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Induction of tolerance in organ recipients by hematopoietic stem cell transplantation
International Immunopharmacology 9 (2009) 694–700
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Induction of tolerance in organ recipients by hematopoietic stem cell transplantation☆ Eran Ophir, Yair Reisner ⁎ Weizmann Institute of Science, Dept. of Immunology, Rehovot 76100, Israel
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Article history: Received 11 November 2008 Received in revised form 19 November 2008 Accepted 10 December 2008 Keywords: Mixed chimerism Megadose transplant Veto Anti 3rd-party CTLs
a b s t r a c t The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+lin-hematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Induction of immune tolerance towards a specific set of donor antigens (Ags), which enables the subsequent engraftment of donor cells or tissues without the need for deleterious continuous nonspecific immunosuppressive therapy, represents a seminal goal in transplantation biology. Although several regimens for inducing tolerance have been demonstrated in rodents (reviewed in [1]), the translation of such protocols to the clinic has been more difficult. To date, proof of concept has been demonstrated in humans only for tolerance induction by means of hematopoietic stem cell transplantation (HSCT) [2,3]. In general, upon induction of mixed hematopoietic chimerism, donor and host hematopoietic stem cells continuously give rise to both host-derived and donor-derived antigen presenting cells (APCs), which home to the recipient thymus and, through the process of negative selection, mediate deletion of host and donorreactive T cell clones [4–6]. The robustness and long term durability of
☆ Supported by: the Gabriella Rich Center for Transplantation Biology; Mrs. Erica Drake; National Institutes of Health Grants 5PO1 CA049639 and 5 U19 CA100265-03; The European Community grants: ALLOSTEM and RISET, and the Legacy Heritage Fund. ⁎ Corresponding author. Tel.: +972 8 9344023; fax: +972 8 9344145. E-mail address: [email protected] (Y. Reisner). 1567-5769/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.12.009
this process, which provides continuous maintenance of the tolerance, represent a major advantage of HSCT over most other approaches. However, the protocols used to induce chimerism are associated with risk for transplant-related mortality due to GVHD or infections. Clearly, in non-cancer patients such as organ transplant recipients, HSCT can only be justified when used with RIC, but such safer protocols fail to sufficiently reduce host immune cells. Thus, overcoming rejection of donor hematopoietic stem cells represents a major challenge for the induction of chimerism as a prelude to organ transplantation. This challenge is particularly difficult when using purified donor-derived CD34 stem cells or TDBMT in order to avoid GVHD [7,8]. 2. GVHD and graft rejection as mirror images The role of T cell depletion in HSCT was initially demonstrated in the context of haploidentical (three HLA loci mismatched) transplants in severe combined immunodeficiency (SCID) patients. The problem of GVHD, which is almost uniformly lethal in haploidentical patients, was completely prevented by three-log T cell depletion using soybean lectin and E-rosetting [9–12]. By now, more than 200 SCID patients have been treated with such transplants and, as shown in Fig. 1, longterm survival in a 16-year follow-up is around 80% [13,14]. Interestingly, analysis of the patients in these early studies revealed, for the
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related mortality (mainly due to infections) of around 20% [21]. While in high-risk leukemia patients, such transplant-related mortality is acceptable, it would be intolerable if applied to induce hematopoietic chimerism in patients with a long life expectancy, such as organ transplant recipients. Therefore, developing new strategies to achieve engraftment of T cell depleted HSTC following RIC, which spares a substantial level of host immunity, is warranted. Moreover, by using such non-myeloablative reduced conditioning, mixed chimerism can be achieved, as opposed to the full donor chimerism achieved when using supra-lethal conditioning. Mixed chimerism was suggested to be associated with improved immunity, compared to full allogeneic chimerism [22,23], probably due to the presence in the mixed chimera of APCs expressing host MHC, which may enable more effective presentation of Ags to the T cells that have undergone positive selection and maturation on host type thymic epithelial cells. Thus, patients treated with the less drastic conditioning may be less prone to infection, not only by virtue of enhanced speed of immune reconstitution, but also as a consequence of the presence of host APCs. Nonetheless, the marked levels of host lymphocytes surviving such mild preparatory regimens, represents a difficult barrier for the engraftment of T cell depleted HSCT. Fig. 1. Long term survival of SCID patients treated by transplantation of human T cell depleted bone marrow. Results of O'Reilly, et al. (A) (Sloan Kettering) [13] and Buckley et al, (B). (Duke University Medical Center) [14].
first time, that in humans this protocol induces permanent immune tolerance towards host type Ags [9]. As shown in Table 1, the paternal donor T cells, which exhibited a marked response in an in-vitro mixed lymphocyte reaction (MLR) against the recipient cells prior to transplantation, were completely inactive toward the patient cells when collected from the patient's blood after transplantation. Nevertheless, their ability to respond against the other parental haplotype or against 3rd-party stimulators was preserved. Following the encouraging results in SCID patients, it was reasonable to assume that in leukemia patients, pre-treated with supralethal radiotherapy and chemotherapy, the remaining immunity at the time of the transplant would be dramatically reduced, reaching levels similar to those found in SCID patients. Therefore, it was assumed that in these patients graft rejection should not represent a major problem. However, early results using T-depleted haploidentical donor cells suggested that this was not the case, and a high rate of graft rejection was documented [7,8]. The rejection was shown to be mediated by residual radiotherapy and chemotherapy resistant host-derived T cells [15,16]. Thus, graft rejection and GVHD represent mirror images in these patients; both are mediated by T cells, and overcoming one enhances the risk for the other [7]. Subsequently, murine models have indeed shown that donor T cells, although causing GVHD, also play an important role in the engraftment of HSCT [17–19]. One way to overcome this host T cell-mediated rejection of TDBMT is to perform HSCT following supra-lethal conditioning and functional inactivation of host T cells using immunosuppressive drugs and ‘mega doses’ of purified CD34 stem cells [20]. Using these protocols, long term survival in acute leukemia patients and especially in patients with AML, transplanted while in remission, is around 40%, with transplant-
Table 1 Posttransplantation MLC of SCID patient receiving paternal TDBM graft. Responders Paternal T lymphocytes Engrafted paternal T lymphocytes
Stimulators Patient cells
Father's cells
Mother's cells
Unrelated cells
5,843 350
685 80
9,038 8,950
7,773 3,242
Data show in vitro incorporation of 14C-thymidine [net cpm])[9].
