Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.

It has been known since the early observations of Owen and Medawar and collegues (1, 2, 3, 4) that a state of hematopoietic chimerism is associated with donor-specific transplantation tolerance. However, inducing hematopoietic chimerism in an immunocompetent adult is a challenge that has thus far precluded the use of this approach to inducing tolerance in humans. A relatively nontoxic conditioning regimen consisting of host treatment with depleting anti-CD4 and anti-CD8 mAbs, 3 Gy total body irradiation (TBI),4 and 7 Gy local irradiation to the thymus can reliably permit the engraftment of fully allogeneic marrow, which leads to the induction of mixed hematopoietic chimerism and transplantation tolerance (5). Tolerance in such animals is induced and maintained largely by an intrathymic deletional mechanism (6, 7, 8). Subsequently, we observed that thymic irradiation could be avoided in this regimen by giving repeated treatments with T cell-depleting mAbs to the recipient (9). However, this approach had the disadvantage of being associated with prolonged T cell depletion in the recipient, a state that is not desirable in adult humans, in whom limited thymic reserve might greatly delay the recovery of a functional immune system. More recently, we observed that the requirement for either thymic irradiation or repeated T cell-depleting (TCD) mAb injection could be obviated by treating recipient mice with one injection of either anti-CD40 ligand (CD40L) mAb on day 0 or with an injection of CTLA4Ig on day 2 (10). Furthermore, the use of a combination of both of these costimulatory blockers, each given once, completely replaced the requirement for recipient T cell depletion with mAbs and for thymic irradiation (11). Unfortunately, this very benign conditioning regimen (anti-CD40L day 0, CTLA4Ig day 2, and 3 Gy TBI day 0) has recently become less reliable as we have switched to a new CTLA4Ig preparation.

Studies with our original TCD nonmyeloablative bone marrow transplantation (BMT) regimen had demonstrated a role for both CD4 and CD8 T cells of the recipient in resisting engraftment of fully allogeneic marrow (5, 12). The goals of the present studies were to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to alloengraftment in mice receiving costimulatory blockade without TCD mAbs, and to use this information to develop a regimen for induction of mixed chimerism and transplantation tolerance that is minimally toxic and 100% reliable, and, therefore, potentially clinically applicable. The results demonstrate that anti-CD40L treatment is sufficient to completely overcome CD4 cell-mediated resistance to fully mismatched allogeneic marrow engraftment, and that a conditioning regimen consisting of depleting anti-CD8 mAb on day −1, a single injection of anti-CD40L mAb on day 0, and 3 Gy TBI is sufficient to reliably allow engraftment of fully allogeneic marrow and CD4 T cell tolerance in every strain combination tested. This protocol is associated with robust donor-specific tolerance to solid tissue grafted on day 1, and hence has considerable relevance to cadaveric organ transplantation.

Eight- to 12-wk-old female C57BL/6 (B6: H-2b), B10.A (B10.A: H-2a), A.SW (H-2s), B10.BR (H-2k), BALB/c (H-2d) and B10.RIII (H-2r) mice were purchased from Frederick Cancer Research Center (Frederick, MD) or from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a specific pathogen-free microisolator environment, as previously described (13).

Age-matched (8- to 12-wk-old) mice received 3 Gy TBI and were injected i.v. on the same day (day 0) with unseparated BM harvested from MHC-mismatched donors (8–12 wk old). Mice were injected i.p. with the indicated doses of rat IgG2b anti-mouse CD4 mAb GK1.5 and anti-mouse CD8 mAb 2.43 on day −1. Hamster anti-mouse CD40L mAb (MR1) was injected i.p. on day 0 (0.5 mg or 2 mg). Murine CTLA4Ig was injected i.p. as a single dose (0.5 mg) on day +2. The MR1 hybridoma was kindly provided to us by Randolph J. Noelle (Dartmouth Medical School, Lebanon, NH). CTLA4Ig (14) was prepared in our laboratory from a cell line transfected with CTLA4Ig (kindly provided by Terry Strom, Beth Israel/Deaconess Hospital, Boston, MA).

