tolerance in mice. Treg cells low frequency in peripheral blood will necessitate its ex vivo expansion to enable adaptive immunotherapy. Recently, it has been reported that rapamycin, an immunosuppressive agent, inhibits T-cell proliferation while selectively increasing the number of Treg cells. Based on this additional mode of action, rapamycin can be used to expand Treg cells for ex vivo cellular therapy in T-cell–mediated diseases and in transplantation. We have reported the ex vivo expansion of baboon Treg cells, using irradiated pig peripheral blood mononuclear cell (PBMC) and interleukin (IL)-2, and have demonstrated the suppression of autologus CD4 +CD25 neg T-cell proliferation in response to pig PBMCs. In the present study, we have expanded baboon CD4 + T cells in the presence or absence of rapamycin (0.1–10 nmol/L) using irradiated pig PBMCs and IL-2 to enrich the regulatory T cells. CD4 +CD25 +FoxP3 + Treg cells were increased up to 2 times in the presence of rapamycin versus without rapamycin in vitro. However, a higher dose of rapamycin (≥10 nmol/L) considerably decreases the number of Treg cells. Furthermore, purified CD4 +CD25 + Treg cells enriched from CD4 + cells in the presence of rapamycin were able to suppress the baboon anti-porcine xenogeneic immune responses in vitro up to 93% at a 1:1 ratio (Treg cells:T effector cells) and suppression ability exists even at a 1:256 ratio, whereas freshly isolated natural Treg cells suppress only 70% at 1:1 and lose their suppressive ability (>50%) at 1:16. Our results demonstrate that the addition of rapamycin to the culture enriches the Treg phenotype and induces functional regulatory T cells. This method may allow the production of large numbers of regulatory cells for the preclinical testing of Treg cell therapy in a non-human primate model.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Prevention of Allograft Tolerance by Bacterial Infection with Listeria monocytogenes

Exposure to certain viruses and parasites has been shown to prevent the induction of transplantation tolerance in mice via the generation of cross-reactive memory T cell responses or the induction of bystander activation. Bacterial infections are common in the perioperative period of solid organ allograft recipients in the clinic, and correlations between bacterial infections and acute allograft rejection have been reported. However, whether bacterial infections at the time of transplantation have any effect on the generation of transplantation tolerance remains to be established. We used the Gram-positive intracellular bacterium Listeria monocytogenes (LM) as a model pathogen because its effects on immune responses are well described. Perioperative LM infection prevented cardiac and skin allograft acceptance induced by anti-CD154 and donor-specific transfusion in mice. LM-mediated rejection was not due to the generation of cross-reactive T cells and was largely independent of signaling via MyD88, an adaptor for most TLRs, IL-1, and IL-18. Instead, transplant rejection following LM infection was dependent on the expression of the phagosome-lysing pore former listeriolysin O and on type I IFN receptor signaling. Our results indicate that bacterial exposure at the time of transplantation can antagonize tolerogenic regimens by enhancing alloantigen-specific immune responses independently of the generation of cross-reactive memory T cells.

Nitric Oxide and Indoleamine 2,3-Dioxygenase Mediate CTLA4Ig-Induced Survival in Heart Allografts in Rats

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) leads to transplantation tolerance in mice depending on indoleamine 2,3-dioxygenase (IDO). We have shown that CTLA4Ig induces indefinite heart allograft survival in rats and that nitric oxide (NO) was implicated in the in vitro active tolerogenic mechanisms mediated by dendritic cells (DCs). Here we studied the in vivo tolerogenic mechanisms by which CTLA4Ig induces graft survival in rats receiving a cardiac allograft. Treatment of recipients with the IDO inhibitor 1-methyltryptophan (1-MT) did not abrogate the indefinite graft survival observed with CTLA4Ig alone. This was also the case after administration of the inducible nitric oxide synthase inhibitor aminoguanidine when again, indefinite allograft survival was maintained. However, administration of both inhibitors led to acute rejection. We show that IDO and NO are responsible for the impaired capacity of DCs from CTLA4Ig-treated rats to stimulate allogeneic T cells. In conclusion, we show that NO and IDO mediate CTLA4Ig-induced tolerance in rat allograft recipients.

