Megan Sykes

HLA-Mismatched Renal Transplantation without Maintenance Immunosuppression

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.

Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys.

We have developed a nonmyeloablative preparative regimen that can produce mixed chimerism and renal allograft tolerance between MHC-disparate nonhuman primates. The basic regimen includes ATG, nonmyeloablative total-body irradiation (TBI, 300 rads), thymic irradiation (TI, 700 rads), and donor bone marrow infusion. Kidney allografts from MHC-mismatched donors were transplanted with various manipulations of the preparative regimen. Monkeys treated with the basic regimen alone (n = 2) rejected allografts by day 15. With the addition of cyclosporine (CsA) for one month (n = 3), one monkey developed multilineage mixed chimerism and renal allograft tolerance thereafter (> 430 days). To reduce the toxicity of the preparative regimen, TBI was fractionated to 150 rads on two successive days in subsequent studies. All monkeys receiving this modified regimen (n = 4) developed multilineage chimerism with fewer side effects and accepted renal allografts long-term with no further immunosuppression (196 days, 198 days, > 150 days, and > 40 days). In long-term survivors, donor-specific nonreactivity was confirmed by MLR and skin transplantation. Three monkeys treated with the basic regimen plus CsA but with only 150 rads of TBI (n = 1) or no TBI (n = 2) did not develop multilineage chimerism and grafts were rejected (day 40-50) soon after the CsA discontinuation. Monkeys treated with the same regimen, but without DBM (n = 2), rejected kidney allografts by day 52. Therefore, at least transient engraftment of DBM appears to be essential for induction of donor specific tolerance in this monkey model.

Allogeneic bone marrow transplantation with co-stimulatory blockade inducesmacrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques (‘macrochimerism’ or ‘mixed chimerism’). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.

Marked prolongation of porcine renal xenograft survival in baboons through the use of α1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue

The use of animal organs could potentially alleviate the critical worldwide shortage of donor organs for clinical transplantation. Because of the strong immune response to xenografts, success will probably depend upon new strategies of immune suppression and induction of tolerance. Here we report our initial results using α-1,3-galactosyltransferase knockout (GalT-KO) donors and a tolerance induction approach. We have achieved life-supporting pig-to-baboon renal xenograft survivals of up to 83 d with normal creatinine levels.

Mixed lymphohaemopoietic chimerism and graft-ver suslymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation

BACKGROUND HLA-mismatched donor bone-marrow transplantation after standard myeloablative conditioning therapy for haematological malignant disorders has been limited by severe graft-versus-host disease (GVHD) and graft failure. We tested a new approach to find out whether lymphohaemopoietic graft-versus-host reactions could occur without excessive GVHD in mixed haemopoietic chimeras produced across HLA barriers with non-myeloablative conditioning. METHODS Five patients with refractory non-Hodgkin lymphoma underwent bone-marrow transplantation from haploidentical related donors sharing at least one HLA A, B, or DR allele on the mismatched haplotype. Conditioning included cyclophosphamide and thymic irradiation before transplantation, and antithymocyte globulin before and after transplantation. The only other GVHD prophylaxis was cyclosporin. FINDINGS Four of five patients were evaluable and showed engraftment. Mixed haemopoietic chimerism was established, with a predominance of donor lymphoid tissue and varying degrees of myeloid chimerism. Two patients were in GVHD-free states of complete and partial clinical remission at 460 and 103 days after bone-marrow transplantation. INTERPRETATION Mixed chimerism can be induced in adult recipients of HLA-mismatched bone-marrow transplantation by a non-myeloablative conditioning regimen. The antilymphoma responses seen in two patients suggest that allogeneic bone-marrow transplantation without myeloablative conditioning might have potent immunotherapeutic benefits.

Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease : The induction of allograft tolerance through mixed lymphohematopoietic chimerism