FCM of multilineage chimerism was performed as previously described (9). In brief, forward angle and 90° light scatter properties were used to distinguish lymphocytes, monocytes, and granulocytes in peripheral white blood cells. Two-color FCM was used to distinguish donor and host cells of particular lineages, and the percentage of donor cells was calculated as previously described (9), by subtracting control staining from quadrants containing donor and host cells expressing a particular lineage marker, and by dividing the net percentage of donor cells by the total net percentage of donor plus host cells of that lineage. Dead cells were excluded using propidium iodide staining. Nonspecific FcR binding was blocked by anti-mouse FcR mAb 2.4G2 (15). FITC-conjugated mAbs included anti-CD4, anti-CD8, anti-B220 (all purchased from PharMingen, San Diego, CA), and anti-MAC1 (Caltag, San Francisco, CA). Negative control mAb HOPC1-FITC, with no reactivity to mouse cells, was prepared in our laboratory. Biotinylated anti-H-2Dd mAb 34-2-12, anti-H-2Kk mAb 36-7-5 (PharMingen) and control mAb HOPC1 were developed with PE-streptavidin.

PBLs were stained with FITC-conjugated anti-Vβ5.1/2, Vβ11, and Vβ8.1/2 or control mAbs vs PE-conjugated anti-CD4 mAb (all purchased from PharMingen). Nonspecific PE-conjugated rat IgG2a (PharMingen) served as a negative control. Two-color FCM analysis was performed on gated CD4+ cells. Splenocytes were stained with FITC-conjugated anti-Vβ5.1/2, Vβ11, and Vβ8.1/2 or control mAbs vs PE-conjugated anti-CD4 mAb (or anti-CD8 mAb; PharMingen). Three-color FCM analysis was performed on gated host-type class I (KH95)-high, CD8-negative (CD4), or CD4-negative (CD8) cells, and the percentage of Vβ-positive cells in this gate was corrected for the percentage of TCRβ-high cells in the same gate, as previously described (6). Thymocytes were stained with FITC-conjugated anti-TCRβ- (PharMingen), or anti-Vβ5.1/2, Vβ11, and Vβ8.1/2 vs BIO-conjugated KH95 (anti-Db, PharMingen) developed with CyChrome-streptavidin (PharMingen). For B10.A controls, gated 34-2-12-high cells were analyzed in a similar fashion. Background staining (as determined by nonreactive mAb HOPC-FITC) in the same gate was subtracted from the percentage of cells staining with each anti-Vβ mAb.

Full thickness tail skin (∼1.0 cm2) from B10.A or B10.BR (donor-specific) and fully MHC-mismatched B10.RIII (third party) mice were grafted on the dorsal thoracic wall, sutured with 5–0 silk, bandaged, and followed by daily visual inspection. Grafts were defined as rejected when <10% of the graft remained viable.

Splenocytes were cultured in triplicate wells containing 4 × 105 responders with 4 × 105 stimulators (30 Gy) in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 15% (v/v) controlled processed serum replacement (Sigma, St. Louis, MO), 0.09 mM nonessential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.05 mM 2-ME, and 0.01 M HEPES buffer at 37°C in 5% CO2 for 3–4 days before they were pulsed with [3H]thymidine and harvested ∼18 h later. Stimulation index was calculated by dividing mean cpm from allogeneic responses by mean cpm from anti-self (or anti-host in the case of BMT recipients) responses, which were similar to background cpm (i.e., cpm with no stimulator cell population).

Splenocytes from controls, BMT recipients and normal mice were resuspended in RPMI 1640 (Mediatech, Herndon, VA) containing 10% FBS (Sigma), 0.09 mM nonessential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.05 mM 2-ME, and 0.01 M HEPES buffer. Responder and stimulator cells (30 Gy) were diluted to a concentration of 8 × 106 cells/ml, and 100 μl responder cells were cocultured with 100 μl stimulator cells per well. Cultures were set up in two rows of three replicates each, and after 5 days of incubation in 5% CO2 at 37°C, 2-fold serial solutions were prepared from the second row of triplicates, so that cytolytic capacity could be examined at five different responder-to-target ratios. A total of 8000 51Cr-labeled, 2-day concanavalin-A-stimulated lymphoblasts were added to each well and incubated for four hours in 5% CO2 at 37°C before they were harvested. The percent of specific lysis was calculated with the following formula: percent of specific lysis = ((experimental release − spontaneous release)/(maximum release − spontaneous release)) × 100%.