3. Tolerance induction using ‘megadose’ stem cell transplantation As mentioned above, rejection of allogeneic HSCT can be overcome by large doses of hematopoietic stem cells. This was initially demonstrated in murine studies [24–26], but a significant increase in the BM inoculum has been difficult to achieve in humans. One approach to overcome this problem was based on the observation that granulocyte colony stimulating factor (G-CSF) facilitates mobilization of CD34 hematopoietic stem cells from the BM, enabling the collection of a large number of CD34 cells from the peripheral blood [27,28]. Thus, the goal of stem cell escalation was achieved in humans by supplementing the conventional TDBMT with purified progenitors collected from the blood after administration of G-CSF to the donor. Using this method, it became possible in 1993 to test the concept of stem cell dose escalation in humans. A pilot study carried out between 1993 to 1995 showed, for the first time, that in humans as in mice, cell dose escalation facilitated engraftment of T cell-depleted mismatched hematopoietic transplants [29,30].
Fig. 2. The regulatory activity of CD34 cells: evidence for target specificity. The average CTL response (SD) in the presence (black bars) or absence (white bars) of CD34+ cells at a veto-to-responder cell ratio of 0.5. The veto effect was tested by a limiting dilution assay as follows: equal numbers (1 × 106/mL) of responder cells and irradiated allogeneic stimulator cells from the donor of the CD34 cells and a third party donor were co-cultured for 5 days. The responder cells were then cultured again for 7 days under limiting dilution, and the CTL activity was determined by 51Cr-release assay. Data represent the average ± standard deviation of 11 independent experiments using different donor and third party pairs. A significant difference (P b.001 on t-test compared with control cultures without CD34 cells) between control cultures and those including CD34 cells was found upon stimulation against donor cells [59].
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rates of GVHD was demonstrated in more than 93% of the patients, in the absence of post transplantation GVHD prophylaxis [20]. The few patients who failed to engraft achieved engraftment following a second transplant. Similar results were obtained by other centers in adults [31,32] and in children [33–35]. Thus, by using megadose purified stem cell transplants, it is now possible to increase the donor pool and use readily available haplo-identical family members for BM transplantation. 4. The veto concept
Fig. 3. The veto ‘concept’. A CTLp specifically recognizes the veto cell by the binding of its T cell receptor (TCR) to the MHC class I molecule on the veto cell. Once the cells interact, instead of triggering stimulation and expansion of the CTLp, the veto cell induces the transduction of a death signal (apoptosis) in the CTLp. The veto activity is specific, as a CTLp, which bears TCR against third party MHC molecule, does not recognize the veto cell, and thus survives. In CD8 + veto cells, the binding of the CD8 molecule to the α3 domain of the MHC class I molecule on the CTLp, plays a role in the apoptosis signal.
Beginning in 1995, T cell-depletion was replaced by positive selection of the CD34 hematopoietic stem cells using magnetic beads. After several modifications, an optimized protocol, based on the use of CD34 cells isolated by Miltenyi magnetic beads, was tested in 1999 in Perugia. The clinical experience in Perugia, using this methodology in high-risk leukemia patients, was summarized in 2005. Primary engraftment of the haplo-identical megadose transplants with low
The intriguing question of how the CD34 cells overcome the barrier presented by host T cells was first addressed by Rachamim et al. who demonstrated that cells within the CD34 fraction are endowed with potent veto activity (Fig. 2) [36]. Subsequently, Gur et al. suggested that this veto activity is mediated through a TNF-α based mechanism [37]. Veto activity was defined in 1980 by Miller [38] as the capacity to specifically suppress CTL precursors (CTLp), directed against Ags recognized by the veto cells themselves, but not against 3rd-party Ags (Fig. 3). Thus, the recognizing T cell, with specificity directed against the veto cell, is killed upon binding to its veto target. This inherent specificity of veto cells, eliminating only host CTLp directed against the donor Ags, while sparing other CTLp, which can further persist and fight infectious pathogens, has suggested that veto cells could offer a specific and effective modality for the induction of transplantation tolerance. 5. The use of megadose stem cell transplants and other veto cell populations in nonmyeloablative transplants Since CD34 cells are endowed with veto activity, it would be highly attractive to use a megadose of CD34 purified cells to achieve engraftment, without GVHD, under non-myeloablative conditioning.
Fig. 4. Sca-1 + Lin- cells can induce mixed chimerism in sublethally irradiated mice. Split chimerism following transplantation of C57BL/6 Sca-1 + Lin- cells (H2b) into sublethally irradiated (7 Gy TBI) C3H/HeJ recipients (H2k). Percentages of donor and host T cells (determined by gating of CD3 stained cells, upper right of the panel) and non-T cells are shown in the appropriate FACS dot plots. Peripheral blood chimerism was determined 30 days post-transplant by cytofluorimetry. Similar results (not shown) were obtained 7 months post transplantation, showing the persistence of the chimeric state. Representative chimerism analysis of one mouse, out of 23 mice engrafted in 8 independent experiments, is displayed. [39].
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Fig. 5. Veto CTLs induce apoptosis in the effector T cells by the Fas-FasL mechanism. Upon engagement between the TCR of the effector cell and class I of the veto cell, the effector cell is activated and Fas is upregulated. However, the presence of FasL on the veto CTL is not sufficient to trigger apoptosis, as FLIP is also upregulated. The high affinity interaction between the CD8 on the veto cell and the α3 domain on the effector cell likely maintains the contact long enough (60–72 h) for FLIP and other inhibitory molecules to be downregulated, and for Fas-FasL killing to be completed [60].
