THYMIC DEPENDENCE OF LOSS OF TOLERANCE IN MIXED ALLOGENEIC... : Transplantation (original) (raw)
The major mechanisms of donor-specific tolerance induction identified in experimental transplantation models include central thymic deletion of donor-reactive T cells (1), peripheral clonal deletion (2), clonal anergy (3), and suppression (4). Different mechanisms may predominate, depending on the method used to induce tolerance.
Bone marrow transplantation provides an effective way of inducing donor-specific tolerance (5). However, the toxicity of myeloablative conditioning regimens used to permit MHC-mismatched bone marrow engraftment has precluded its use in clinical organ transplantation. We have recently described a relatively safe and reliable method of inducing donor-specific tolerance across complete major histocompatibility MHC barriers in mice (6). This is achieved by administering depleting anti-CD4 and anti-CD8 mAbs to recipients before treatment with 3-Gy whole body irradiation (WBI*) and 7-Gy thymic irradiation (TI) on the day of bone marrow transplantation (BMT). Identification of the mechanism of tolerance induction in this model would allow precisely directed host conditioning, thus making the conditioning process even less toxic and, consequently, more readily applicable to human transplantation.
In this BMT model, deletion of donor-reactive T cells has been demonstrated by following the course of host-type Vβ11+ T cells. Vβ11+ T cells react against Mtv-8- and Mtv-9-derived superantigens, Dvb11-1 and Dvb11-2, respectively, which are encoded in the B10 background genome (7-9). These superantigens are expressed in association with I-E, and cause intrathymic deletion of Vβ11+ cells in I-E+ B10.A mice, but not in I-E- B10 mice (10,11). B10 mice, after conditioning with the above-described regimen and injection of B10.A bone marrow cells, achieve mixed hematopoietic chimerism and show marked intrathymic deletion of mature host Vβ11+ T cells. This deletion is apparent as early as 10 days after BMT, when the T-cell repertoire first recovers, and persists for the life of the animal. As a result, the long-term peripheral T-cell repertoire shows deletion of these T cells. However, in the early weeks after BMT, a small population of host-type Vβ11+ T cells is detectable in the spleen only. These cells appear to be anergic, as they do not respond to stimulation with anti-Vβ11 antibody (12). These results suggested a possible role for anergy in the tolerization of residual host T cells that were not depleted by anti-T cell monoclonal antibody treatment.
Other studies in mixed allogeneic chimeras prepared with our nonmyeloablative regimen showed that donor hematopoietic cells could be eliminated by giving high doses of anti-donor class I MHC mAb (anti-Dd, 34-2-12) in vivo, and that such treatment was associated with loss of tolerance to the donor (13). This phenomenon could be due to a loss of either central or peripheral tolerance, or both. With respect to peripheral tolerance, two major possibilities could be envisioned: first, once donor-type antigen has been removed, anergic T cells may recover their capacity to reject donor grafts; second, suppressive T cells may lose their ability to suppress donor-specific responses when donor antigen is removed. Regarding central tolerance, it is possible that removal of donor-type antigen from the thymus allows the maturation and emigration to the periphery of donor-reactive host T cells, which then reject donor-type skin grafts. We have now performed studies to distinguish among these possibilities. The results suggest that donor antigen-dependent intrathymic deletion is the only significant mechanism maintaining tolerance in stable mixed allogeneic chimeras.
MATERIALS AND METHODS
Animals. Female C57BL/10ScNCR (B10:H2b; KbAbE-Db), B10.A (H2a; KkAkEkDdLd), A/J (H2a), BALB/c (H2d), B10.D2 (H2d), SJL (H2s), and DBA/2 mice were purchased from Jackson Laboratories (Bar Harbor, ME) or from Harlan Sprague-Dawley, via Frederick Cancer Research Facility (FCRF, Frederick, MD). C57BL/6 (B6) nu/nu mice were purchased from Taconic (Germantown, NY), and bred in our own colony. Recipient mice were used at 10-12 weeks, and donors were used at 10-22 weeks of age. Recipient C57BL/6 nu/nu mice were used at 5-9 weeks of age. All mice were maintained in sterilized microisolator cages, in which they received autoclaved feed and autoclaved acidified drinking water.
Conditioning and BMT. BMT was performed with modifications of the previously described regimen (6). Briefly, B10 or B10.A mice received 100 μl of ascites containing 2 mg of anti-CD4 mAb GK1.5 (14) and 70 μl (0.7 mg) of purified anti-CD8 mAb 2.43 (15) intraperitoneally on days -6 and -1. On day 0, 3 Gy (0.94 Gy/min with cesium 137 irradiator, J.L. Shepherd, Inc., San Fernando, CA) of WBI and 7 Gy (cobalt 60 irradiator, at 0.54 Gy/min) of selective TI were administered to mAb-treated animals. Fifteen million untreated bone marrow cells (BMC) from B10.A, B10, or A/J mice were administered intravenously on the same day, as described (6).