BACKGROUND Experimental and clinical evidence has demonstrated that the establishment of allogeneic chimerism after bone marrow transplantation may provide donor-specific tolerance for solid organ allografts. METHODS Based on the preliminary results of a clinical trial using nonmyeloablative preparative therapy for the induction of mixed lymphohematopoietic chimerism, we treated a 55-year-old woman with end stage renal disease secondary to multiple myeloma with a combined histocompatibility leukocyte antigen-matched bone marrow and renal transplant after conditioning with cyclophosphamide, antithymocyte globulin, and thymic irradiation. RESULTS The posttransplant course was notable for early normalization of renal function, the absence of acute graft-versus-host disease, and the establishment of mixed lymphohematopoietic chimerism. Cyclosporine, which was the only posttransplant immunosuppressive therapy, was tapered and discontinued on day +73 posttransplant. No rejection episodes occurred, and renal function remains normal on day + 170 posttransplant (14 weeks after discontinuing cyclosporine). Although there is presently no evidence of donor hematopoiesis, there is evidence of an ongoing antitumor response with a recent staging evaluation showing no measurable urine kappa light chains. The patient remains clinically well and is off all immunosuppressive therapy. CONCLUSION This is the first report of the deliberate induction of mixed lymphohematopoietic chimerism after a nonmyeloablative preparative regimen to treat a hematological malignancy and to provide allotolerance for a solid organ transplant.

Mixed chimerism and transplant tolerance.