Statistical significance was determined with a two-tailed Student’s t test for comparison of means with unequal variances. A p value of <0.05 was considered to be statistically significant.

We have previously demonstrated that treatment of B6 mice with depleting doses of anti-CD4 mAb GK1.5 (1.76 mg) and anti-CD8 mAb 2.43 (1.4 mg) on day −1, in combination with anti-CD40L mAb MR1 on day 0 and 3 Gy TBI on day 0, allowed engraftment of B10.A fully MHC-mismatched marrow and acceptance of donor skin grafted in the peritransplant period (10). Because complete peripheral T cell depletion is more difficult to achieve in large animals and humans than in mice, we wished to determine whether a similar outcome could be achieved if less than fully depleting doses of anti-CD4 and anti-CD8 mAbs were given in this regimen. B6 mice were treated with various doses of these mAbs on day −1, followed on day 0 by 0.5 mg of MR1 and 3 Gy TBI, and injection of 20 × 106 unseparated BM cells from fully MHC-mismatched B10.A donors. Donor hematopoiesis was then assessed at multiple time points after BMT by FCM analysis of peripheral white blood cells.

Injection of MR1 alone (0.5 mg/mouse) to 3 Gy-irradiated mice permitted induction of lasting mixed chimerism in only two of six animals (Fig. 1, a and b). Consistent with previous results (10), administration of a standard dose of TCD mAbs (1.76 mg GK1.5 and 1.4 mg 2.43) on day −1 to mice receiving 3 Gy TBI and 0.5 mg MR1 on day 0 allowed induction of high levels of lasting, multilineage mixed chimerism in six of six mice (data not shown). Reduction of the dose of TCD mAbs to 1/4 of the standard dose still allowed lasting multilineage chimerism to be achieved in all mice (n = 6; Fig. 1, a and b). We evaluated a variety of progressively lower doses of TCD mAbs in combination with MR1 and 3 Gy TBI, and, as is shown in Fig. 1, a and b, administration of only 1/64 of the standard TCD mAb dose (0.025 mg GK1.5 and 0.02 mg 2.43) was sufficient to allow induction of lasting chimerism in six of six animals also receiving 0.5 mg of MR1 and 3 Gy TBI on day 0. Similar results were obtained in a repeat experiment.

FIGURE 1.

Long-term multilineage chimerism in peripheral blood of mice receiving reduced doses of TCD mAbs plus MR1. B6 mice were treated with 3 Gy TBI and received 20 × 106 unseparated BM cells from fully MHC-mismatched B10.A donors. All the groups are from one experiment. a, Percentage of long-term chimeras (>25 wk) among the mice receiving various mAb treatments are presented. TCD mAbs were given on day −1, and MR1 (0.5 mg) was given on day 0. A single injection of MR1 (n = 6) allowed induction of lasting mixed chimerism in only two of six mice. When the mice were treated with a single dose of 1/4 or 1/64 of the standard TCD mAbs dose on day −1 and MR1 (0.5 mg) on day 0, all mice (n = 6 per group) became long-term chimeras. b, The mean percentage of donor cells among peripheral blood granulocytes as determined by two-color FCM at various times post-BMT. The mice treated with 1/4 or 1/64 of the standard doses of TCD mAbs plus MR1 (1/4 TCD, MR1 and 1/64 TCD, MR1, respectively) showed high levels of lasting chimerism. Two of six mice treated with MR1 alone (MR1) became long lasting chimeras, whereas four mice failed to achieve lasting mixed chimerism. Results are shown as mean ± SEM in each group. Similar results were obtained in all of the lineages tested (CD4 and CD8 T cells, monocytes, B cells), and granulocytes are shown as being representative of all lineages.