Phenotyping of chimeras. Chimerism was evaluated by flow cytometric (FCM) analysis of peripheral white blood cells (WBC) and spleen cells (SC). FCM analyses were performed on a FACScan (Becton Dickinson & Co., Mountain View, CA). WBC were prepared by hypotonic shock, as described (16). Cells were stained with fluoresceinated (fluorescein isothiocyanate [FITC]) anti-αβTCR (Pharmingen, San Diego, CA) and biotinylated anti-H2Dd mAb 34-2-12 (17) for 30 min at 4°C, then washed twice. In order to block nonspecific FcγR binding of labeled antibodies, 10 μl of undiluted culture supernatant of 2.4G2 (rat anti-mouse FcγR mAb) (18) were added before the first incubation. Cell-bound biotinylated mAb was detected with phycoerythrin-streptavidin (Pharmingen), with which cells were incubated for 15 min at 4°C. FITC-conjugated murine mAb IgG2a HOPC1, with no reactivity to mouse cells, and biotinylated HOPC1 mAb were used as non-staining irrelevant antibodies. In some studies, two-color staining with FITC-labeled anti-Kb (host-specific) mAb 5F1 (19) versus donor-specific anti-Dd mAb 34-2-12-biotin/phycoerythrin-streptavidin was performed. Two-color data were analyzed as dot plot diagrams with logarithmically increasing intensities of green (FITC) fluorescence plotted on the x axis versus logarithmically increasing intensities of orange phycoerythrin fluorescence on the y axis. Forward versus side scatter analysis was used to distinguish lymphocytes, monocytes, and granulocytes. In all experiments, the percentage of cells staining with each monoclonal antibody was determined by comparison with that obtained from normal donor and host-type animals, which were used as positive and negative controls. The percentage of cells considered positive after staining with a monoclonal antibody was determined using a cutoff chosen as the fluorescence level at the beginning of the positive peak for the positive control strain, and by subtracting the percentage of cells in that region after staining with an irrelevant mAb.
Skin grafting. Skin grafting was performed as described (20, 21). Square full-thickness skin (1 cm2) grafts were prepared from the trunk or tail skin of donors. Graft beds were prepared on the right and left lateral thoracic wall of recipient mice. Grafts were fixed to the beds with 2-8 interrupted sutures of 5-0 silk. The first inspection was carried out on the 7th day, followed by daily inspection. After approximately 3 weeks, grafts showing no sign of rejection were followed on a weekly basis. Grafts were defined as rejected at the time of complete sloughing or when they formed a dry scab. Recipient mice were considered to be tolerant of donor antigens when donor skin grafts were in perfect condition for at least 100 days.
Analysis of Vβ11+ and Vβ8.1/2+ TCR expression. For analysis of TCR expression, peripheral blood lymphocytes (PBL) were prepared as described (5). For two-color FACS analysis, PBLs were blocked by 2.4G2 (FcγR blockade), and then incubated for 30 min with fluoresceinated anti-Vβ11 or Vβ8.1/2 antibodies (Pharmingen). The second incubation was with phycoerythrin-conjugated anti-CD4 and anti-CD8 antibodies (Pharmingen). Murine mAb HOPC1-FITC, with no reactivity to mouse cells, and nonspecific rat IgG2a-phycoerythrin were used as negative controls. Either 5000, or as many as were available, CD4+ plus CD8+ or CD4+ T cells were gated to determine the percentage of Vβ11+ and Vβ8.1/2+ T cells.
Thymectomy. Animals were anesthetized with 50 mg/kg pentobarbital and ketamine/xylazine (11 mg/kg and 1.1 mg/kg mixture), administered intraperitoneally, or methoxyflurane inhalation. After a partial sternotomy, thymectomy was performed by en bloc excision using two forceps. The absence of thymic tissue was always confirmed when thymectomized animals were killed, and animals showing the presence of residual thymic tissue were excluded from analysis.
Adoptive transfer of SC from chimeras to C57BL/6 nu/nu mice. To deplete donor B10.A cells from spleens of mixed chimeras, 1×107 SC were incubated with 1 ml of an antibody mixture containing 34-2-12 ascites (1/600 dilution), a C3H.SW (H2b) anti-C3H/HeJ (H2k) antiserum (1/600 dilution; cytotoxic titer 1:1200), and ascites of anti-H2Ld mAb 30-5-7 (1/600 dilution: cytotoxic titer 1:2300) (22) at 4°C for 30 min. The cells were washed twice, then incubated with rabbit complement (1/10 dilution) for 45 min at 37°C, and then washed twice. After depletion, 107 cells were injected intravenously into 3-Gy irradiated B6 nu/nu mice. Depletion was continued in vivo with three weekly injections of purified (2 mg/injection) anti-Dd mAb 34-2-12 (a mouse IgG2a).