Tolerance induced by mixed chimerism in place of chronic immunosuppressive therapy would be a drastic departure from current clinical practice in allograft recipients. Thus, efforts to translate from the above rodent models into clinical trials must be interceded by preclinical, large animal studies to document the safety and efficacy of the approach. Chimerism has generally been more difficult to induce in large animals than in rodents, in large part due to differences in the types and amounts of irradiation and reagents used in the different species. For example, T cell–depleting antibodies that accomplish the level of T cell depletion that has been achieved in mouse models for mixed chimerism induction have not been evaluated in primate models. Stable mixed chimerism has recently been achieved in MHC-identical (but not MHC-mismatched) dogs using a non-myeloablative irradiation protocol involving a limited course of pharmacological immunosuppression after BMT (Storb et al., 1997xStable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Storb, R., Yi, C., Wagner, J.L., Deeg, J., Nash, R.A., Kiem, H.-P., Leisenring, W., and Shulman, H. Blood. 1997; 89: 3048–3054PubMedSee all References(Storb et al., 1997). Recent results in a porcine model (Fuchimoto et al. 2000xMixed chimerism and tolerance without whole body irradiation in a large animal model. Fuchimoto, Y., Huang, C.A., Yamada, K., Shimizu, A., Kitamura, H., Colvin, R.B., Ferrara, V., Murphy, M.C., Sykes, M., White-Scharf, M. et al. J. Clin. Invest. 2000; 105: 1779–1789Crossref | PubMedSee all References, Huang et al. 2000xStable mixed chimerism and tolerance using a non-myeloablative preparative regimen in a large animal model. Huang, C.A., Fuchimoto, Y., Sheir-Dolberg, R., Murphy, M.C., Neville, D.M. Jr., and Sachs, D.H. J. Clin. Invest. 2000; 105: 173–181Crossref | PubMedSee all References) have applied approaches developed in mice to achieve mixed chimerism across MHC barriers. Cynomolgus monkeys conditioned with anti-thymocyte globulin (ATG), fractionated TBI (3 Gy), local TI (7 Gy), and splenectomy before transplantation of MHC-mismatched bone marrow and kidney grafts, followed by 4 weeks of CyA treatment, achieve transient multilineage chimerism in association with long-term kidney graft acceptance (Kawai et al., 1995xMixed allogeneic chimerism and renal allograft tolerance in cynomologous monkeys. Kawai, T., Cosimi, A.B., Colvin, R.B., Powelson, J., Eason, J., Kozlowski, T., Sykes, M., Monroy, R., Tanaka, M., and Sachs, D.H. Transplantation. 1995; 59: 256–262Crossref | PubMedSee all References(Kawai et al., 1995). While these studies provide important proof of principle, in both large animal studies the level of T cell depletion achieved with available reagents was far inferior to that achieved in the murine model upon which they are based.Donor lymphocyte infusions (DLI) convert mixed chimeras to full chimeras without causing GVHD (Sykes et al., 1988xGraft-versus-host-related immunosuppression is induced in mixed chimeras by alloresponses against either host or donor lymphohematopoietic cells. Sykes, M., Sheard, M.A., and Sachs, D.H. J. Exp. Med. 1988; 168: 2391–2396Crossref | PubMedSee all References(Sykes et al., 1988). This observation provided an opportunity to evaluate a protocol for mixed chimerism induction with non-myeloablative conditioning in patients with hematologic malignancies (Spitzer et al., 1999bxThe intentional induction of mixed chimerism and achievement of anti-tumor responses following non-myeloablative conditioning therapy and HLA-matched and mismatched donor bone marrow transplantation for refractory hematologic malignancies. Spitzer, T.R., MCafee, S., Sackstein, R., Colby, C., Toh, H.C., Multani, P., Saidman, S., Weymouth, D., Preffer, F., Poliquin, C. et al. Biol. Blood Marrow Transplant. 1999; 6: 309–320Abstract | Full Text PDFSee all References(Spitzer et al., 1999b), in an effort to exploit lymphohematopoietic GVH reactions induced by DLI in mixed chimeras to achieve graft-versus-tumor effects without GVHD. The potential of this approach to induce transplantation tolerance was evaluated in a patient who had a hematologic malignancy, multiple myeloma, and consequent renal failure. She received a simultaneous bone marrow and renal allograft from her HLA-identical sister, and has now accepted her kidney graft without any immunosuppression for over 2 years. Similar to the primate model described above, in which BMT has been shown to be essential for tolerance induction (Kawai et al., 1995xMixed allogeneic chimerism and renal allograft tolerance in cynomologous monkeys. Kawai, T., Cosimi, A.B., Colvin, R.B., Powelson, J., Eason, J., Kozlowski, T., Sykes, M., Monroy, R., Tanaka, M., and Sachs, D.H. Transplantation. 1995; 59: 256–262Crossref | PubMedSee all References(Kawai et al., 1995), chimerism in this patient was only transient (Spitzer et al., 1999axCombined HLA-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease (the induction of allograft tolerance through mixed lymphohematopoietic chimerism) . Spitzer, T.R., Delmonico, F., Tolkoff-Rubin, N., MCafee, S., Sackstein, R., Saidman, S., Colby, C., Sykes, M., Sachs, D.H., and Cosimi, A.B. Transplantation. 1999; 68: 480–484Crossref | PubMed | Scopus (307)See all References(Spitzer et al., 1999a), suggesting that the kidney graft itself may participate in tolerance induction and/or maintenance after chimerism has played its initial role. Because T cell depletion is only partial in these models, it is clear that the long-term central, deletional tolerance described above in murine models has not yet been achieved with non-myeloablative conditioning in monkeys or humans.Other clinical trials have involved the administration of donor bone marrow from HLA-mismatched cadaveric or living renal or liver allograft donors, without host myelosuppressive or lymphoablative treatment, with the use of conventional chronic immunosuppressive therapy posttransplant. These studies were based on the observation that many long-term organ allograft recipients spontaneously exhibit lasting microchimerism (Starzl et al., 1992xCell migration, chimerism and graft acceptance. Starzl, T.E., Demetris, A.J., Murase, N., Ildstad, S., Ricordi, C., and Trucco, M. Lancet. 1992; 339: 1579–1582Abstract | PubMed | Scopus (799)See all References(Starzl et al., 1992), which is only measurable by highly sensitive techniques. “Microchimerism” should be distinguished from the mixed chimerism discussed above, in which multilineage chimerism is readily measurable by flow cytometry. Numerous attempts have been made to demonstrate a relationship between microchimerism and tolerance. While under certain circumstances microchimerism may contribute to a state of operational tolerance by poorly defined mechanisms, tolerance is by no means assured in the presence of microchimerism (Schlitt et al., 1994xSystemic microchimerism of donor-type associated with irreversible acute liver graft rejection eight years after transplantation. Schlitt, H.J., Hundreiser, J., Ringe, B., and Pichlmayr, R. N. Engl. J. Med. 1994; 330: 646–647Crossref | PubMed | Scopus (114)See all References(Schlitt et al., 1994). The administration of donor marrow with solid organ allografts with standard chronic immunosuppressive therapy has augmented microchimerism, but no significant impact on acute rejection episodes or immunosuppressive medication doses has yet been reported (Rugeles et al., 1997xEvidence for the presence of multilineage chimerism and progenitors of donor dendritic cells in the peripheral blood of bone marrow-augmented organ transplant recipients. Rugeles, M.T., Aitouche, A., Zeevi, A., Fung, J.J., Watkins, S.C., Starzl, T.E., and Rao, A.S. Transplantation. 1997; 64: 735–741Crossref | PubMed | Scopus (23)See all References(Rugeles et al., 1997).The recent development of depleting, humanized monoclonal antibodies against human T cells and against costimulatory molecules raises hope that the advances in rodent models described above in the development of minimal, nontoxic host conditioning regimens for mixed chimerism induction will soon be applied in large animal models and humans. Such efforts are likely to require more than one of these new reagents and thus will depend on the willingness of biotechnology firms to share technologies in order to optimize progress toward this goal.

