Prospects and strategies for clinical tolerance

Prospects and Strategies for Clinical Tolerance A.P. Monaco ABSTRACT The morbidity and mortality associated with chronic immunosuppression provide a strong motivation for development of clinical tolerance. This paper discusses the definition(s) of clinical (operational) tolerance, the role of chimerism in experimental and clinical tolerance, and the special role of bone marrow in tolerance induction. The states of microchimerism and macrochimerism are defined and related to certain clinical observations in solid organ transplantation. Current clinical strategies already being tested in the clinic are briefly reviewed. Certain principles for induction of clinical (operational) tolerance are elaborated.

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HE ENORMOUS progress made in clinical organ transplantation is one of the great medical success stories. This fact has been accomplished by exponential growth in our understanding of the immunobiology of transplantation and the development of increasingly effective and less toxic methods of immunosuppression. In spite of such progress, there is strong motivation and justification for developing methods to induce clinical transplantation tolerance; current methods of immunosuppression are associated with infection, spontaneous neoplasm, undesirable metabolic effects, drug toxicity, and long-term failure to control rejection. Thus, although the 1-year success rate for renal allografts has increased from 60% to 90% over the past three decades, the annual rate of graft loss through graft failure or death has been relatively constant.1 After 1 year, 50% of graft losses are due to chronic or acute allograft rejection or chronic toxic effects of calcineurin inhibitors and 50% are due to death from cardiovascular or oncologic etiologies; these results reflect the inadequacy of current chronic immunosuppressive therapy in terms of effectiveness and/or toxicity. The toxic effect of calcineurin inhibitors is clearly evident in the documented loss of renal function in uvcitis patients treated with cyclosporine,2 the dramatically increased incidence of chronic renal insufficiency and end-stage renal disease in extrarenal organ transplant recipients,3 and the finding of histologic damage on protocol renal allograft biopsies in both cyclosporineand tacrolimus-treated patients.4 Furthermore, the cumulative incidence of some type of spontaneous neoplasia has been estimated to be 40% to 60% 20 years after successful transplantation.5 Strategies to minimize the toxic effects of chronic immunosuppression have emphasized the development of less © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 227⫺231 (2004)

toxic, more effective immunosuppressive agents, which could permit avoidance of agents such as calcineurin inhibitors and steroids. Other approaches focus on minimalization of immunosuppression requirements by utilizing protocols to induce donor specific hyporesponsiveness—a concept introduced by Calne6—so-called prope tolerance—to achieve essentially a minimal immunosuppressive tolerant state. The ultimate strategy is to induce donor specific allo-antigen unresponsiveness; that is, a state of true or operational tolerance. CLINICAL (OPERATIONAL) TOLERANCE

Although there have been numerous definitions of tolerance from an immunologic standpoint, a scientific pragmatic definition of clinical (operational) tolerance is a state of prolonged (indefinite) survival of a solid organ allograft with stable function without requirement of maintenance immunosuppression. Such a tolerant state, if complete, would imply normal (indefinite) graft survival with normal function and graft histology without any requirement of immunosuppression. In addition, the recipient would have normal immune responses and have no immunosuppression related-infection, neoplasia, or other side effects. The state of tolerance in the recipient might be incomplete with only partial specific immunosuppression, there would be improved (but not perfect) graft survival, function, and histology with a reduced requirement for maintenance immunoFrom Harvard Medical School, Boston, Massachusetts. Address reprint requests to Claire L. Kelly, The Transplant Center, Beth Israel Deaconess Medical Center 110 Francis Street - 7th Floor Boston, MA 02215. E-mail: ckelly@bidmc. harvard.edu 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2003.11.047 227

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suppression. The recipient immune responses would be improved (over standard immunosuppression) but not normal, and there would be reduced immunosuppressionrelated infection, neoplasia, and so on. This partial, incomplete, donor-specific hyporesponsiveness would be the state of near or almost (prope) tolerance6 or minimal immunosuppression tolerance. At the present time identification of a complete or partial donor-specific tolerance state can only be confirmed by doing elimination/withdrawal or reduction/ minimization of maintenance immunosuppression. No in vitro, in vivo, or intragraft monitoring assays for tolerance are available.