In vivo depletion of donor antigen in B10.A→B10 chimeras. This was achieved with four weekly intraperitoneal injections of anti-Dd mAb 34-2-12 (1 mg [experiment 1] or 2 mg [experiment 2] per injection). The presence or absence of remaining donor-type B10.A cells was determined by incubating WBC from chimeras with 0.5 μg of 34-2-12, and then staining with fluoresceinated rat-anti-mouse (RαM) IgG2a mAb (Pharmingen), and comparing the profile to that of cells from normal B10 and B10.A mice.
Determination of persistent 34-2-12 mAb in serum of treated chimeras. After treatment of B10.A→B10 chimeras with depleting anti-Dd mAb 34-2-12 and before skin grafts were applied, the absence of 34-2-12 in the serum was verified in one of two experiments. Blood was collected and centrifuged for 2 min at 14,000 rpm. The serum was aspirated, and 10 μl (adjusted for the presence of a small volume of heparin in the original blood) of undiluted serum were incubated for 30 min with B10.A spleen cells to which mAb 2.4G2 had been added (see above). After washing, the cells were incubated with RαM IgG2a-FITC for 30 min, washed, then analyzed on a Becton Dickinson FACScan. Cells stained with 0.5 μg of 34-2-12, or with serum from a normal B10 mouse, plus RαM IgG2a-FITC served as positive, or negative, controls, respectively. Serum was considered to contain residual 34-2-12 if the percentage of cells staining was greater than that observed with normal serum.
RESULTS
Treatment of thymectomized or euthymic chimeras with anti-donor antibody leads to loss of chimerism. B10 (H2b; KbAbE-Db) mice were conditioned with the regimen described above, then received 1.5×107 allogeneic B10.A (H2a; KkAkEkDd) BMC. Multilineage chimerism (lymphocytes and granulocytes) was first evaluated at least 6 weeks after BMT, before treatment with anti-donor class I (Dd-specific) mAb 34-2-12. As shown in Table 1, a high level of lymphoid and myeloid chimerism was established.
Previous studies have demonstrated that treatment of chimeras with anti-donor class I (Dd-specific) mAb 34-2-12 results in a loss of chimerism and of donor-specific tolerance (13). To determine whether loss of donor-specific tolerance required the presence of a host thymus, the mice presented in Table 1 were divided into four groups, which were treated as follows: Groups A and B were thymectomized 7 weeks after BMT; groups C and D were not. Six weeks after thymectomy, groups B and D were injected with anti-Dd mAb 34-2-12 weekly for 4 weeks. Multilineage chimerism was determined using indirect staining (see Materials and Methods) from 2 to 11 weeks after the last dose of anti-donor antibody. As expected, groups receiving anti-donor antibody (groups B and D) showed no residual donor-type repopulation in any lineage. On the other hand, groups A and C, which did not receive anti-donor antibody, retained their high levels of donor-type repopulation in all hematopoietic lineages (Table 1), consistent with the presence of engrafted donor pluripotent hematopoietic stem cells.
Only donor antigen-depleted chimeras with an intact thymus reject donor-type grafts. To determine whether the loss of donor-specific tolerance previously observed when donor cells were depleted in vivo with anti-Dd mAb depends on the presence of a host thymus, we grafted all four groups of mice with donor-type (B10.A) and third-party (B10.D2 or DBA/2) tail skin 13 weeks after administration of the last dose of anti-Dd mAb. Similar to previous results (13), the anti-Dd mAb-treated euthymic mice (group D) rejected donor-type grafts (Fig. 1), demonstrating a loss of tolerance. In contrast, thymectomized, anti-Dd mAb-treated mice (group B) demonstrated long-term acceptance of donor-type skin grafts. These mice rejected third-party skin grafts, indicating that they were immunocompetent. Therefore, in the absence of a thymus, depletion of donor hematopoietic cells did not alter the state of donor-specific tolerance (Fig. 1). Non-monoclonal-antibody-treated euthymic (group C) or thymectomized (group A) mice also remained specifically tolerant to B10.A skin. These results show that loss of donor-specific tolerance upon depletion of donor antigen is dependent on the presence of a host thymus. To ensure that skin rejection was not a result of remaining circulating anti-Dd mAb in these studies, in one of two experiments combined in Figure 1, all groups were grafted only after serum anti-Dd mAb levels were shown to be undetectable.