Indeed, in 1999, it was demonstrated that BM cells within the Sca-1+Lincell fraction, previously shown to be enriched for early hematopoietic progenitors, are specifically capable of reducing anti-donor CTLp frequency in vitro and in vivo, and can induce mixed chimerism in sublethally (7Gy) irradiated recipient mice across major MHC barriers (Fig. 4). The immune tolerance induced by the Sca-1+Lin- cells was also associated with specific tolerance toward donor-type skin grafts [39]. However, primate studies suggested that further reduction of the conditioning to levels acceptable for organ transplantation requires stem cell numbers which cannot be realistically collected from human donors (Gan et al., unpublished results). Therefore, the availability of other populations of veto or other immunoregulatory cells is crucial for further application of allogeneic purified stem cell transplantation under RIC. Various cell types have been shown to mediate veto activity, including T lymphocytes, natural killer cells and dendritic cells. A very strong veto activity was documented for some CD8+ CTL lines or clones [40–42].
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Fig. 7. Anti 3rd-party CTLs are depleted of GVH reactivity. Donor (Balb/c) anti 3rd-party (C57BL/6) CTLs were depleted of GVH reactivity against the host (C3H/HeJ). Host mice were conditioned with supralethal irradiation (11Gy TBI) and radio-protected with 2 × 106 Balb/c-nude BM cells. The GVH reactivity of the CTLs or naive CD8+ T cells is reflected by the percent survival of the animals following infusion of 107 cells. One representative experiment, out of a total of 3 experiments, with 7 animals in each group is shown. Statistical analysis by t-test revealed a significant difference (P b 0.05) between survival rates of mice receiving naïve CD8+ T cells compared to mice receiving anti 3rd-party CTLs (figure adapted from [47]).
Direct comparison of the veto activity of various cell types revealed that CTLs exhibit the strongest veto reactivity [43]. Previous insights on the veto mechanism of CD8+ veto CTLs, combining anti-CD8 blockade and FASL-mutated veto cells, have suggested that co-expression of CD8 and FASL is required for the veto activity of these cells [44,45]. Such a mechanism involves initial recognition of the veto cell by the TCR of the effector T cells, leading to expression of Fas by the effector T cell upon activation, and thereby enabling Fas-FasL mediated apoptosis to take place, once inhibitory molecules such as FLICE-inhibitory protein (FLIP) are down regulated in the effector cell (Fig. 5). The extra affinity required to maintain the interaction between the effector cell and the veto cell might be provided through binding between CD8 on the veto cell and class I α3 domain on the effector cell, but some form of signaling via this interaction might also occur. In vivo, host non-reactive F1 CD8+ T cells were shown to enhance engraftment of TDBMT [18,46], indicating that BM engraftment by
Fig. 6. Generation of host-nonreactive anti 3rd party CTLs by stimulation of donor CD8+ T cells against third party stimulators under IL-2 deprivation [47].
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Fig. 8. Enhancement of BM allografting by anti 3rd-party CTLs in sublethally irradiated mice. Donor type chimerism was assessed in peripheral blood of sublethally (7Gy TBI) irradiated recipients (C3H/HeJ) of 5 × 106 allogeneic BM cells (Balb/c-nude). Statistical analysis by t-test showed a significant difference (P b 0.05) between donor chimerism levels of mice receiving only a BM allograft compared to mice receiving a BM allograft supplemented with veto CTLs [47].
CD8+ T cells is not only mediated by alloreactivity against host cells, but rather may be generated by other mechanisms, such as veto activity. However, despite their remarkable and potent veto activity, CD8+ CTLs can not be used for tolerance induction in allogeneic stem cell transplantation because of their marked GVH reactivity. One approach developed in our laboratory to generate veto CTLs without GVH reactivity is to induce stimulation against 3rd-party stimulators in the absence of exogenous IL-2 (Fig. 6). This approach is based on the observation that only activated anti 3rd-party CTLp are capable of surviving the IL-2 deprivation in the primary culture, while most other non-activated CTLp (including anti host CTLp) are eliminated during the primary culture due to death by neglect, which results from the lack of exogenous IL-2 and TCR engagement. The anti 3rd-party CTLs were shown to be depleted of GVHD reactivity (Fig. 7) and their capacity to enhance engraftment was demonstrated in sublethally irradiated mice [47]. Thus, as can be seen in Fig. 8, while infusion of 5 × 106 nude mouse BM cells leads to a very low level of chimerism, engraftment was markedly enhanced when veto anti 3rd-party CTLs were added. This enhancement of engraftment mediated by veto cells was found to be synergetic with shortterm treatment with rapamycin [47]. Thus, when the BM cells dose was reduced by 20%, the anti 3rd-party veto CTLs alone failed to overcome rejection, while the chimerism level was markedly enhanced upon the addition of both veto CTLs and rapamycin (Fig. 9). It is important to note that a similar level of tolerance induction was induced when the CTLs were generated from (hostXdonor)F1 mice, further strengthening the suggestion that their tolerizing activity is not mediated by residual anti host alloreactivity. Furthermore, in accordance with the veto concept, the tolerizing effect of the CTLs was found to be H-2 specific. Thus, a CTL line originating from a strain other than that of the BM donor failed to prevent graft rejection [47]. Another very important attribute of the anti 3rd-party veto CTLs is their ability to eliminate not only host anti-donor naive cells, but also host anti-donor memory cells. Memory T cells, derived from prior exposure to alloantigen or generated by heterologous immunity or lymphopenia-induced proliferation, are believed to be an important part of the barrier preventing the translation of tolerance induction protocols from inbred rodent strains to the clinic [48–50]. Memory cells may be resistant to T cell depleting antibodies [48] and less prone to costimulatory inhibitors [51]. Moreover, naturally occurring CD4+CD25+ regulatory T cells, which are required for transplantation tolerance in many model systems [52,53], fail to
