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Blood, 1 June 2004, Vol. 103, No. 11, pp. 4336-4343. Prepublished online as a Blood First Edition Paper on February 12, 2004; DOI 10.1182/blood-2003-08-2642.
TRANSPLANTATION Mechanisms of early peripheral CD4 T-cell tolerance induction by anti-CD154 monoclonal antibody and allogeneic bone marrow transplantation: evidence for anergy and deletion but not regulatory cellsFrom the Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston; Cellular and Molecular Immunology (CMI) Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston.
Anti-CD154 (CD40L) monoclonal antibody (mAb) plus bone marrow transplantation (BMT) in mice receiving CD8 cell-depleting mAb leads to long-term mixed hematopoietic chimerism and systemic donor-specific tolerance through peripheral and central deletional mechanisms. However, CD4+ T-cell tolerance is demonstrable in vitro and in vivo rapidly following BMT, before deletion of donor-reactive CD4 cells is complete, suggesting the involvement of other mechanisms. We examined these mechanisms in more detail. Spot enzyme-linked immunosorbent (ELISPOT) analysis revealed specific tolerization (within 4 to 15 days) of both T helper 1 (Th1) and Th2 cytokine responses to the donor, with no evidence for cytokine deviation. Tolerant lymphocytes did not significantly down-regulate rejection by naive donor-reactive T cells in adoptive transfer experiments. No evidence for linked suppression was obtained when skin expressing donor alloantigens in association with third-party alloantigens was grafted. T-cell receptor (TCR) transgenic mixing studies revealed that specific peripheral deletion of alloreactive CD4 T cells occurs over the first 4 weeks following BMT with anti-CD154. In contrast to models involving anti-CD154 without BMT, BMT with anti-CD154 leads to the rapid induction of anergy, followed by deletion of pre-existing donor-reactive peripheral CD4+ T cells; the rapid deletion of these cells obviates the need for a regulatory cell population to suppress CD4 cell-mediated alloreactivity. (Blood. 2004;103:4336-4343)
The combination of costimulatory blockade with bone marrow transplantation (BMT) allows the induction of high levels of long-term stable donor hematopoietic chimerism across full major histocompatibility complex (MHC) and minor histoincompatibility barriers.1-4 When recipient CD8+ T cells are depleted, mixed chimerism can be reliably established in mice receiving only 3 Gy total body irradiation (TBI) and one injection of anti-CD154 monoclonal antibody (mAb).5 Peripheral CD4+ T cells are rapidly tolerized by donor marrow when CD40/CD154 interactions are blocked.6 This tolerization protocol is reliably successful in even the most "resistant" recipient strains1,2,5 and leads to systemic tolerance, as measurable in vitro and by the most stringent in vivo test, skin allografting. In these models, donor antigen presented by skin grafts does not play a requisite role in tolerance induction and does not itself induce tolerance, because durable mixed chimerism and systemic tolerance are achieved regardless of whether skin is grafted early, and third-party skin is rejected even when grafted at the time of conditioning.1,2,5
In studies of BMT with costimulatory blockade, thymus-independent early deletion of donor-reactive CD4 T cells in the peripheral lymphoid tissues has been demonstrated.1,6,7 Both activation-induced cell death and "passive cell death" initially contribute to this peripheral CD4 cell deletion,8 but only passive cell death may be critical for tolerance induction.9 However, these studies of CD4 cell deletion in recipients of bone marrow (BM) transplants with anti-CD154 mAb relied on the analysis of T cells with T-cell receptor (TCR) V
Cell populations with immune regulatory properties may play a role in the induction of tolerance with the use of costimulatory blockade. In vitro, the combination of anti-CD154 mAb with suboptimal levels of anti-CD3 stimulates production of interleukin 10 (IL-10), interferon- We have further evaluated the mechanisms involved in the early induction and the long-term maintenance of donor-specific tolerance in mixed chimeras prepared with anti-CD154. In a TCR transgenic model, we demonstrate that specific peripheral deletion of alloreactive T cells occurs in recipients of BM transplants with anti-CD154 over a period of 4 weeks following BMT. In contrast to regimens not involving BMT, no evidence implicated either cytokine deviation or regulatory cell populations in tolerance induction or maintenance. The early mechanisms of CD4 cell tolerance in recipients of BM transplants and anti-CD154 may involve anergy followed by peripheral deletion. Similar to other mixed hematopoietic chimera models, the robust, long-term, donor-specific tolerance in these recipients is ultimately maintained through central deletion. These observations are consistent with the concept that cell populations that down-regulate T-cell responses are not maintained in an environment in which effective deletion of T cells with that specificity occurs.