FIGURE 1.

Long-term multilineage chimerism in peripheral blood of mice receiving reduced doses of TCD mAbs plus MR1. B6 mice were treated with 3 Gy TBI and received 20 × 106 unseparated BM cells from fully MHC-mismatched B10.A donors. All the groups are from one experiment. a, Percentage of long-term chimeras (>25 wk) among the mice receiving various mAb treatments are presented. TCD mAbs were given on day −1, and MR1 (0.5 mg) was given on day 0. A single injection of MR1 (n = 6) allowed induction of lasting mixed chimerism in only two of six mice. When the mice were treated with a single dose of 1/4 or 1/64 of the standard TCD mAbs dose on day −1 and MR1 (0.5 mg) on day 0, all mice (n = 6 per group) became long-term chimeras. b, The mean percentage of donor cells among peripheral blood granulocytes as determined by two-color FCM at various times post-BMT. The mice treated with 1/4 or 1/64 of the standard doses of TCD mAbs plus MR1 (1/4 TCD, MR1 and 1/64 TCD, MR1, respectively) showed high levels of lasting chimerism. Two of six mice treated with MR1 alone (MR1) became long lasting chimeras, whereas four mice failed to achieve lasting mixed chimerism. Results are shown as mean ± SEM in each group. Similar results were obtained in all of the lineages tested (CD4 and CD8 T cells, monocytes, B cells), and granulocytes are shown as being representative of all lineages.

Close modal

Although significant numbers (>0.3%) of T cells were not detected by FCM in the blood of mice that received the standard dose of TCD mAbs, at dilutions of one-sixteenth and more of the standard dose of TCD mAb, T cells were readily detectable (>0.3%) in the peripheral blood by 1 wk (data not shown). Although T cell concentrations were significantly lower (p < 0.001) than those in mice receiving MR1 without TCD mAbs, those receiving 1/64 of the standard TCD mAb dose had substantial numbers of host T cells (range 11.81–22.63% of PBL vs 38.7% in normal B6) in their blood by 1 wk. Donor T cells were not detectable. Because it is unlikely that this level of reconstitution occurred from the thymus within 1 wk, we concluded that injection of a single dose of MR1 on day 0 reliably allowed the induction of lasting, multilineage mixed chimerism in mice receiving an incompletely depleting dose of TCD mAbs and 3 Gy TBI followed by fully MHC-mismatched allogeneic BMT.

To determine whether or not donor-specific tolerance was achieved in mice receiving BMT with the above protocols, skin grafting was performed 1 day following BMT. All mice that displayed lasting mixed chimerism, regardless of whether completely or incompletely depleting doses of TCD mAb were administered along with MR1 and 3 Gy TBI, accepted donor skin grafts for the duration of follow-up (>200 days, data not shown). In mice treated with MR1 and 3 Gy TBI alone, the two of six mice that showed sustained chimerism accepted donor skin grafts, whereas mice that did not develop lasting chimerism (chimerism undetectable by 6–10 wk) rejected donor skin. Mice treated with 1/4 of the standard TCD dose and 3 Gy TBI alone rejected donor skin by 47 days. Third-party (B10.RIII) skin was rejected by day 47 in all treatment groups, regardless of whether or not chimerism was induced (data not shown). The delayed third-party skin graft rejection observed in some mice (especially those receiving the higher dose of TCD mAbs in combination with MR1) reflects the temporary immunosuppressive effect of the conditioning regimens. The regimen involving 1/64 TCD mAbs, MR1, and 3 Gy TBI was only slightly immunosuppressive, with all third-party skin grafts completely rejected by 20 days posttransplant, whereas donor-specific grafts were accepted for the duration of follow-up (34 wk; data not shown).