interfere with allograft rejection mediated by memory T cells, as opposed to their ability to modulate and tolerize naive T cells [54]. In contrast, the anti 3rd-party veto CTLs were recently shown to be equally effective in overcoming rejection mediated either by naive or by memory host T cells, both in-vitro and in vivo [55]. Therefore, veto CTLs are likely excellent candidates for tolerance induction in humans. Recently, human anti 3rd-party veto CTLs were generated using a similar methodology to that used in rodent experiments, except that a prolonged IL-2 deprivation period of 2 weeks, instead of 6 days, was employed [56]. This prolonged period was necessary due to the relative resilience of human T cells compared to mouse T cells. Such human veto CTLs are currently under clinical investigation, together with Champlin et al, at MD Anderson Cancer Center in Houston, for their capacity to enable engraftment without GVHD in elderly multiple myeloma and B-CLL patients undergoing HSCT under RIC. The use of anti 3rd-party CTLs in these patients is highly attractive because, in addition to their prominent tolerizing activity demonstrated in mouse models, human anti 3rd-party CTLs also exhibit antitumor reactivity against B-CLL [57] and other B cell malignancies [58]. 6. Concluding remarks Induction of hematopoietic mixed chimerism by means of BMT offers a promising approach for tolerance induction as a prelude to organ transplantation. This approach is already being evaluated in patients. Kawai et al. recently published a study in the New England Journal of Medicine, in which the authors impressively demonstrate that they could discontinue all immunosuppressive therapy, without significantly affecting transplant function, a few months following combined BM and kidney transplants from HLA single-haplotype mismatched donors [3]. However, using their protocol, they achieved only transient hematopoietic chimerism, which was lost 21 days post transplant. They believe that the mechanism of tolerance induction in their system may switch from central tolerance to a peripheral mechanism that might include regulatory T cells. However, protocols which favor long term mixed chimerism may support a more stable tolerance towards donor tissues. Indeed, the transient chimerism achieved by Kawai et al. was also accompanied by reversible capillary leak syndrome, which might indicate minor rejection episodes. A similar study published in parallel in the same issue, using a different protocol in fully HLA matched recipients [2] demonstrated stable mixed chimerism in one patient. However, in this protocol, the
Fig. 9. Synergistic enhancement of BM allografting by anti 3rd-party CTLs and rapamycin in sublethally irradiated mice. Donor type chimerism was assessed in peripheral blood of sublethally (7Gy TBI) irradiated recipients (C3H/HeJ) of 4 × 106 allogeneic BM cells (Balb/c-nude). Statistical analysis by t-test showed a significant difference (P b 0.05 ) between chimerism levels of mice receiving a BM allograft alone and mice receiving BM allograft supplemented with veto CTLs and rapamycin [47].
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[39] Bachar-Lustig E, Li HW, Gur H, Krauthgamer R, Marcus H, Reisner Y. Induction of donor-type chimerism and transplantation tolerance across major histocompatibility barriers in sublethally irradiated mice by Sca-1(+)Lin(-) bone marrow progenitor cells: synergism with non-alloreactive (host × donor)F(1) T cells. Blood 1999;94(9):3212–21. [40] Claesson MH, Miller RG. Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. III. Direct correlation between development of syngeneic cytotoxicity and loss of veto activity; implications for the mechanism of veto action. Scand J Immunol 1989;29(4):493–7. [41] Claesson MH, Ropke C. Antiself suppressive (veto) activity of responder cells in mixed lymphocyte cultures. Curr Top Microbiol Immunol 1986;126:213–23. [42] Fink PJ, Rammensee HG, Benedetto JD, Staerz UD, Lefrancois L, Bevan MJ. Studies on the mechanism of suppression of primary cytotoxic responses by cloned cytotoxic T lymphocytes. J Immunol 1984;133(4):1769–74. [43] Reich-Zeliger S, Bachar-Lustig E, Gan J, Reisner Y. Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. I. Relative reactivity of different veto cells. J Immunol 2004;173(11):6654–9. [44] Reich-Zeliger S, Gan J, Bachar-Lustig E, Reisner Y. Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. II. Deletion of effector cells by Fas-Fas ligand apoptosis. J Immunol 2004;173(11):6660–6. [45] Reich-Zeliger S, Zhao Y, Krauthgamer R, Bachar-Lustig E, Reisner Y. Anti-third party CD8+ CTLs as potent veto cells: coexpression of CD8 and FasL is a prerequisite. Immunity 2000;13(4):507–15. [46] Lapidot T, Faktorowich Y, Lubin I, Reisner Y. Enhancement of T-cell-depleted bone marrow allografts in the absence of graft-versus-host disease is mediated by CD8+CD4 − and not by CD8−CD4+ thymocytes. Blood 1992;80(9):2406–11. [47] Bachar-Lustig E, Reich-Zeliger S, Reisner Y. Anti-third-party veto CTLs overcome rejection of hematopoietic allografts: synergism with rapamycin and BM cell dose. Blood 2003;102(6):1943–50. [48] Adams AB, Williams MA, Jones TR, Shirasugi N, Durham MM, Kaech SM, et al. Heterologous immunity provides a potent barrier to transplantation tolerance. J Clin Invest 2003;111(12):1887–95. [49] Pantenburg B, Heinzel F, Das L, Heeger PS, Valujskikh A. T cells primed by Leishmania major infection cross-react with alloantigens and alter the course of allograft rejection. J Immunol 2002;169(7):3686–93.