Animals
Female C57BL/6 (B6; H-2b), B10.A (H-2a), B10.RIII (H-2r), A.SW (H-2s), A/J (H-2a), C57BL/6-IFN- Conditioning and BMT Age-matched (7-14 weeks old) recipient mice were treated with a nonmyeloablative dose of TBI (3 Gy) followed by injection of 15 to 25 x 106 bone marrow cells (BMCs) as described. Antimouse CD154 mAb (MR1; 0.5 mg or 2.0 mg) was administered on day 0, and anti-CD8 mAb (2.43; 1.44 mg/mouse) was administered on day -1 as described.5 Flow cytometric analysis of multilineage chimerism in white blood cells
Flow cytometric (FCM) analysis of multilineage chimerism in peripheral white blood cells (WBCs) was performed as previously described,15 using fluorescein isothiocyanate (FITC)-conjugated anti-CD4, anti-CD8, anti-B220 (PharMingen, San Diego, CA), and anti-Mac1 (Caltag, San Francisco, CA) with biotinylated anti-H-2Dd mAb 34-2-12 plus phycoerythrin-streptavidin (PEA). In the AND experiments involving A.SW (H2s) donors, chimerism was detected by staining of recipient (B10) cells with KH95-FITC. Nonspecific Fc
FCM analysis of T-cell receptor V
Peripheral blood lymphocytes (PBLs) were stained with FITC-conjugated anti-V Mixed lymphocyte reaction (MLR) assay MLR reactions were performed as described17 by using serum-free medium and triplicate wells containing 4 x 105 responder cells with 4 x 105 stimulators (irradiated with 3000 cGy). Recombinant human IL-2 (rhIL-2; R & D Systems, Minneapolis, MN) was added as at various concentrations. ELISPOT assay
Ninety-six-well spot enzyme-linked immunosorbent (ELISSPOT) plates (Polyfiltronics, Rockland, MA) were coated with a capture mAb in sterile phosphate-buffered saline (PBS) overnight. Anti-IFN- Skin grafting
Full-thickness tail skin ( Adoptive transfer of AND tg CD4+ T cells
CD4+ T cells were positively selected from C57BL/10-AND tg spleens using magnetic beads (MACS; Miltenyi Biotech, Auburn, CA) per the manufacturer's protocol. The purity of the V
Deletion of alloreactive CD4+ T cells following BMT and anti-CD154
Allogeneic BMT with costimulatory blockade induces peripheral deletion of CD4+ T cells recognizing endogenous superantigens (SAs) presented by donor MHC.1,8 However, as CD8+ cells play a critical role in producing the Mtv determinants required to delete V
All BM transplant recipients developed long-term multilineage chimerism at similar levels. One week after BMT, B10-AND recipients of A.SW (n = 7) or B10.A (n = 4) marrow showed 14.91% ± 2.33% and 15.15% ± 1.51% B-cell chimerism, respectively (data not shown). Because an anticlonotypic mAb is not available for the AND transgenic TCR, transgenic cells were identified on the basis of CD4 expression with both V
Addition of exogenous IL-2 is unable to restore in vitro response to donor antigens
In the above-mentioned experiments, we did not detect expansion of the AND CD4+ T cells prior to their subsequent deletion, similar to previous results assessing deletion of donor-reactive CD4 T cells expressing V
Lack of a role for IFN-
In several models using costimulatory blockade without BMT, IFN-
To determine whether Th2 cytokine deviation might contribute to early CD4+ T-cell tolerance, we performed ELISPOT analysis on splenocytes from animals killed at 2, 4, 8, 15, and 36 days after BMT to compare IFN-