Requirement for host thymus to generate Vβ11+ T cells after elimination of donor hematopoietic cells. Similar to previous results (12), deletion of Vβ11+ T cells was observed among PBL of B10.A→B10 mixed chimeras before anti-Dd mAb treatment. Absence of deletion in another Vβ family, Vβ8.1/2+, was used as a control to demonstrate that deletion is specific for Vβ11+ T cells. In long-term euthymic chimeras not receiving anti-Dd mAb treatment (Table 1, group C), Vβ11 deletion persisted for the duration of the study period. In both groups of chimeras that underwent thymectomy, group A (thymectomy, not anti-Dd mAb treated) and group B (thymectomy, anti-Dd mAb treated), no change in the deleted levels of Vβ11+ T cells in PBL was observed over time (Table 1), indicating that Vβ11+ T cells do not recover in the absence of a thymus, regardless of the presence or absence of donor-type antigen. In contrast, euthymic mice receiving anti-Dd mAb treatment (group D) demonstrated a marked increase in the percentage of Vβ11+ T cells in PBL over time (Table 1). Similar results were obtained when peripheral blood CD4+ cells were specifically evaluated for Vβ11 expression 27 weeks after the last anti-Dd mAb treatment. By this time, an average of 3.7% of CD4+ cells in euthymic, anti-Dd mAb-treated chimeras expressed Vβ11, compared with a mean of 0.27% in thymectomized, similarly treated chimeras (data not shown). Therefore, depletion of donor hematopoietic cells with anti-Dd mAb 34-2-12 resulted in the thymus-dependent development of Vβ11+ T cells.
SC from chimeras, when parked in B6 nu/nu mice and depleted of donor antigen, remain tolerant of the original marrow donor. To verify, in another model, that tolerance persists in the absence of antigen when the host lacks a thymus, SC from long-term mixed B10.A→B10 chimeras were isolated and depleted of donor-type cells ex vivo using anti-donor antibodies and complement. Then, 107 of these depleted SC were injected into 3-Gy irradiated B6 nu/nu mice, and these mice were treated with weekly injections of anti-donor antibody for 3 weeks. Elimination of donor B10.A cells was verified using FCM 4 weeks after the last dose of anti-Dd mAb. These mice were grafted with donor-type (B10.A) and third-party (B10.D2) tail skin 13 weeks after the last dose of anti-donor class I antibody, i.e., when it was shown that no circulating anti-Dd mAb remained. As shown in Figure 2, these mice accepted donor-type skin grafts but vigorously rejected third-party skin grafts, thus verifying that the presence of a thymus is required for the loss of donor antigen to lead to loss of tolerance.
The ability of B6 nu/nu mice receiving donor cell-depleted SC, but not of control nude mice, to reject third-party skin grafts provides evidence for engraftment of T cells from chimeras. To verify this engraftment, we measured percentages of T cells in spleens and lymph nodes when the animals were killed 18 weeks after skin grafting. While the percentages of CD4+ and CD8+ αβTCR+ T cells (2.6-7.5% CD4 and 4.6-7.5% CD8 cells in spleens; 5.6-11.3% CD4 and 9.3-12.7% CD8 cells in lymph nodes; n=4) were lower than those of a normal B10 mouse (15.6% CD4 and 8.1% CD8 cells in spleen; 38.0% CD4 and 23.5% CD8 cells in lymph nodes), percentages of these cells were significantly higher in animals that received SC from chimeras than in those that did not (0.1-0.2% CD4 and 1.2-2.8% CD8 cells in spleen, P<0.05 and _P_=0.07 for CD4s and CD8s, respectively, compared with spleens of adoptive recipients; 0.0-0.1% CD4 and 1.6% CD8 cells in lymph nodes, P<0.01 and P<0.001, respectively, compared with lymph nodes of adoptive recipients), thus demonstrating the long-term persistence of T cells from these inocula. However, we cannot rule out the possibility that some of the T cells in adoptive recipients of SC from chimeras were cells that developed extrathymically in nude mice and expanded in the presence of cytokines produced by transferred mature CD4 cells. We also stained BMC, spleens, and lymph nodes with donor (Dd) and host-specific monoclonal antibodies (anti-Kb mAb 5F1 (19) and were unable to detect any residual B10.A (Dd+ cells in these tissues of any of the mice (data not shown).
Injection of naive host-type T cells into established chimeras can break tolerance. While the above results suggested central deletion mechanism as the major mechanism of tolerance in mixed chimeras, we wished to verify the absence of a role for active suppression in maintaining tolerance. We performed studies to address this question in several different mouse strain combinations. Four B10 mice were given A/J (H2a) allogeneic BMC after receiving the conditioning regimen described above, and multilineage chimerism was verified using FACS analysis (Table 2). Two of these long-term chimeras (> 1 year after BMT) were then given intravenous injections of 30×106 naive host-type (B10) SC, and 1 week later all mice received grafts of donor-type and third-party skin. The results (Table 2) show that mice that received naive host-type SC rejected donor-type skin grafts, whereas chimeras not receiving naive host-type SC retained their skin grafts for greater than 98 days. Mice that received naive host-type cells lost detectable chimerism, indicating that the naive SC rejected all donor-type cells (Table 2).