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[50] Wu Z, Bensinger SJ, Zhang J, Chen C, Yuan X, Huang X, et al. Homeostatic proliferation is a barrier to transplantation tolerance. Nat Med 2004;10(1):87–92. [51] Chen Y, Heeger PS, Valujskikh A. In vivo helper functions of alloreactive memory CD4+ T cells remain intact despite donor-specific transfusion and anti-CD40 ligand therapy. J Immunol 2004;172(9):5456–66. [52] Sanchez-Fueyo A, Sandner S, Habicht A, Mariat C, Kenny J, Degauque N, et al. Specificity of CD4+CD25+ regulatory T cell function in alloimmunity. J Immunol 2006;176(1):329–34. [53] Lee MKt, Moore DJ, Jarrett BP, Lian MM, Deng S, Huang X, et al. Promotion of allograft survival by CD4+CD25+ regulatory T cells: evidence for in vivo inhibition of effector cell proliferation. J Immunol 2004;172(11):6539–44. [54] Yang J, Brook MO, Carvalho-Gaspar M, Zhang J, Ramon HE, Sayegh MH, et al. Allograft rejection mediated by memory T cells is resistant to regulation. Proc Natl Acad Sci U S A 2007;104(50):19954–9. [55] Reich-Zeliger S, Bachar-Lustig E, Bar-Ilan A, Reisner Y. Tolerance induction in presensitized bone marrow recipients by veto CTLs: effective deletion of host antidonor memory effector cells. J Immunol 2007;179(10):6389–94.
[56] Aviner S, Yao X, Krauthgamer R, Gan Y, Goren-Arbel R, Klein T, et al. Large-scale preparation of human anti-third-party veto cytotoxic T lymphocytes depleted of graft-versus-host reactivity: a new source for graft facilitating cells in bone marrow transplantation. Hum Immunol 2005;66:644–52. [57] Arditti FD, Aviner S, Dekel B, Krauthgamer R, Gan J, Nagler A, et al. Eradication of BCLL by autologous and allogeneic host nonreactive anti-third-party CTLs. Blood 2005;105(8):3365–71. [58] Lask Assaf, Goihberg Polina, Goren-Arbel Rinat, Milstein Oren, Aviner Shraga, Feine Ilan, Klein Tirza, Berrebi Alain, Reisner Yair. Eradication of B cell malignancies by anti3rd party CTLs: a novel role for MHC class I. Blood 2007;10(11):a2759. [59] Gur H, Krauthgamer R, Berrebi A, Klein T, Nagler A, Tabilio A, et al. Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34(+) cells. Blood 2002;99(11):4174–81. [60] Reisner Y, Reich-Zeliger S, Bachar-Lustig E. The role of veto cells in bone marrow transplantation. Curr Opin Organ Transplant 2006;11:366–72.
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Induction of tolerance in organ recipients by hematopoietic stem cell transplantation☆ Eran Ophir, Yair Reisner ⁎ Weizmann Institute of Science, Dept. of Immunology, Rehovot 76100, Israel
a r t i c l e
i n f o
Article history: Received 11 November 2008 Received in revised form 19 November 2008 Accepted 10 December 2008 Keywords: Mixed chimerism Megadose transplant Veto Anti 3rd-party CTLs
a b s t r a c t The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+lin-hematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Induction of immune tolerance towards a specific set of donor antigens (Ags), which enables the subsequent engraftment of donor cells or tissues without the need for deleterious continuous nonspecific immunosuppressive therapy, represents a seminal goal in transplantation biology. Although several regimens for inducing tolerance have been demonstrated in rodents (reviewed in [1]), the translation of such protocols to the clinic has been more difficult. To date, proof of concept has been demonstrated in humans only for tolerance induction by means of hematopoietic stem cell transplantation (HSCT) [2,3]. In general, upon induction of mixed hematopoietic chimerism, donor and host hematopoietic stem cells continuously give rise to both host-derived and donor-derived antigen presenting cells (APCs), which home to the recipient thymus and, through the process of negative selection, mediate deletion of host and donorreactive T cell clones [4–6]. The robustness and long term durability of
☆ Supported by: the Gabriella Rich Center for Transplantation Biology; Mrs. Erica Drake; National Institutes of Health Grants 5PO1 CA049639 and 5 U19 CA100265-03; The European Community grants: ALLOSTEM and RISET, and the Legacy Heritage Fund. ⁎ Corresponding author. Tel.: +972 8 9344023; fax: +972 8 9344145. E-mail address: [email protected] (Y. Reisner). 1567-5769/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.12.009
this process, which provides continuous maintenance of the tolerance, represent a major advantage of HSCT over most other approaches. However, the protocols used to induce chimerism are associated with risk for transplant-related mortality due to GVHD or infections. Clearly, in non-cancer patients such as organ transplant recipients, HSCT can only be justified when used with RIC, but such safer protocols fail to sufficiently reduce host immune cells. Thus, overcoming rejection of donor hematopoietic stem cells represents a major challenge for the induction of chimerism as a prelude to organ transplantation. This challenge is particularly difficult when using purified donor-derived CD34 stem cells or TDBMT in order to avoid GVHD [7,8]. 2. GVHD and graft rejection as mirror images The role of T cell depletion in HSCT was initially demonstrated in the context of haploidentical (three HLA loci mismatched) transplants in severe combined immunodeficiency (SCID) patients. The problem of GVHD, which is almost uniformly lethal in haploidentical patients, was completely prevented by three-log T cell depletion using soybean lectin and E-rosetting [9–12]. By now, more than 200 SCID patients have been treated with such transplants and, as shown in Fig. 1, longterm survival in a 16-year follow-up is around 80% [13,14]. Interestingly, analysis of the patients in these early studies revealed, for the
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related mortality (mainly due to infections) of around 20% [21]. While in high-risk leukemia patients, such transplant-related mortality is acceptable, it would be intolerable if applied to induce hematopoietic chimerism in patients with a long life expectancy, such as organ transplant recipients. Therefore, developing new strategies to achieve engraftment of T cell depleted HSTC following RIC, which spares a substantial level of host immunity, is warranted. Moreover, by using such non-myeloablative reduced conditioning, mixed chimerism can be achieved, as opposed to the full donor chimerism achieved when using supra-lethal conditioning. Mixed chimerism was suggested to be associated with improved immunity, compared to full allogeneic chimerism [22,23], probably due to the presence in the mixed chimera of APCs expressing host MHC, which may enable more effective presentation of Ags to the T cells that have undergone positive selection and maturation on host type thymic epithelial cells. Thus, patients treated with the less drastic conditioning may be less prone to infection, not only by virtue of enhanced speed of immune reconstitution, but also as a consequence of the presence of host APCs. Nonetheless, the marked levels of host lymphocytes surviving such mild preparatory regimens, represents a difficult barrier for the engraftment of T cell depleted HSCT. Fig. 1. Long term survival of SCID patients treated by transplantation of human T cell depleted bone marrow. Results of O'Reilly, et al. (A) (Sloan Kettering) [13] and Buckley et al, (B). (Duke University Medical Center) [14].