Mixed chimerism was not associated with IL-4 production in response to donor antigens (Table 2). By day 4, the antidonor IL-4 response was significantly lower than that to third-party antigens in mixed chimeras. Higher antidonor IL-4 responses were seen on day 4 in a conditioned control, and in a mouse that received BMT without anti-CD154 (Table 2). By day 8, 3 of 3 mixed chimeras showed a specific lack of IL-4 production to the donor compared with third-party antigens, and the antidonor responses were markedly lower than that of the conditioned control animal. BMT without anti-CD154 led to a sensitized antidonor IL-4 response by this time (Table 2). Thus, tolerance of IL-4-producing cells was achieved in mixed chimeras between 4 and 8 days after BMT. A specific lack of an IL-5 response to donor antigens was also observed in mixed chimeras by day 15 (data not shown), further ruling out a Th2 shift. Similar results were obtained in additional experiments involving ELISPOT analyses of early (1-3 weeks) and long-term chimeras (data not shown). Thus, both Th1- and Th2-producing cytokine responses were rapidly and specifically tolerized in mixed allogeneic chimeras, and increased Th2 cytokine production cannot be implicated in tolerance induction. Lack of evidence for a role for regulatory/suppressive mechanisms in the maintenance of tolerance
To investigate whether regulatory cells maintained tolerance in mixed chimeras, we injected 30 x 106 naive host-type (B6) splenocytes 17 weeks after BMT into chimeras prepared with anti-CD8 mAb, anti-CD154, and 3 Gy TBI. In mixed chimeras prepared with lethal TBI, administration of 20 to 30 x 106 nontolerant host-type spleen cells rejects donor BMCs and breaks tolerance.25 In contrast, in models involving suppressive mechanisms, tolerance is resistant to higher doses of nontolerant host T cells.26-29 As shown in Figure 4, mixed chimeras prepared with anti-CD154, anti-CD8, and 3 Gy TBI lost measurable chimerism within 3 weeks following infusion of naive splenocytes (AdopTrans 1-3). Control chimeras remained stable. Donor-reactive V
The above-mentioned studies could not rule out the possibility that a regulatory cell population might contribute to initial tolerance induction prior to the deletion of donor-reactive CD4 T cells. Because anti-CD154 mAb is still present in the sera of these mice at 4 weeks after BMT (data not shown), the variable ability to break tolerance with the transfer of naive splenocytes might reflect the influence of this circulating mAb or the activity of a regulatory cell population. To distinguish between these possibilities, we removed the tolerant splenocytes from the mAb-containing environment early following BMT. Immunoincompetent recipient mice (B6-SCID or B6-Rag1-/-) were grafted with both donor and third-party skin grafts and then reconstituted 8 days later with spleen cells from either tolerant chimeric recipients 2 weeks after BMT, nontolerant controls, or a combination of both. In the first experiment (shown in Figure 5) B6 (CD45.2) mice received anti-CD8 mAb, 3 Gy TBI, and anti-CD154, followed by B10.A BMT. Two weeks later, chimeric mice (determined by FCM analysis of WBCs prior to killing, data not shown) and B6 (CD45.1) mice that had received only 3 Gy TBI and CD8-depleting mAb (without anti-CD154 or BMT) were killed, and their splenocytes were harvested. FCM analysis showed that, among splenocytes of BM transplant recipients, less than 0.7% of CD4+ cells were donor derived and 27.3% of B cells were donor derived, consistent with previous results,5,6 and that less than 0.1% CD8+ cells were present in chimeras or treated B6-CD45.1 mice. Splenocytes were washed (to prevent transfer of residual anti-CD154 mAb from the chimeric splenocytes) and injected intravenously into the grafted immunoincompetent recipient mice. Controls received only spleen cells (containing 5 x 106 CD4+ T cells) from chimeric mice (tolerant) or 5 x 106 CD4+ T cells from the B6 (CD45.1) mice (naive). Two different mixtures containing different ratios of tolerant and naive CD4+ T cells were injected into additional groups of B6-SCID mice: One group received a 1:1 ratio of tolerant to naive (5 x 106 of each) CD4+ splenocytes, and the other received a 3:1 ratio (15 x 106 tolerant to 5 x 106 naive CD4+ splenocytes). As shown in Figure 5, all SCID mice that received CD4+ splenocytes, regardless of origin, rejected fully MHC-mismatched third-party grafts within 19 days, whereas SCID controls accepted both donor and third-party grafts indefinitely. Recipients of naive CD4+ T cells, with or without an equal or 3-fold greater number of tolerant CD4+ T cells, rejected donor-type skin within 28 days. In contrast, mice that received only tolerant CD4+ T cells accepted donor skin grafts for more than 60 days. Thus, recently tolerized CD4+ T cells did not suppress the rejection of donor-type skin grafts by naive, nontolerant CD4+ T cells. Similar results were obtained in a repeat experiment involving adoptive transfer into B6 Rag1-/- recipients (data not shown).
After 60 days, mice that received only splenocytes from chimeric animals slowly rejected the donor grafts (Figure 5). No donor cells were detectable in WBCs of these adoptive recipients 6 weeks after transfer. In addition to confirming the incomplete deletion of donor-reactive CD4 cells in chimeras 2 weeks after BMT, this result demonstrates that donor antigens expressed on skin grafts are insufficient to maintain the tolerance of CD4 cells obtained from mixed chimeras at this point. To assess whether BMT with anti-CD154 might induce regulatory cells that mediate "linked suppression,"12 we prepared mixed chimeras and grafted them on day +1 after BMT with donor-type (B10.A) and third-party skin grafts that were either fully MHC- and minor antigen-mismatched to the BMC donor strain (A.SW), MHC-matched to the donor but expressing minor histocompatibility differences (A/J), or MHC class II and minor antigen matched to the donor but class I mismatched (B10.BR) (Table 3). As shown in Figure 6 and Table 3, all chimeras accepted donor skin more than 100 days, while eventually rejecting the various third-party grafts. Fully mismatched A.SW grafts were rejected quickly, within 20 days. Grafts sharing the entire donor MHC, but mismatched at multiple minor histocompatibility loci, were all rejected within 55 days. Four of 5 chimeras rejected grafts sharing donor MHC class II but mismatched for class I within 80 days (B10.BR). Thus, linked suppression did not induce tolerance to skin grafted immediately after BMT. Because tolerance mediated by regulatory cells may take several months to develop after transplantation,30 similarly prepared mixed chimeras (n = 4) received skin grafts 13 weeks after BMT from B10.A donors. The mixed chimeras all accepted the B10.A skin grafts indefinitely (> 100 days), while rapidly rejecting B10.BR grafts (days 15, 19, 27, and 30). Thus, BMT with anti-CD154 does not induce a cell population that tolerizes T cells with other specificities encountering their antigenic ligands on antigen-presenting cells (APCs) that also express donor antigens.