ROLE OF CHIMERISM

Billingham, Brent, and Medawar7 discovered the phenomenon of actively acquired immunologic tolerance to foreign cells. They noted that naturally chimeric freemartin cattle described by Owen8 tolerated each other’s skin grafts. Medawar and colleagues7 injected allogeneic spleen cells into naturally immunologically incompetent neonatal mice; such mice were chimeras as adults and tolerated donorspecific skin grafts indefinitely as adults but normally rejected third-party grafts. Infusion of normal syngeneic recipient lymphoid cells into tolerant recipients abrogated chimerism and destroyed tolerance. Persistence of donor strain chimerism was associated with persistent tolerance. The concept that establishment of chimerism was synonymous with establishment of tolerance was introduced into transplantation biology. However, many years later Streilein and colleagues9 studied neonatally induced tolerance with modern immunologic methods. They noted that, depending on differing histocompatibility between donors and recipients, neonatally injected mice could be operationally tolerant (perfect skin grafts with indefinite survival) but still show anti-donor activity in vitro by mixed lymphocyte cultures (MLC) and cytoxicity (CML). More importantly, certain combinations became chimeras as adults but still rejected grafts. They concluded that chimerism did not equal tolerance and tolerance was not necessarily a total absence of donor-specific alloimmunity. A number of studies have shown that the operationally tolerant state can be achieved and/or maintained by a number of immunologic mechanisms sometimes operating in a sequential fashion. Tolerance can be associated with anergy (immune cells are not capable of elaborating an immune response to their specific antigen when stimulated), suppression (cells can respond to their antigen but this response is donor-regulated by suppressor [regulatory] cells), clonal deletion (immune cells capable of responding to a specific antigen are absent or deleted), costimulation blockade (two pathways are required for T-cell activation and response; blockade of one pathway and stimulation of the other leads to tolerance), and chimerism (persistence of replicating donorspecific cells in a modified recipient).

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BONE MARROW AS A BIOLOGICAL REAGENT TO INDUCE TOLERANCE

Shortly after Medawar’s studies,7 Main and Prehn10 showed that successful skin allograft survival could be achieved in rodents ablated with total body irradiation and rescued with donor-specific bone marrow to become radiation-induced donor cell chimeras. Monaco and colleagues11,12 later demonstrated that bone marrow was the optimal replicating lymphoid cell population to induce specific prolonged survival of donor skin grafts in mice whose immune system was transiently ablated with polyclonal antilymphocyte serum. Graft survival was specifically, but not indefinitely, prolonged. Animals with prolonged survival were not chimeras in the standard sense, but some evidence of microchimerism was later demonstrated. Addition of a limited course of post bone marrow cyclosporine dramatically augmented the tolerance achieved.13 Subsequently, Hale et al14 showed that addition of a single injection of rapamycin to antilymphocyte serum-treated, bone marrow-infused mice produced 100% tolerance in class I and II histo-incompatible combinations but not in complete mismatch combinations. Escalation of the bone marrow dose from 25 to 150 –200 ⫻ 106 cells achieved robust, indefinite tolerance in complete mismatches.15 Tolerance was affirmed by acceptance of second donor strain skin graft at 150 days with simultaneous rejection of third-party grafts. Likewise, CML in vitro activity was absent against donor-specific targets but normal to third-party targets at 150 days. Of interest was the finding that there was a progressive increase in the percent of donor cell chimerism with escalating doses of bone marrow, which correlated with the degree and duration of tolerance. Low degrees of chimerism (1%) were present for 50 to 100 days only with 25 ⫻ 106 bone marrow cells, but were 8% to 10% (⬎200 days) when 150 ⫻ 106 cells or more were infused. The unique effect of rapamycin in promoting tolerance may be due to its ability to facilitate activationinduced apoptosis of T cells.16 Of interest was the finding that the chimerism was all B cell, monocyte–macrophage phenotype (no T cells). Extensive studies by Maki and colleagues17,18 using various knockout mice as bone marrow cell donors have confirmed that in this model using ALS-induced lymphocyte ablation, the critical bone marrow cell for tolerance induction is a class II positive cell; bone marrow from CD4, CD8, B-cell, and T-cell knockout mice all induced tolerance while BM from class II knockouts did not. Also, some animals given class II knockout BM exhibited chimerism even though they were not tolerant. Thus, chimerism was no guarantee of tolerance induction. CHIMERISM AND CLINICAL ORGAN TRANSPLANTATION