first time, that in humans this protocol induces permanent immune tolerance towards host type Ags [9]. As shown in Table 1, the paternal donor T cells, which exhibited a marked response in an in-vitro mixed lymphocyte reaction (MLR) against the recipient cells prior to transplantation, were completely inactive toward the patient cells when collected from the patient's blood after transplantation. Nevertheless, their ability to respond against the other parental haplotype or against 3rd-party stimulators was preserved. Following the encouraging results in SCID patients, it was reasonable to assume that in leukemia patients, pre-treated with supralethal radiotherapy and chemotherapy, the remaining immunity at the time of the transplant would be dramatically reduced, reaching levels similar to those found in SCID patients. Therefore, it was assumed that in these patients graft rejection should not represent a major problem. However, early results using T-depleted haploidentical donor cells suggested that this was not the case, and a high rate of graft rejection was documented [7,8]. The rejection was shown to be mediated by residual radiotherapy and chemotherapy resistant host-derived T cells [15,16]. Thus, graft rejection and GVHD represent mirror images in these patients; both are mediated by T cells, and overcoming one enhances the risk for the other [7]. Subsequently, murine models have indeed shown that donor T cells, although causing GVHD, also play an important role in the engraftment of HSCT [17–19]. One way to overcome this host T cell-mediated rejection of TDBMT is to perform HSCT following supra-lethal conditioning and functional inactivation of host T cells using immunosuppressive drugs and ‘mega doses’ of purified CD34 stem cells [20]. Using these protocols, long term survival in acute leukemia patients and especially in patients with AML, transplanted while in remission, is around 40%, with transplant-
Table 1 Posttransplantation MLC of SCID patient receiving paternal TDBM graft. Responders Paternal T lymphocytes Engrafted paternal T lymphocytes
Stimulators Patient cells
Father's cells
Mother's cells
Unrelated cells
5,843 350
685 80
9,038 8,950
7,773 3,242
Data show in vitro incorporation of 14C-thymidine [net cpm])[9].
3. Tolerance induction using ‘megadose’ stem cell transplantation As mentioned above, rejection of allogeneic HSCT can be overcome by large doses of hematopoietic stem cells. This was initially demonstrated in murine studies [24–26], but a significant increase in the BM inoculum has been difficult to achieve in humans. One approach to overcome this problem was based on the observation that granulocyte colony stimulating factor (G-CSF) facilitates mobilization of CD34 hematopoietic stem cells from the BM, enabling the collection of a large number of CD34 cells from the peripheral blood [27,28]. Thus, the goal of stem cell escalation was achieved in humans by supplementing the conventional TDBMT with purified progenitors collected from the blood after administration of G-CSF to the donor. Using this method, it became possible in 1993 to test the concept of stem cell dose escalation in humans. A pilot study carried out between 1993 to 1995 showed, for the first time, that in humans as in mice, cell dose escalation facilitated engraftment of T cell-depleted mismatched hematopoietic transplants [29,30].
Fig. 2. The regulatory activity of CD34 cells: evidence for target specificity. The average CTL response (SD) in the presence (black bars) or absence (white bars) of CD34+ cells at a veto-to-responder cell ratio of 0.5. The veto effect was tested by a limiting dilution assay as follows: equal numbers (1 × 106/mL) of responder cells and irradiated allogeneic stimulator cells from the donor of the CD34 cells and a third party donor were co-cultured for 5 days. The responder cells were then cultured again for 7 days under limiting dilution, and the CTL activity was determined by 51Cr-release assay. Data represent the average ± standard deviation of 11 independent experiments using different donor and third party pairs. A significant difference (P b.001 on t-test compared with control cultures without CD34 cells) between control cultures and those including CD34 cells was found upon stimulation against donor cells [59].
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rates of GVHD was demonstrated in more than 93% of the patients, in the absence of post transplantation GVHD prophylaxis [20]. The few patients who failed to engraft achieved engraftment following a second transplant. Similar results were obtained by other centers in adults [31,32] and in children [33–35]. Thus, by using megadose purified stem cell transplants, it is now possible to increase the donor pool and use readily available haplo-identical family members for BM transplantation. 4. The veto concept
Fig. 3. The veto ‘concept’. A CTLp specifically recognizes the veto cell by the binding of its T cell receptor (TCR) to the MHC class I molecule on the veto cell. Once the cells interact, instead of triggering stimulation and expansion of the CTLp, the veto cell induces the transduction of a death signal (apoptosis) in the CTLp. The veto activity is specific, as a CTLp, which bears TCR against third party MHC molecule, does not recognize the veto cell, and thus survives. In CD8 + veto cells, the binding of the CD8 molecule to the α3 domain of the MHC class I molecule on the CTLp, plays a role in the apoptosis signal.