We performed similar studies in a second strain combination, B10→B10.A. After multilineage chimerism had persisted for longer than 1 year (Table 2), three mice received 30×106 naive host-type SC intravenously, and 1 week later all six mice received donor-type and third-party skin grafts. Injection of host SC again resulted in rejection of donor-type skin grafts and loss of chimerism. Control mice not injected with naive donor-type SC retained their chimerism and accepted donor-type skin grafts, yet promptly rejected third-party grafts, indicating persistent donor-specific tolerance. Therefore, tolerance was readily broken by the infusion of 3×107 nontolerant host-type SC.
DISCUSSION
We have investigated the mechanisms maintaining tolerance in mixed allogeneic bone marrow chimeras prepared with a nonmyeloablative conditioning regimen. Host treatment with anti-CD4 and anti-CD8 mAbs, TI, and a low dose of WBI allows fully allogeneic marrow engraftment with permanent, mixed multilineage chimerism and long-term donor-specific skin graft tolerance in mice (6, 23). Tolerance is also observed in vitro at the level of CTL and mixed leukocyte reaction activity (13). We have recently shown that the induction of tolerance in these mice is associated with the early presence of donor class II+ cells with dendritic morphology in the thymus, and that the presence of these cells correlates with deletion of mature host thymocytes bearing Vβ11+ TCR that can react to superantigens presented by donor I-E in B10.A (I-E+)→B10 (I-E-) chimeras (12). Thus, we have obtained evidence for intrathymic clonal deletion of donor-reactive TCR among the earliest recovering thymocytes (day 10). The repertoire of T cells subsequently repopulating the periphery of these mice also showed marked and permanent deletion of Vβ11+ host-type T cells (12). However, a small population of Vβ11+ T cells was detectable in the spleens, but not the other lymphoid tissues of chimeras in the early months after BMT, and subsequently declined to undetectable levels, possibly due to dilution by tolerant T cells emerging from the thymus or to superantigen-driven expansion and deletion (2). These Vβ11+ T cells were anergic to stimulation through their TCR (12). Since anergy and suppressive mechanisms have been reported to maintain tolerance in other BMT models (24-29), it was essential to determine whether or not these additional mechanisms played a role in maintaining tolerance in our mixed chimeras.
Our results are consistent with the interpretation that intrathymic clonal deletion in the thymus is the only significant mechanism by which tolerance is maintained in these mixed chimeras. Two separate experimental models provided concordant results demonstrating that maintenance of tolerance is not dependent on the presence of antigen in the periphery. First, adoptive transfer of tolerant host-type lymphocytes from mixed chimeras to nude mice H2-matched to the original recipients conferred immunocompetence to the nude recipients, as evidenced by third-party skin graft rejection, while maintaining tolerance to the original donor in the absence of donor antigen (Fig. 2). Second, removal of the host thymus prevented the loss of donor-specific tolerance that is normally associated with administration of donor class I-specific mAb 34-2-12 (Table 1 and Fig. 1). These results strongly suggest that tolerance in the periphery is not maintained by an anergy mechanism, since maintenance of specific anergy has depended on the persistence of donor antigen in other tolerance models (30-32).
In both of these studies, host T cells not exposed to donor-type antigen for at least 13 weeks remained specifically tolerant to donor skin grafts in the absence of a thymus. However, we have not ruled out the formal possibility that the time it takes for anergic cells to regain their reactivity, in the absence of donor-type antigen, is greater than 13 weeks. Nevertheless, it is likely that the time required for loss of tolerance by anergic cells would be less than 13 weeks. Ramsdell and Fowlkes (32) demonstrated reversal of anergy within 10-20 days after removal of chimeric T cells from an antigen containing environment, and Morecki et al. (30) showed reversal of CTL tolerance within 4 days of “parking” chimeric T cells in an antigen-free environment.