It is increasingly apparent that some type of chimerism may be associated with clinical organ transplantation. Microchimerism refers to a state in which replicating donor-specific cells occur at sites distant to that of the solid organ graft; such cells are usually class II dendritic cells and are at

CLINICAL TOLERANCE

extremely low levels, detectable frequently only by molecular techniques. Microchimerism occurs naturally after solid organ or cellular transplantation due to trafficking of passenger leukocytes. Lymphoablative conditioning is not required for this to occur and hematopoietic stem cells (HSC) do not engraft. A role for microchimerism and clinical allograft tolerance was suggested by the observations of Starzl et al19 that long-term hepatic and renal allograft recipients requiring minimal or no maintenance immunosuppressive therapy frequently have persistent donor cells in peripheral tissue. A number of studies of development, stability, and correlation with outcome of allogeneic microchimerism after solid organ transplantation have failed to confirm this. Microchimerism was frequently detectable after heart or liver transplantation but rejection did not correlate with the presence or absence of microchimerism20 and failed to provide any diagnostic or predictive value to the individual patient. Similarly, the presence or absence of microchimerism did not provide any predictive value as to whether long-term stable liver allograft recipients could be completely or partially withdrawn from immunosuppressive drug therapy.21 In addition, acute rejection leading to violent graft loss can occur many years after liver transplantation in spite of documented donor cell type microchimerism.22 At the present time, microchimerism is considered to be a epiphenomoenon but not a causative factor in allograft survival. Macrochimerism is a condition that occurs after bone marrow or other hematologic stem cell transplantation. Lymphoablative conditioning is required, and HSC engraftment occurs giving rise to multi-lineage chimerism. Donor-specific cell levels are higher, that is, 2% to 100%, and are detectable by flow cytometric techniques. It is well established that chimeric recipients of allogeneic bone marrow grafts for hematologic malignancy tolerate donor-specific kidney grafts without immunosuppression.23,24 Although induction of macrochimerism is often associated with tolerance, this is not always the case. Experimental studies in rodents18,25 and dogs (S. Strober, personal communication), especially when lymphoablation preparation utilizes irradiation, have demonstrated isolated instances where macrochimerism (detected by flow cytometry) was achieved but tolerance was not induced. Clinical studies26 have shown that transient, but not permanent, macrochimerism is required for tolerance induction. CURRENT STRATEGIES Prope and Minimal Immunosuppression Tolerance

These strategies emphasize the concept that lymphoablative therapy at the time of solid organ transplant followed by reduced or minimized maintenance immunosuppression therapy will permit evolution of a partial or quasitolerant state by one or more tolerant mechanisms such as generation of immunoregulatory or suppressor cells, clonal deletion, and so on. Strategies utilized thus far are Compath IH monoclonal antibody and cyclosporine (Calne et al6), Compath mAB and Sirolimus (Knechtle et al27), thymoglobulin

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and sirolimus (Swanson and colleagues28) and most recently thymoglobulin and tacrolimis (Starzl et al29). It is very clear that these strategies provide excellent short-term results with excellent survival and minimized immunosuppression requirements, mostly with single-drug immunotherapy. Rejection reactions, when they occur, are relatively mild and usually easily reversible. In the report by Starzl et al,29 the cumulative incidence of rejection reactions is relatively high but such reactions were easily reversible for the most part. Whether such reactions promote tolerance as postulated by these authors or will lead to chronic rejection remains to be seen. Obviously, as emphasized by Kirk30 their strategies are attractive in that they represent a logical first step toward tolerance induction without doing any apparent harm. Costimulation Blockade

The use of various monoclonal antibodies or other biological reagents to achieve costimulation blockade has demonstrated effectiveness in inducing tolerance in both rodents and primates. The studies with primates have identified significant toxicities associated with such monoclonal antibodies as well as very large monoclonal antibody requirements.31 Also, antagonistic effects exerted by calcineurin inhibitors on the tolerance induced has provided an additional obstacle for clinical application of this strategy. Nevertheless, if suitable nontoxic reagents for costimulation blockade can be identified, this avenue remains worthy of intense investigation. Immunologic Ablation (Cytoreduction) With Hematologic Reconstitution