Organ transplantation—how much of the promise has been realized?

Since the introduction of organ transplantation into medical practice, progress and optimism have been abundant. Improvements in immunosuppressive drugs and ancillary care have led to outstanding short-term (1–3-year) patient and graft survival rates. This success is mitigated by several problems, including poor long-term (>5-year) graft survival rates, the need for continual immunosuppressive medication and the discrepancy between the demand for organs and the supply. Developing methods to induce transplant tolerance, as a means to improve graft outcomes and eliminate the requirement for immunosuppression, and expanding the pool of organs for transplantation are the major challenges of the field.

Tolerance and Cancer: Mechanisms of Tumor Evasion and Strategies for Breaking Tolerance

The development of malignant disease might be seen as a failure of immune surveillance. However, not all tumors are naturally immunogenic, and even among those that are immunogenic, the uncontrolled rapid growth of a tumor may sometimes out-run a robust immune response. Nevertheless, recent evidence suggests that mechanisms of tolerance that normally exist to prevent autoimmune disease may also preclude the development of an adequate antitumor response and that tumors themselves have the ability to thwart the development of effective immune responses against their antigens. A major challenge has been to develop approaches to breaking this tolerance in tumor-bearing hosts, and recent advances in our understanding of antigen presentation and tolerance have led to some promising strategies. An alternative approach is to use T cells from nontumor-bearing, allogeneic hosts in the form of lymphocyte infusions, with or without hematopoietic cell transplantation. Immunotherapy may occur in this setting via the response of nontolerant, tumor antigen-specific T cells from nontumor-bearing hosts or via the powerful destructive effect of an alloresponse directed against antigens shared by malignant cells in the recipient. Approaches to exploiting this beneficial effect without the deleterious consequence of graft-versus-host disease in allogeneic hematopoietic cell recipients are discussed.

Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies.

Mixed lymphohematopoietic chimerism can be induced in mice with bone marrow transplantation (BMT) after a nonmyeloablative preparative regimen that includes cyclophosphamide, anti-T-cell antibody therapy, and thymic irradiation. These mixed chimeras are resistant to the induction of graft-versus-host disease (GVHD) after delayed donor leukocyte infusions (DLIs), despite a potent lymphohematopoietic graft-versus-host reaction that converts the mixed chimeric state to a full donor one. Based on this animal model, we initiated a trial of nonmyeloablative therapy with HLA-matched or -mismatched donor BMT and DLI for refractory hematologic malignancies. Twenty-one of 36 patients enrolled in this trial received a genotypically (n = 20) or phenotypically (n = 1) HLA-matched donor transplant; results reported here are for those patients only. Preparative therapy consisted of cyclophosphamide in doses of 150 to 200 mg/kg; peritransplant antithymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal radiation therapy); and cyclosporine. Eighteen of 20 evaluable patients developed persistent mixed lymphohematopoietic chimerism as defined by >1% donor peripheral white blood cells until at least day 35 posttransplantation. Ten patients received prophylactic DLI beginning 5 to 6 weeks after BMT for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-leukemia effect. Fourteen of 20 evaluable patients (70%) achieved an antitumor response; 8 of these responses were complete, and 6 were partial. Of the 8 evaluable patients who received prophylactic DLI, 6 showed conversion to full donor chimerism. Five of the 9 evaluable patients (56%) who received prophylactic DLI achieved a complete response, compared with 3 of 11 patients (27%) who did not receive prophylactic DLI. Currently 11 patients are alive, and 7 of these are free of disease progression at a median follow-up time of 445 days (range, 105-548 days) posttransplantation. Transplantation-related complications included cyclophosphamide-induced cardiac toxicity in 3 of 21 patients (14%) and grade II or greater GVHD in 6 patients (29%). One patient (5%) died from a complication of BMT, and 1 patient (5%) died from GVHD after 2 prophylactic DLIs were given for conversion of chimerism. In summary, mixed lymphohematopoietic chimerism was reproducibly induced after a novel nonmyeloablative preparative regimen incorporating chemotherapy, peritransplant antithymocyte globulin, and thymic irradiation, allowing for early administration of DLI in 10 of 21 patients. After treatment, striking antitumor responses were observed in the majority of patients with chemotherapy-refractory hematologic malignancies.