In contrast to results in mice lacking a thymus, maintenance of tolerance was dependent on the persistence of donor antigen in euthymic mice (Fig. 1). This observation suggests that, even in long-term chimeras that had previously been treated with high-dose TI, the thymus is functional and able to generate and release to the periphery functional host T cells, despite the relatively advanced age of the recipients (>6 months old at first injection of anti-donor antibody). Although we have not directly examined thymic tissue for the timing of loss of intrathymic chimerism, it is likely that depletion of donor marrow progenitors results in an absence of donor antigen in the thymus, thus allowing the maturation of host thymocytes with reactivity to donor antigen. Consistent with this possibility, we observed a recovery of host Vβ11+ T cells, which recognize endogenous superantigens presented by donor I-E molecules, only in euthymic animals in which donor antigen had been depleted (Table 1). Thus, the appearance of these cells in the periphery was thymus dependent and only occurred in the absence of donor antigen. The recovery of such donor-reactive Vβ11+ T cells to a mean of 2.7% by 8 weeks and to 3.7% by 27 weeks after the last dose of anti-donor class I antibody, compared with an average of 5.6% for normal B10 mice, suggests that these donor-reactive T cells may emerge relatively slowly. This may be due in part to the relatively advanced age of the chimeras, since the rate of thymic export of naive T cells decreases with age (33).
The loss of tolerance observed in euthymic mice depleted of donor antigen with anti-class I mAb argues against a role for a peripheral suppressive mechanism in maintaining tolerance. The presumably small number of donor-reactive T cells emerging from the host thymus after depletion of donor antigen resulted in rapid rejection of donor-specific skin grafts, which suggests that significant regulatory mechanisms were not present in the periphery of the mice to inhibit this anti-donor alloreactivity. Consistent with the absence of potent peripheral suppressive mechanisms, animals receiving relatively small numbers of naive host-type SC lost chimerism and rapidly rejected donor-specific grafts (Table 2). This contrasts with results in other tolerance models in which active suppression has been implicated, and in which tolerance has been much more difficult to break with the administration of naive host-type lymphocytes (24, 34-36).
The difference between our results and those obtained in other models (4, 37, 38), in which clonal anergy and/or suppression appear to be major mechanisms of tolerance, may reflect the manner in which the host is conditioned before BMT. Whereas our mAb/3-Gy WBI treatment eliminates the vast majority of preexisting peripheral host T cells before BMT (6), A. Khan, Y. Tomita, and M. Sykes, manuscript in preparation) and then allows intrathymic development of a new T-cell repertoire in the presence of donor antigen, these other models involve modification of responses rather than depletion of preexisting T cells (4, 37, 38). For example, Qin et al. (4) used nondepleting anti-CD4 and anti-CD8 antibodies in the presence of donor-type antigen, whereas Matriano et al. (38) and Dorsch and Roser (37) introduced donor-type antigen to neonates. Because donor-reactive host T cells are present in these animals at the time of transplantation, they must be tolerized by a peripheral mechanism. On the other hand, our conditioning regimen depletes the vast majority of the host T cells. Similarly, we demonstrated previously that, in allogeneic or mixed allogeneic chimeras prepared by transplantation of T cell-depleted marrow after host conditioning with lethal irradiation, tolerance could be readily broken by the infusion of nontolerant host-type T cells (39). Presumably, host T-cell depletion from lethal irradiation also precludes the development of suppressive mechanisms of donor-specific tolerance.
While our previous studies of superantigen plus donor I-E-recognizing Vβ11+ cells strongly suggested that clonal deletion was the major mechanism of tolerance in mixed allogeneic chimeras, confirmation by the functional studies we have now performed was essential, since superantigens are not generally known to serve as transplantation antigens (40, 41).
Clonal deletion may be potentially more durable and resilient than other mechanisms of tolerance, since T-cell clones reactive to the donor are absent. Therefore, bystander activation of donor-reactive T cells, as may occur after infection with organisms containing epitopes that cross-react with donor antigens, could not occur. Tolerance induction protocols that rely on suppressive and anergic mechanisms have the inherent disadvantage of being associated with persistence of donor-reactive T-cell clones whose lack of function might be overcome under certain proinflammatory conditions. Indeed, it was shown recently that clonal anergy induced by Staphylococcus enterotoxin B was broken by infection with Nippstrongylus brasiliensis infection (42). An additional study showed similar disruption of peripheral tolerance (43).
In summary, our studies have demonstrated that in mixed allogeneic bone marrow chimeras prepared by BMT after recipient T-cell depletion, low-dose WBI, and TI, lifelong intrathymic clonal deletion is the only significant mechanism involved in the maintenance of T-cell tolerance. The recent success in applying this approach to the induction of tolerance in a primate model (44) brings this goal closer to clinical application, and has provided the impetus for understanding the mechanisms involved in the induction and maintenance of tolerance when a minimally toxic level of host conditioning is used prior to allogeneic BMT.
Acknowledgments. We thank Drs. David H. Sachs and Henry J. Winn for helpful review of the manuscript; Dr. Sachs for providing us with monoclonal antibody reagents; Denise A. Pearson, Justin J. Sergio, and Gregory L. Szot for expert technical assistance; Phuong Tran for excellent animal husbandry; and Pamela Roderick for assistance with the manuscript.