Methods of immunoablation (cytoreduction) utilize radiation-based protocols (total body irradiation, subtotal irradiation, fractional total body irradiation, or thymic irradiation) with or without supplemental immunosuppression or nonradiation-based strategies (polyclonal ALS, immunotoxin, multiple monoclonal antibodies, Compath mAB) also with or without additional immunosuppression. This author feels strongly that nonradiation-based protocols using thymoglobulin cytoreduction are extremely attractive. Thymoglobulin can be viewed as a multivalent reagent with multiple target antigens that depletes lymphocytes in the peripheral vascular and tissue lymphocyte compartments by complement-dependent cytotoxicity, apoptosis, and opsonization.32 It has special ability in achieving apoptosis of activated anti-donor T cells during acute rejection in the graft and in the periphery. For hematopoietic reconstitution after cytoreduction and solid organ transplantation, one may use whole donor bone marrow cells or cytokinemodified peripheral blood cells obtained by leukopheresis. Eventually, in vitro cultured cytokine expanded bone marrow and/or purified stem cells could be used. Strober and colleagues33 attempted tolerance induction in four HLA-mismatched kidney allograft recipients treated with total lymphoid irradiation (800c Gy given in 10 doses),

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anti-thymocyte globulin (1.5 mg/kg five times over 14 days) along with cyclosporine and steroids combined with an infusion of cytokine-modified donor-specific CD34⫹ cells (day 14). Of four patients, three became chimeric and two met study criteria for cyclosporine and steroid withdrawal, i.e. development of chimerism within 3 months, failure to show serologic and histologic evidence of rejection, and failure to show anti-donor cell reactivity in MLR during drug withdrawal. Both patients became drug free for nearly a year, but then had a rejection episode at 17 months and were removed from the study but retained grafts on reinstituted immunosuppression. This report emphasizes that rejection reactions can occur off drugs but are self-limited and do not necessarily lead to graft loss and that current in vitro assays do not accurately identify a tolerant state.

rejection was dramatically reduced in bone marrow-infused patients. Studies of donor cell chimerism levels in the peripheral blood and bone marrow demonstrated that bone marrow-infused patients with no rejection had significantly higher levels of donor cell chimerism in their marrow aspirates and these levels increased with time. This study is remarkable in that it demonstrates a salutary effect of donor bone marrow infusion, which becomes evident over time. Although long-term bone marrow recipients are on quite low doses of tacrolimus (J. Miller, personal communication), they remain on immunosuppressive therapy. Whether any of these patients could be safely withdrawn from drug therapy has not been addressed.

Chimerism

As with the introduction of any new medical therapy or procedure, so too the implementation of tolerance-inducing protocols or strategies must be achieved with a positive or favorable risk/benefit ratio. No significant net harm or risk should occur to the transplant patients that submit to a tolerance strategy. The eventual success of the transplant must be equivalent or better to that achieved by the standard of care immunosuppression absent the tolerance protocol. Most tolerance strategies would best satisfy this principle by being imposed on standard immunosuppressive regimens eventually followed by some type of staged reduction or withdrawal, the speed of which would vary with a number of factors. This author does recognize that a tolerance protocol involving complete, relatively early immunosuppressive drug withdrawal could certainly be appropriate if the indications for transplant were strong, but the risk of even low-dose immunosuppression was unacceptable for any reason. Because the outcome of any tolerance strategy is by definition unknown except in the most specific circumstances, it would seem intuitive that non–life-saving organ transplants (such as kidney or islet transplants) would be the logical candidates for tolerance protocols at present. Furthermore, it also seems intuitive that graft dysfunction from other nonimmunologic factors (ischemia, reperfusion injury, etc) should be minimized by the use of living donors whenever possible. At the present time there is no in vivo or in vitro assay or test that can conclusively identify a state of clinical tolerance. The only tolerance test currently available is the ability to withdraw or significantly reduce maintenance immunotherapy with careful monitoring of graft function and histology. Confounding this problem is the fact that patients can be completely off immunosuppressive therapy (as in noncompliance) and not express graft dysfunction or loss for many years. Therefore, any tolerance induction protocol must take into consideration the relationship of time in any definition of operational tolerance. Strategies directed at achieving a minimal immunosuppressive regimen (rather than complete immunosuppression withdrawal) might be more appropriate when the concept of time and tolerance are considered. On the other hand, in