The combination of anti-CD154 mAb and BMT permits the rapid establishment of specific CD4 T-cell tolerance to the donor,5,6 as demonstrated by the acceptance of donor-type skin grafted 1 day after BMT and by donor-specific MLR nonresponsiveness by 1 week after BMT6,9 (Tables 1, 2). However, analyses of donor-reactive V  5- and V11-bearing CD4+ T cells in these experiments showed no measurable deletion of these cells before 2 weeks after BMT,5,6 suggesting that donor-specific tolerance develops before completion of deletion of donor-reactive T cells. However, the superantigen-mediated deletion of CD4+ T cells depends, to some extent, on the presence of CD8+ T cells, which are depleted in this model,5 and may not necessarily reflect the deletion of conventional alloreactive CD4+ T cells. In the present studies, we demonstrate significant deletion of alloreactive AND TCR transgenic CD4+ T cells during the first week after BMT in mice treated with anti-CD8 and anti-CD154, but show that the deletion is not complete until 3 to 4 weeks after BMT. Therefore, mechanisms in addition to deletion must play a role in establishing initial peripheral donor-specific CD4 T-cell tolerance.
In various models, costimulatory blockade may induce T-cell anergy,31 Th2 deviation,32 suppression,12 or apoptosis.33-36 The failure to observe early expansion of donor-specific CD4 T cells in these (Figure 1) or our previous studies using V
Whereas IFN-
Th2 cytokine deviation has been described in regimens using costimulatory blockade,32 and it has been reported that CD154 blockade is less effective in a predominantly Th1 environment.39 We have previously shown that by day 4 after BMT, animals receiving our BMT regimen already show specific tolerance of IL-2-producing cells to the donor.9 We now examined the possible role of cytokine deviation in the induction of tolerance in this model. Our ELISPOT analyses showed no increase in donor-reactive IFN- A body of literature has implicated CD25+CD45RBlowCD4+ regulatory T cells in the maintenance of peripheral tolerance to organ-specific self-antigens.40,41 Regulatory T cells have also been implicated in various models using anti-CD154 for tolerance induction. CD4+ T cells stimulated with alloantigens ex vivo in the presence of anti-CD154 included a population of CD4+CD25+ T cells that down-modulated alloresponses in vitro and in vivo.11 CD4+ T-cell-dependent regulatory mechanisms have been implicated in models of linked suppression and infectious tolerance using anti-CD154 and anti-CD8 mAbs in a minor antigen-mismatched skin graft model.12,13 We assessed the potential role of regulatory mechanisms in the development of early CD4+ T-cell tolerance in recipients of BM transplants with anti-CD154. In long-term mixed chimeras prepared with anti-CD8 and anti-CD154 mAbs and 3 Gy TBI, the administration of 30 x 106 naive host-type cells led to the rapid loss of chimerism and rejection of donor skin grafts, suggesting that potent suppression of antidonor responses is not a major mechanism maintaining tolerance in these chimeras. However, these studies did not rule out the possibility that suppression contributes to initial CD4 tolerance. This was difficult to assess by infusing nontolerant recipient lymphocytes at early time points, because persisting anti-CD154 mAb and donor BMC-derived cells might tolerize these cells through the same mechanisms as those affecting pre-existing host T cells. Therefore, we adoptively transferred recently tolerized lymphocytes (2 weeks after BMT) into immunodeficient mice to test their potential to suppress rejection by naive CD4 cells. These recently tolerized CD4+ T cells did not markedly suppress the rejection of donor-type skin grafts by naive, nontolerant CD4+ T cells, even when they were administered in a 3:1 tolerant/naive ratio. Although it might be argued that homeostatic proliferation of infused T cells in immunodeficient adoptive recipients precludes the ability to detect suppression in this assay,42 adoptive transfer to T-cell-deficient mice has been used successfully to demonstrate the presence of regulatory cells in mice receiving allografts with costimulatory blockade and other mAbs.43-46 The rapid rejection of third-party skin grafts by adoptive recipients of tolerant chimeric CD4+ T cells alone demonstrates that the marked donor skin prolongation is not due to generalized hyporesponsiveness. However, recipients of tolerant spleen cells eventually