Requirement for a host thymus for loss of tolerance to occur after depletion of donor hematopoietic cells in mixed chimeras. Survival is shown for donor-type (B10.A; left panels) and third-party (DBA/2 or B10.D2; right panels) skin grafts in B10.A→B10 mixed chimeras receiving anti-Dd mAb treatment (-----) (groups D and B) or no further treatment (―) (groups A and C) with (bottom panels) or without (top panels) having been thymectomized 7 weeks after BMT. Results of two experiments are combined (n=5 in group A, n=8 in group B, n=4 in group C, and n=10 in group D). Mice were grafted 13 weeks after the last anti-Dd mAb treatment was given. Grafts were followed for 122 days in one experiment and for 155 days in the other experiment. The one euthymic anti-Dd mAb-treated chimera showing long-term graft survival was found dead 128 days after grafting with its B10.A graft intact. One euthymic control chimera was found dead at 29 days with its B10.A graft intact.
Donor-specific tolerance in B6 nude mouse recipients of donor-depleted SC from B10.A→B10 chimeras. SC from four long-term chimeras were depleted in vitro with anti-H2a antibodies and complement, and 107 cells were injected into B6 nude mice (n=4; chimera→nude). To deplete residual donor cells, anti-Dd mAb was given in vivo every week for 3 weeks. Thirteen weeks after the last anti-Dd mAb injection, the nude recipients were grafted with B10.A donor (•-----•) and with third-party B10.D2 (▴-----▴) skin. Control nude mice in the same cohort (n=2) accepted both B10.A (▵―▵) and B10.D2 (○―○) skin grafts.
Footnotes
This study was supported by National Institutes of Health grant RO1 HL49915.
Abbreviations: BMC, bone marrow cells; BMT, bone marrow transplantation; FCM, flow cytometry; FITC, fluorescein isothiocyanate; PBL, peripheral blood lymphocytes; SC, spleen cells; TI, thymic irradiation; WBC, white blood cells; WBI, whole body irradiation.
REFERENCES
1. Kappler JW, Roehm N, Marrack P. T cell tolerance by clonal elimination in the thymus. Cell 1987; 49: 273.
2. Webb S, Morris C, Sprent J. Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell 1990; 63: 1249.
3. Burkly LC, Lo D, Kanagawa O, Brinster RL, Flavell RA. T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E. Nature 1989; 342: 564.
4. Qin S, Cobbold SP, Pope H, et al. “Infectious” transplantation tolerance. Science 1993; 259: 974.
5. Ildstad ST, Wren SM, Bluestone JA, Barbieri SA, Sachs DH. Characterization of mixed allogeneic chimeras. Immunocompetence, in vitro reactivity, and genetic specificity of tolerance. J Exp Med 1985; 162: 231.
6. Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a non-lethal preparative regimen. J Exp Med 1989; 169: 493.
7. Acha-Orbea H, Palmer E. Mls-a retrovirus exploits the immune system. Immunol Today 1991; 12: 356.
8. Tomonari K, Fairchild S. The genetic basis of negative selection of Tcrβ-V11+ T cells. Immunogenetics 1991; 33: 157.
9. Dyson PJ, Knight AM, Fairchild S, Simpson E, Tomonari K. Genes encoding ligands for deletion of Vβ11 T cells cosegregate with mammary tumour virus genomes. Nature 1991; 349: 531.
10. Bill J, Kanagawa O, Woodland D, Palmer E. The MHC molecule I-E is necessary but not sufficient for the clonal deletion of Vβ11 bearing T cells. J Exp Med 1989; 169: 1405.
11. Tomonari K, Lovering E. T cell-receptor-specific monoclonal antibodies against a Vβ11+ mouse T cell-clone. Immunogenetics 1988; 28: 445.
12. Tomita Y, Khan A, Sykes M. Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a non-myeloablative regimen. J Immunol 1994; 153: 1087.
13. Sharabi Y, Abraham VS, Sykes M, Sachs DH. Mixed allogeneic chimeras prepared by a non-myeloablative regimen: requirement for chimerism to maintain tolerance. Bone Marrow Transplant 1992; 9: 191.
14. Dialynas DP, Quan ZS, Wall KA, et al. Characterization of murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to human Leu3/T4 molecule. J Immunol 1983; 131: 2445.
15. Sarmiento M, Glasebrook AL, Fitch FW. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt2 antigen block T cell-mediated cytolysis in the absence of complement. J Immunol 1980; 125: 2665.
16. Tomita Y, Sachs DH, Sykes M. Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow. Blood 1994; 83: 939.
17. Ozato K, Mayer NM, Sachs DH. Monoclonal antibodies to mouse major histocompatibility complex antigens IV. A series of hybridoma clones producing anti-H-2d antibodies and an examination of expression of H-2d antigens on the surface of these cells. Transplantation 1982; 34: 113.