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The Massachusetts General Hospital Group has utilized nonmyeloablative host conditioning and bone marrow transplant to produce chimerism for kidney transplantation from the same donor without requirement for maintenance immunosuppression. Patients with multiple myeloma and attendant ESRD were prepared with their protocol of thymic irradiation (700c Gy) and cyclophosphamide (60 mg/kg ⫻ 2) during the week prior to HLA matched kidney transplant and bone marrow (2.7 ⫻ 108 cells/kg) infusion followed by thymoglobulin (1.5 mg/kg ⫻ 4) during the first postoperative week and cyclosporine and steroids for only 90 days thereafter. Patients became chimeric for 3 to 4 months and then lost chimerism. Myeloma went into remission with 90% or more reduction in kappa light chain urinary execretion. Most importantly, creatinine levels were normal with no evidence of rejection up to 3 years off immunosuppressive drugs. These authors concluded that combined bone marrow and kidney transplant is definitive therapy for multiple myeloma and associated ESRD. Bone marrow transplant combined with kidney transplant can be done safely without graft-versus-host disease. Operational tolerance was achieved despite contrary results of in vitro assays and loss of peripheral chimrism. Bone Marrow Infusion

Ciancio and colleagues34 have provided a 6-year follow-up of the effect of donor bone marrow infusions in renal allograft patients prepared with antilymphocyte antibody induction and standard tacrolimus and steroid therapy. Sixty-three cadaveric renal transplant recipients who received one or two postoperative donor-specific bone marrow infusions (DBMC) were prospectively compared with noninfused controls given equivalent immunosuppression. Graft survival was no different at 3 years, but at 6 years posttransplant graft survival (censored for death) was clearly superior in the bone marrow-infused group. Serum creatinine levels were superior in the bone marrow group. The incidence of acute rejection reactions was the same in both the bone marrow and control groups, but chronic

PRINCIPLES OF CLINICAL (OPERATIONAL) TOLERANCE INDUCTION

CLINICAL TOLERANCE

patients in whom complete drug withdrawal has been achieved after a tolerance protocol, late rejections have been relatively easily controlled and graft survival achieved with reinstitution of immunosuppression.33 Finally, there is emerging evidence both experimentally and now clinically26,34 that donor specific bone marrow infusion can exert a salutary tolerogenic effect without establishment of permanent macrochimerism. Depending on the tolerance strategy the effect may be discernible early26 or late34 and not be associated with significant risk for graft-versus-host disease. Incorporation of donor-specific bone marrow infusions in tolerance strategies seems a rational approach. REFERENCES 1. Cecka JM, Terasaki PI (eds): Clinical Transplants. Los Angeles, Calif: UCLA Tissue Typing Laboratory; 1997 p 237 2. Palestine AG, Austin HA 3rd, Nussenblatt RB: Renal histopathologic alterations in patients treated with cyclosporine for uveitis. N Engl J Med 15:314, 1986 3. Hornberger J, Best J, Geppert J, et al: Risks and costs of end-stage renal disease after heart transplantation. Transplantation 66:1763, 1998 4. Solez K, Vincenti F, Filo RS: Histopathologic findings from 2-year protocol biopsies from a U.S. multicenter kidney transplant trial comparing tacrolimus versus cyclosporine. Transplantation 66:1736, 1998 5. Sheil AG, Disney AP, Mathew TH, et al: De novo malignancy emerges as a major cause of morbidity and late failure in renal transplantation. Transplant Proc 25:1383, 1993 6. Calne R, Moffatt SD, Friend PJ, et al: Prope tolerance, perioperative compath 1H, and low-dose cyclosporin monotherapy in renal allograft recipients. Lancet 351:1701, 1998 7. Billingham RE, Brent L, Medawar PB: Actively acquired tolerance of foreign cells. Nature 4379:603, 1953 8. Owen RD: Immunogenetic consequences of vascular anastomosis between bovine twins. Science 102:400, 1945 9. Streilein JW: Neonatal tolerance of H-2 alloantigens. procuring graft acceptance the “old-fashioned” way. Transplantation 52:1, 1991 10. Main JM, Prehn RT: Successful skin homografts after administration of high dosage Xradiation and homologous bone marrow. JNCI 15:1023, 1955 11. Monaco AP, Wood ML: Studies on heterologous antilymphocyte serum in mice. VII. Optimal cellular antigen for induction of immunologic tolerance with antilymphocyte serum. Transplant Proc 2:489, 1970 12. Gozzo JL, Wood ML, Monaco AP: Use of allogeneic, homozygous bone marrow cells for the induction of specific immunologic tolerance in mice treated with antilymphocyte serum. Surg Forum 21:243, 1970 13. Wood ML, Gottschalk R, Monaco AP: The effect pf cyclosporine on the induction of unresponsiveness in ALS-treated, marrow-injected mice. Transplantation 46:449, 1988 14. Hale DA, Gottschalk R, Maki T, et al: Superiority of serolimus over cyclosporine in augmenting allograft and xenograft survival in mice treated with ALS and donor specific bone marrow. Transplantation 65:473, 1998