Beginning in 1995, T cell-depletion was replaced by positive selection of the CD34 hematopoietic stem cells using magnetic beads. After several modifications, an optimized protocol, based on the use of CD34 cells isolated by Miltenyi magnetic beads, was tested in 1999 in Perugia. The clinical experience in Perugia, using this methodology in high-risk leukemia patients, was summarized in 2005. Primary engraftment of the haplo-identical megadose transplants with low
The intriguing question of how the CD34 cells overcome the barrier presented by host T cells was first addressed by Rachamim et al. who demonstrated that cells within the CD34 fraction are endowed with potent veto activity (Fig. 2) [36]. Subsequently, Gur et al. suggested that this veto activity is mediated through a TNF-α based mechanism [37]. Veto activity was defined in 1980 by Miller [38] as the capacity to specifically suppress CTL precursors (CTLp), directed against Ags recognized by the veto cells themselves, but not against 3rd-party Ags (Fig. 3). Thus, the recognizing T cell, with specificity directed against the veto cell, is killed upon binding to its veto target. This inherent specificity of veto cells, eliminating only host CTLp directed against the donor Ags, while sparing other CTLp, which can further persist and fight infectious pathogens, has suggested that veto cells could offer a specific and effective modality for the induction of transplantation tolerance. 5. The use of megadose stem cell transplants and other veto cell populations in nonmyeloablative transplants Since CD34 cells are endowed with veto activity, it would be highly attractive to use a megadose of CD34 purified cells to achieve engraftment, without GVHD, under non-myeloablative conditioning.
Fig. 4. Sca-1 + Lin- cells can induce mixed chimerism in sublethally irradiated mice. Split chimerism following transplantation of C57BL/6 Sca-1 + Lin- cells (H2b) into sublethally irradiated (7 Gy TBI) C3H/HeJ recipients (H2k). Percentages of donor and host T cells (determined by gating of CD3 stained cells, upper right of the panel) and non-T cells are shown in the appropriate FACS dot plots. Peripheral blood chimerism was determined 30 days post-transplant by cytofluorimetry. Similar results (not shown) were obtained 7 months post transplantation, showing the persistence of the chimeric state. Representative chimerism analysis of one mouse, out of 23 mice engrafted in 8 independent experiments, is displayed. [39].
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Fig. 5. Veto CTLs induce apoptosis in the effector T cells by the Fas-FasL mechanism. Upon engagement between the TCR of the effector cell and class I of the veto cell, the effector cell is activated and Fas is upregulated. However, the presence of FasL on the veto CTL is not sufficient to trigger apoptosis, as FLIP is also upregulated. The high affinity interaction between the CD8 on the veto cell and the α3 domain on the effector cell likely maintains the contact long enough (60–72 h) for FLIP and other inhibitory molecules to be downregulated, and for Fas-FasL killing to be completed [60].
Indeed, in 1999, it was demonstrated that BM cells within the Sca-1+Lincell fraction, previously shown to be enriched for early hematopoietic progenitors, are specifically capable of reducing anti-donor CTLp frequency in vitro and in vivo, and can induce mixed chimerism in sublethally (7Gy) irradiated recipient mice across major MHC barriers (Fig. 4). The immune tolerance induced by the Sca-1+Lin- cells was also associated with specific tolerance toward donor-type skin grafts [39]. However, primate studies suggested that further reduction of the conditioning to levels acceptable for organ transplantation requires stem cell numbers which cannot be realistically collected from human donors (Gan et al., unpublished results). Therefore, the availability of other populations of veto or other immunoregulatory cells is crucial for further application of allogeneic purified stem cell transplantation under RIC. Various cell types have been shown to mediate veto activity, including T lymphocytes, natural killer cells and dendritic cells. A very strong veto activity was documented for some CD8+ CTL lines or clones [40–42].
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Fig. 7. Anti 3rd-party CTLs are depleted of GVH reactivity. Donor (Balb/c) anti 3rd-party (C57BL/6) CTLs were depleted of GVH reactivity against the host (C3H/HeJ). Host mice were conditioned with supralethal irradiation (11Gy TBI) and radio-protected with 2 × 106 Balb/c-nude BM cells. The GVH reactivity of the CTLs or naive CD8+ T cells is reflected by the percent survival of the animals following infusion of 107 cells. One representative experiment, out of a total of 3 experiments, with 7 animals in each group is shown. Statistical analysis by t-test revealed a significant difference (P b 0.05) between survival rates of mice receiving naïve CD8+ T cells compared to mice receiving anti 3rd-party CTLs (figure adapted from [47]).
Direct comparison of the veto activity of various cell types revealed that CTLs exhibit the strongest veto reactivity [43]. Previous insights on the veto mechanism of CD8+ veto CTLs, combining anti-CD8 blockade and FASL-mutated veto cells, have suggested that co-expression of CD8 and FASL is required for the veto activity of these cells [44,45]. Such a mechanism involves initial recognition of the veto cell by the TCR of the effector T cells, leading to expression of Fas by the effector T cell upon activation, and thereby enabling Fas-FasL mediated apoptosis to take place, once inhibitory molecules such as FLICE-inhibitory protein (FLIP) are down regulated in the effector cell (Fig. 5). The extra affinity required to maintain the interaction between the effector cell and the veto cell might be provided through binding between CD8 on the veto cell and class I α3 domain on the effector cell, but some form of signaling via this interaction might also occur. In vivo, host non-reactive F1 CD8+ T cells were shown to enhance engraftment of TDBMT [18,46], indicating that BM engraftment by
Fig. 6. Generation of host-nonreactive anti 3rd party CTLs by stimulation of donor CD8+ T cells against third party stimulators under IL-2 deprivation [47].
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Fig. 8. Enhancement of BM allografting by anti 3rd-party CTLs in sublethally irradiated mice. Donor type chimerism was assessed in peripheral blood of sublethally (7Gy TBI) irradiated recipients (C3H/HeJ) of 5 × 106 allogeneic BM cells (Balb/c-nude). Statistical analysis by t-test showed a significant difference (P b 0.05) between donor chimerism levels of mice receiving only a BM allograft compared to mice receiving a BM allograft supplemented with veto CTLs [47].