18. Unkeless JC. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J Exp Med 1979; 150: 580.
19. Sherman LA, Randolph CP. Monoclonal anti-H-2Kb antibodies detect serological differences between H-2Kb mutants. Immunogenetics 1981; 12: 183.
20. Muller-Rucholtz W, Muller-Hermelink HK, Wottge HU. Induction of lasting hematopoietic chimerism in a xenogeneic (rat→mouse) model. Transplant Proc 1979; 11: 517.
21. Eto M, Mayumi H, Tomita Y, Yoshikai Y, Nomoto K. Intrathymic clonal deletion of Vβ6+ T cells in cyclophosphamide-induced tolerance to H-2-compatible, Mls-disparate antigens. J Exp Med 1990; 171: 97.
22. Ozato K, Hansen TH, Sachs DH. Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex. J Immunol 1980; 125: 2473.
23. Lee LA, Sergio JJ, Sykes M. Natural killer cells weakly resist engraftment of allogeneic long-term multilineage-repopulating hematopoietic stem cells. Transplantation 1996; 61: 125.
24. Roser BJ. Cellular mechanisms in neonatal and adult tolerance. Immunol Rev 1989; 107: 179.
25. Tutschka PJ, Ki PF, Beschorner WE, Hess AD, Santos GW. Suppressor cells in transplantation tolerance. II. Maturation of suppressor cells in the bone marrow chimera. Transplantation 1981; 32: 321.
26. Maki T, Gottshalk R, Wood ML, Monaco AP. Specific unresponsiveness to skin allografts in anti-lymphocyte serum-treated, marrow-injected mice: participation of donor marrow-derived suppressor T cells. J Immunol 1981; 127: 1433.
27. Morecki S, Leshem B, Weigensberg M, Bar S, Slavin S. Functional clonal deletion versus active suppression in transplantation tolerance induced by total lymphoid irradiation. Transplantation 1985; 40: 201.
28. Ramsdell F, Fowlkes BJ. Clonal deletion versus clonal anergy: the role of the thymus in inducing self tolerance. Science 1990; 248: 1342.
29. Scully R, Qin S, Cobbold S, Waldmann H. Mechanisms in CD4 antibody-mediated transplantation tolerance: kinetics of induction, antigen dependency and role of regulatory T cells. Eur J Immunol 1994; 24: 2383.
30. Morecki S, Leshem B, Eid A, Slavin S. Alloantigen persistence in induction and maintenance of transplantation tolerance. J Exp Med 1987; 165: 1468.
31. Rocha B, Tanchot C, Von Boehmer H. Clonal anergy blocks in vivo growth of mature T cells and can be reversed in the absence of antigen. J Exp Med 1993; 177: 1517.
32. Ramsdell F, Fowlkes BJ. Maintenance of in vivo tolerance by persistence of antigen. Science 1992; 257: 1130.
33. Sprent J. T lymphocytes and the thymus. In: Paul WE, ed. Fundamental immunology, 3rd ed. New York: Raven, 1993: 75.
34. Ramseier H. Immunization against abolition of transplantation tolerance. Eur J Immunol 1973; 3: 156.
35. Silvers WK. Studies on the apparent serial passage of transplantation immunity in tolerant mice. Transplantation 1970; 10: 538.
36. Billingham RE, Brent L, Medawar PB. Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance. Philos Trans R Soc 1955; 239 (Ser. B): 44.
37. Dorsch S, Roser B. T cells mediate transplantation tolerance. Nature 1975; 2580: 233.
38. Matriano JA, Socarras S, Streilein JW. Cellular mechanisms that maintain neonatally-induced tolerance of class 11 alloantigens. J Immunol 1994; 153: 1505.
39. Sykes M, Sheard MA, Sachs DH. Effects of T cell depletion in radiation bone marrow chimeras II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance. J Exp Med 1988; 168: 661.
40. Korngold R, Sprent J. Lethal graft-versus-host disease after bone marrow transplantation across minor histocompatibility barriers in mice. J Exp Med 1978; 148: 1687.
41. Salaun J, Bandeira A, Khazaal I, et al. Transplantation tolerance is unrelated to superantigen-dependent deletion and anergy. Proc Natl Acad Sci USA 1992; 89: 10420.
42. Rocken M, Urban JF, Shevach EM. Infection breaks T-cell tolerance. Nature 1992; 359: 79.
43. Ohashi PS, Oehen S, Buerki K, et al. Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 1991; 65: 305.
44. Kawai T, Cosimi AB, Colvin RB, et al. Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys. Transplantation 1995; 59: 256.
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