The influence of cyclosporin A on lymphocyte attenuator expression

B and T lymphocyte attenuator (BTLA), a recently identified immune inhibitory receptor, has been demonstrated to have the ability to maintain self-tolerance and transplant-tolerance in mice. However, little is known about the effects of immunosuppressive drugs on the expression of BTLA. In the present study, we observed that the immunosuppressive drug cyclosporin A (CsA) could significantly reduce BTLA but not CD25 and CD69 expression on CD4 + T cells during activation in vitro, while rapamycin (RPM) had little effect on it. Exogenous interleukin-2 (IL-2) failed to reverse the inhibitory effect that CsA had on BTLA expression. Furthermore, phorbol 12-myristate 13-acetate (PMA) or ionomycin alone could efficiently induce BTLA protein expression on CD4 + and CD8 + T cells, while CsA significantly suppressed BTLA expression in this system. The present data indicate that the regulation of BTLA expression on CD4 + T cells does not depend on IL-2 and T cell activation but depends on calcineurin-dependent and calcineurin-independent pathways. The observation that CsA significantly inhibits BTLA expression on CD4 + T cells during activation, suggests that CsA might block the immune tolerance induced by BTLA and potentially increase the susceptibility to autoimmune diseases and graft rejection.

Mixed chimerism as an approach to transplantation tolerance.

The induction of tolerance to transplanted organs could make transplantation safer and more uniformly successful. One of the most promising approaches currently being investigated involves the induction of deletional tolerance through the establishment of "mixed chimerism." In this laboratory, we first studied mixed chimerism as an approach to transplantation tolerance in mice, using a nonmyeloablative preparative regimen consisting of 300 R whole-body irradiation, 700 R thymic irradiation, and treatment with monoclonal antibodies to CD4 and CD8. This approach has subsequently been extended successfully to the induction of tolerance to renal transplants in fully mismatched cynomolgus monkeys. In addition, the same approach, with minor modifications, has been found effective in producing mixed chimerism and transplantation tolerance in the concordant xenogeneic baboon to cynomolgus monkey species combination. Because pigs have many advantages as a potential xenograft donor for humans, we are also trying to extend our nonmyeloablative regimen for production of mixed chimerism to the discordant pig --> primate combination. We have used absorption of natural antibodies to prevent hyperacute rejection and then proceeded with a mixed chimerism approach. Administration of pig hematopoietic stem cells along with pig recombinant cytokines (SCF and IL-3) to primates has enabled the pig bone marrow to survive in these xenogeneic hosts for over 6 months. This chimerism has apparently been sufficient to markedly diminish T cell immunity and the induction of new T-cell-dependent responses. However, to date we have not succeeded in preventing the return of natural antibodies, which appear to be the cause of eventual loss of organ transplants and are the subject of further intensive investigations.

Autoimmunity, hyporeactivity to T cell mitogens and lymphoproliferative disorders following neonatal induction of transplantation tolerance in mice

We have reported that, in A/J (A) (H-2a) mice, a partial tolerance to C57BL/10ScSn (B10) (H-2b) skin allografts and a high incidence of lethal lymphoproliferative disorders (LPD) can be induced by the neonatal i.v. injection of 2 x 10(7) semiallogeneic (B10 x A)F1 spleen cells (SC) (Végh, P., Baranyi, L. and Jánossy, T., Cell. Immunol. 1990, 129: 56). In this study, we show that the incidence and mortality of LPD were continuously growing from 1 month of age until the end of the experiment at 1 year (64% and 36%, respectively). Based on histology, 27% of the diseased mice suffered from lymphoid malignancies. In the remaining cases (73%), reactive histopathological changes were seen in the spleen, lymph nodes (LN), liver and kidneys. The proportion of CD4+ T cells in the spleen and LN as well as that of splenic B cells decreased, while the percentages of mature and immature myeloid cells doubled. The total cell number of each (sub)population, however, was elevated in both lymphoid organs. The cells taking part in the lymphoproliferation were of host (A) and not of donor (F1) origin. Preceding the development of apparent LPD, the SC, LN cell and thymus cell suspensions of 1-month-old tolerized mice showed reduced in vitro proliferative responses to T cell and T cell-dependent B cell mitogens (Con A or PWM), while their reactivity to a T cell-independent B cell mitogen (lipopolysaccharide) was essentially unimpaired. This hyporeactivity seems to be functional, because neither histology nor immunophenotyping by flow cytometry revealed significant alterations in the spleen and thymus of such animals, apart from a slight reduction in the ratio of CD4+/CD8+ T cell subpopulations in the spleen. The in vivo T cell-mediated immune response of the tolerized mice was practically normal to third party CBA/Ca (H-2k) allografts. Antithymocyte autoantibodies (ATA) were detected in the sera of 76% of the tolerized mice at 1 month of age (i.e., even before the mass appearance of LPD). ATA as well as antinuclear Ab were present in 65% of the adult tolerized mice, independently of the presence of LPD. Taken together, in A mice neonatally injected with (B10 x A)F1 SC, a partial, specific allograft tolerance and a chronic host-vs.-graft disease-like syndrome developed. The latter is manifested in hyporeactivity to T cell mitogens, development of autoantibodies and, subsequently, in progressive LPD and lymphoid malignancies.