CD8+ T cells is not only mediated by alloreactivity against host cells, but rather may be generated by other mechanisms, such as veto activity. However, despite their remarkable and potent veto activity, CD8+ CTLs can not be used for tolerance induction in allogeneic stem cell transplantation because of their marked GVH reactivity. One approach developed in our laboratory to generate veto CTLs without GVH reactivity is to induce stimulation against 3rd-party stimulators in the absence of exogenous IL-2 (Fig. 6). This approach is based on the observation that only activated anti 3rd-party CTLp are capable of surviving the IL-2 deprivation in the primary culture, while most other non-activated CTLp (including anti host CTLp) are eliminated during the primary culture due to death by neglect, which results from the lack of exogenous IL-2 and TCR engagement. The anti 3rd-party CTLs were shown to be depleted of GVHD reactivity (Fig. 7) and their capacity to enhance engraftment was demonstrated in sublethally irradiated mice [47]. Thus, as can be seen in Fig. 8, while infusion of 5 × 106 nude mouse BM cells leads to a very low level of chimerism, engraftment was markedly enhanced when veto anti 3rd-party CTLs were added. This enhancement of engraftment mediated by veto cells was found to be synergetic with shortterm treatment with rapamycin [47]. Thus, when the BM cells dose was reduced by 20%, the anti 3rd-party veto CTLs alone failed to overcome rejection, while the chimerism level was markedly enhanced upon the addition of both veto CTLs and rapamycin (Fig. 9). It is important to note that a similar level of tolerance induction was induced when the CTLs were generated from (hostXdonor)F1 mice, further strengthening the suggestion that their tolerizing activity is not mediated by residual anti host alloreactivity. Furthermore, in accordance with the veto concept, the tolerizing effect of the CTLs was found to be H-2 specific. Thus, a CTL line originating from a strain other than that of the BM donor failed to prevent graft rejection [47]. Another very important attribute of the anti 3rd-party veto CTLs is their ability to eliminate not only host anti-donor naive cells, but also host anti-donor memory cells. Memory T cells, derived from prior exposure to alloantigen or generated by heterologous immunity or lymphopenia-induced proliferation, are believed to be an important part of the barrier preventing the translation of tolerance induction protocols from inbred rodent strains to the clinic [48–50]. Memory cells may be resistant to T cell depleting antibodies [48] and less prone to costimulatory inhibitors [51]. Moreover, naturally occurring CD4+CD25+ regulatory T cells, which are required for transplantation tolerance in many model systems [52,53], fail to
interfere with allograft rejection mediated by memory T cells, as opposed to their ability to modulate and tolerize naive T cells [54]. In contrast, the anti 3rd-party veto CTLs were recently shown to be equally effective in overcoming rejection mediated either by naive or by memory host T cells, both in-vitro and in vivo [55]. Therefore, veto CTLs are likely excellent candidates for tolerance induction in humans. Recently, human anti 3rd-party veto CTLs were generated using a similar methodology to that used in rodent experiments, except that a prolonged IL-2 deprivation period of 2 weeks, instead of 6 days, was employed [56]. This prolonged period was necessary due to the relative resilience of human T cells compared to mouse T cells. Such human veto CTLs are currently under clinical investigation, together with Champlin et al, at MD Anderson Cancer Center in Houston, for their capacity to enable engraftment without GVHD in elderly multiple myeloma and B-CLL patients undergoing HSCT under RIC. The use of anti 3rd-party CTLs in these patients is highly attractive because, in addition to their prominent tolerizing activity demonstrated in mouse models, human anti 3rd-party CTLs also exhibit antitumor reactivity against B-CLL [57] and other B cell malignancies [58]. 6. Concluding remarks Induction of hematopoietic mixed chimerism by means of BMT offers a promising approach for tolerance induction as a prelude to organ transplantation. This approach is already being evaluated in patients. Kawai et al. recently published a study in the New England Journal of Medicine, in which the authors impressively demonstrate that they could discontinue all immunosuppressive therapy, without significantly affecting transplant function, a few months following combined BM and kidney transplants from HLA single-haplotype mismatched donors [3]. However, using their protocol, they achieved only transient hematopoietic chimerism, which was lost 21 days post transplant. They believe that the mechanism of tolerance induction in their system may switch from central tolerance to a peripheral mechanism that might include regulatory T cells. However, protocols which favor long term mixed chimerism may support a more stable tolerance towards donor tissues. Indeed, the transient chimerism achieved by Kawai et al. was also accompanied by reversible capillary leak syndrome, which might indicate minor rejection episodes. A similar study published in parallel in the same issue, using a different protocol in fully HLA matched recipients [2] demonstrated stable mixed chimerism in one patient. However, in this protocol, the
Fig. 9. Synergistic enhancement of BM allografting by anti 3rd-party CTLs and rapamycin in sublethally irradiated mice. Donor type chimerism was assessed in peripheral blood of sublethally (7Gy TBI) irradiated recipients (C3H/HeJ) of 4 × 106 allogeneic BM cells (Balb/c-nude). Statistical analysis by t-test showed a significant difference (P b 0.05 ) between chimerism levels of mice receiving a BM allograft alone and mice receiving BM allograft supplemented with veto CTLs and rapamycin [47].
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purified stem cell graft was supplemented with 1 × 106/kg body weight naive donor T cells. This might expose the recipients to GVHD risk, especially when trying to employ haploidentical donors. The use of TDBMT or purified stem cell grafts could almost completely eliminate the risk of GVHD, but such T-cell-depleted transplants are more easily rejected. Megadose stem cell transplants can effectively overcome residual host immunity surviving the myeloablative conditioning in leukemia patients, but are not capable of overcoming the large number of host T cells remaining after RIC employed in recipients of organ transplantation. Therefore, combining megadose stem cell transplants with anti 3rd-party CTLs, together with short term treatment with rapamycin, following conditioning with RIC, could enable the use of BMT as a safe approach for tolerance induction without GVHD. References [1] Kingsley CI, Nadig SN, Wood KJ. Transplantation tolerance: lessons from experimental rodent models. 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