231 15. Hale DA, Gottschalk R, Umemura A: Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation. Transplantation 69:1242, 2000 16. Wells AD, Li XC, Li Y, et al: Requirement of T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med 5:1303, 1999 17. Umemura A, Monaco AP, Maki T: Donor T cells are not required for induction of allograft tolerance in mice treated with antilymphocyte serum, rapamycin and donor bone marrow cells. Transplantation 70:1005, 2000 18. Umemura A, Monaco AP, Maki T: Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells. J Immunol 164:4452, 2000 19. Starzl TE, Demetris AJ, Murase N, et al: Cell migration, chimerism and graft acceptance. Lancet 339:1579, 1992 20. Hisanaga M, Hundrieser J, Boker K, et al: Deveopment, stability and clinical correlations of allogeneic microchimerism after solid organ transplantation. Transplantation 61:40, 1996 21. Devlin J, Doherty D, Thomson L, et al: Defining the outcome of immunosuppression withdrawal after liver transplantation. Hepatology 27:926, 1998 22. Schlitt HJ, Hundrieser J, Ringe B, et al: Donor-type microchimerism associated with graft rejection eight years after liver transplantation. N Engl J Med 330:346, 1994 23. Sayegh MH, Fine NA, Smith JL, et al: Immunological tolerance to renal allografts after bone marrow transplants from the same donors. Ann Intern Med 114:954, 1991 24. Helg C, Chapuis B, Bolle JF, et al: Renal transplantation without immunosuppression in a host with tolerance induced by allogeneic bone marrow transplantation. Transplantation 58:1420, 1994 25. Maki T, Kanamoto K: Am J Transplant 3:267, 2003 26. Spitzer TR, Delmonico F, Tolkoff-Rubin N, et al: 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. Transplantation 68:480, 1999 27. Knechtle SJ, Pirsch JD, H Fechner J Jr, et al: Compath-1H induction plus rapamycin monotherapy for renal transplantation. results of a pilot study. Am J Transplantation 3:722, 2003 28. Swanson SJ, Hale DA, Mannon RB, et al: Kidney transplantation with rabbit antithymocyte globulin induction and sirolimus monotherapy. Lancet 360:1662, 2002 29. Starzl TE, Murase N, Abu-Elmagd K, et al: Tolerogenic immunosuppression for organ transplantation. Lancet 361:3, 2003 30. Kirk AD: Less is more. maintenance minimization as a step toward tolerance. Am J Transplant 3:643, 2003 31. Kirk AD, Burkly LC, Batty DS, et al: Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat Med 5:686, 1999 32. Preville X, Flacher M, LeMauff B, et al: Mechanisms involved in antithymocyte globulin immunosuppressive activity in a non-human primate model. Transplantation 71:460, 2001 33. Millan MT, Shizuru JA, Hoffmann P, et al: Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation 73:1386, 2002 34. Ciancio G, Miller J, Garcia-Morales RO, et al: Six-year clinical effect of donor bone marrow infusions in renal transplant patients. Transplantation 71:827, 2001