Induction of transplantation tolerance in mice across major histocompatibility barrier by using allogeneic thymus transplantation and total lymphoid irradiation.

The use of allogeneic thymus transplantation as a means of inducing tolerance across MHC barriers was investigated in thymectomized, total lymphoid irradiated BALB/c mice. In 90% of the animals long term outgrowth of histologically normal C57BL thymus grafts was observed. None of the latter animals was chimeric. All thymus graft-bearing mice showed specific nonresponsiveness for C57BL MHC Ag in mixed lymphocyte reaction and cell-mediated lympholysis. Spleen cells of the C57BL thymus-bearing mice were unable to induce lethal graft-vs-host disease in neonatal (BALB/c X C57BL) F1 mice but provoked a vigorous graft-vs-host disease reaction in (BALB/c x C3H) F1 neonates. Tolerant mice permanently accepted C57BL heart and pancreas grafts, but all rejected C3H grafts. Induction of tolerance of BALB/c pre-T cells through allogeneic thymus graft and/or specific suppressor cells seems to be involved. The present model offers new opportunities to study thymocyte maturation in a fully allogeneic environment and may yield applications for clinical organ transplantation.

Management of subdural empyema: a series of 24 cases.

Twenty four cases of subdural empyema are reviewed. The overall mortality was 17%, 18 patients were managed by burr hole and five by craniotomy or craniectomy. Antibiotic therapy was commenced once pus had been evacuated. Infection of the paranasal and mastoid sinuses was the commonest aetiological factor. Aspiration of pus through burr holes is the recommended surgical procedure with low mortality and morbidity, when combined with early diagnosis and aggressive chemotherapy.

Autoimmunity and immune complex disease after neonatal induction of transplantation tolerance in mice.

Mice made neonatally tolerant to alloantigens were found to develop an immunologic disease resembling systemic lupus erythematosus. In BALB/c mice neonatally injected with C57BL/6 X BALB/c F1 hybrid spleen cells, features of autoimmunity were observed first. After 5-24 wk, antinuclear, anti-SS DNA, thymocytotoxic, and rheumatoid factor-like antibodies were detected in association with hypergammaglobulinemia and with the occurrence of circulating immune complexes and cryoglobulins. Some of the antinuclear antibodies were found to be produced by F1 donor B cells persisting in the host. Second, immunopathologic changes were detected in tolerant mice. In the kidneys, an immune complex glomerulonephritis of the membranous type was observed. Immunoglobulin deposits were also found in the choroid plexus and at the dermoepidermal junction. In addition, thrombocytopenia was a common finding, and a positive direct Coomb's test occasionally was detected. Features of autoimmune disease were closely associated with the effective induction of transplantation tolerance, as revealed by the inability of spleen cells to generate in vitro cytolytic responses against C57BL/6 alloantigens. It is suggested that, although transplantation tolerance is associated with a lack of cytolytic reaction of the host against F1 hybrid donor alloantigens, other types of allogeneic interactions could lead in this model to the development of autoimmunity and immunopathology.