rejected donor skin. This rejection may be the result of loss of donor chimerism, as no detectable WBC chimerism (from cotransferred donor spleen cells from chimeras) was observed at 6 weeks after infusion. These donor hematopoietic cells may have been rejected by natural killer (NK) cells in immunodeficient mice. The result suggests that donor skin is an insufficient source of donor antigen to maintain tolerance and highlights the critical role of hematopoietic chimerism in maintaining tolerance of peripheral CD4 T cells in this model. Furthermore, these data also demonstrate that the early mechanisms involved in peripheral CD4+ T-cell tolerance are reversible and that donor-reactive CD4+ T cells that are not deleted by 2 weeks after BMT regain the ability to reject donor grafts in the absence of donor chimerism and/or persisting anti-CD154 mAb. A requirement for persisting chimerism to maintain tolerance in this pre-existing CD4 cell population is consistent with anergy as a mechanism of tolerance before deletion occurs, as other studies have shown dependence on antigen persistence for the maintenance of anergy.47-49 In a minor histocompatibility antigen-mismatched skin graft model, anti-CD154 can induce a state of linked suppression, in which tolerized CD4+ T cells induce tolerance to other antigens expressed on skin grafts sharing the same antigens to which the CD4+ T cells were tolerized.12 Our studies involving early and later grafting of third-party skin expressing both donor and third-party alloantigens on the same cells provides no evidence for linked suppression in this model. The observation that donor MHC class I-or minor histocompatibility antigen-mismatched skin grafted 1 day after BMT was rejected more slowly than the fully MHC-mismatched third-party grafts may be explained by the prolonged (> 4 weeks) peripheral depletion of CD8 T cells observed because of treatment with anti-CD8 mAb, although temporary suppression of alloreactive CD8 T cells by recipient CD4 T cells cannot be ruled out. Studies performed in long-term B10.A to B6 mixed chimeras, in which B10.BR skin grafts were rapidly rejected, demonstrate an absence of linked suppression, consistent with the absence of major suppressive mechanisms in these animals. The combination of costimulatory blockade and BMT induces a robust state of life-long donor-specific tolerance. We have now further defined the mechanisms of specific tolerance induced among peripheral CD4 T cells. Similar to other models of mixed chimerism, the long-term maintenance of donor-specific tolerance relies on intrathymic deletion,5 and no strong peripheral regulatory mechanisms appear to be involved. Furthermore, through the use of an alloreactive transgenic T-cell population, we demonstrated that the deletion of alloreactive CD4+ T cells is complete by about 4 weeks after BMT. In contrast to other regimens that include costimulatory blockade, we could find no evidence for a major role for a suppressive/regulatory mechanism or cytokine deviation in either the long-term maintenance or early induction of donor-specific tolerance. Collectively, our results are consistent with a tolerance mechanism whereby donor-reactive CD4 T cells are initially anergized by donor hematopoietic cells in the presence of CD40 blockade and then deleted. This rapid deletion of donor-reactive CD4 cells precludes the development and/or maintenance of (as well as the need for) a cell population to suppress their alloreactivity. These results have implications for the design of strategies for tolerance induction using BMT in large animal and clinical studies.
We thank Drs Christine Huang and Boris Nikolic for critical review of this manuscript and Ms Robin Laber for expert assistance with manuscript preparation.
Submitted August 7, 2003; accepted February 5, 2004.
Prepublished online as Blood First Edition Paper, February 12, 2004; DOI 10.1182/blood-2003-08-2642.
Supported by National Institutes of Health grant HL49915 and training grant 1T32 AI-07529.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Megan Sykes, Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129; e-mail: megan.sykes{at}tbrc.mgh.harvard.edu.
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Abstract
Introduction
(IFN-
(TNF-
1.0 cm2) was grafted and monitored as described.
cpm = cpm against stimulator - cpm against "self" (recipient in the case of chimeras) stimulation. (A) No IL-2; (B) 2 U/mL IL-2; and (C) 5 U/mL IL-2.