The long-term chimeras that accepted donor skin grafts for the duration of follow-up, regardless of TCD treatment, showed tissue chimerism in the bone marrow, spleen, and thymus, and demonstrated donor-specific unresponsiveness in MLR and CML at 34 wk (data not shown). The two long-term chimeras that were prepared with 3 Gy TBI and MR1 alone also showed donor-specific unresponsiveness in MLR and CML, as well as marrow, spleen, and thymic chimerism at the time of sacrifice (34 wk) (data not shown). Thus, lasting chimerism and systemic donor-specific tolerance was reliably induced across a full MHC barrier in chimeras prepared with an incompletely depleting dose of TCD plus MR1, similar to results seen in other mixed chimeras (10). Although similar results were obtained in a minority of mice receiving MR1 without TCD mAbs, MR1 alone did not reliably allow this outcome to be achieved.

Central deletion has been established as the major mechanism for maintenance of tolerance in mixed chimeras prepared by a variety of regimens (6, 7, 8, 10, 11, 17). We examined whether or not donor-reactive T cells in PBL and thymus were deleted by assessing the usage of certain Vβ subunits within the TCR repertoires. The donor strain B10.A expresses I-E, which is required to present superantigens derived from mammary tumor virus 8 and 9 endogenous retroviruses encoded in the B6/B10 background genome (18, 19, 20). Developing thymocytes whose TCR contain Vβ5 and Vβ11 subunits, which bind to these superantigens, are deleted in I-E-positive B10.A mice, but not in B6 mice, because they do not express I-E (19, 21). The mice that received low-dose TCD mAbs plus MR1 showed profound reductions in the percentage of Vβ5+ CD4 PBL (normal B6, 2.56%; normal B10.A, 0.00%) to 0.18 ± 0.13%, and Vβ11+ CD4 PBL (normal B6, 5.07%; normal B10.A, 0.00%) to 0.17 ± 0.14% at 8 wk post-BMT. At the time of sacrifice 34 wk post-BMT, the chimeras prepared with low-dose (1/64) TCD mAbs plus MR1 showed a profound reduction in the percentage of Vβ5+ and Vβ11+ mature single-positive host-type thymocytes compared with naive mice or BMT recipients prepared with 3 Gy TBI and MR1 alone that did not develop lasting mixed chimerism (data not shown). These data suggest that central deletion of donor-reactive T cells is one of the major mechanisms maintaining tolerance in long-term chimeras prepared with 3 Gy TBI, MR1, and incompletely depleting doses of TCD mAbs.

Because 100% of BMT recipients conditioned with the incompletely depleting 1/64 dilution of the standard TCD mAbs dose in combination with MR1 and 3 Gy TBI developed lasting chimerism and tolerance, we further titrated the TCD mAbs to determine the minimal dose of TCD mAbs required to assure the development of lasting chimerism. In a single experiment, we compared the development of chimerism and tolerance in mice conditioned with MRI, 3 Gy TBI, and a dose of TCD mAbs ranging from 0.055 mg to 0.004 mg of GK1.5 and 0.044–0.003 mg of 2.43 (1/32 to 1/512 of standard dose). Again, all mice receiving the 1/64 dose of TCD mAbs showed lasting chimerism. However, when the dose of TCD mAbs was reduced further, results were more variable, with some animals showing lasting chimerism, and others showing only initial chimerism that declined markedly by 6–10 wk post-BMT. More than half of the mice treated with 1/128 to 1/512 of the standard TCD mAbs dose (plus MR1 and 3 Gy TBI) became lasting chimeras and specifically accepted donor skin grafted 1 day following BMT (data not shown).

We investigated the relationship between recipient T cell recovery and the development of lasting donor chimerism in these groups of mice to see whether recovery of one or the other T cell subset could predict the ultimate loss of chimerism. Substantial levels of recipient CD4 cells were measurable in the blood of most animals by 2 wk post BMT, but there was wide variation, from ∼5% to 30% CD4 cells in PBL of various animals. The higher levels of CD4 cells at this time point were not associated with a failure of chimerism by 6 wk and later post-BMT, as animals with both high and low levels of CD4 cells showed successful and unsuccessful maintenance of chimerism (data not shown). However, substantial host CD8 cell recovery, which became evident in only some animals by 6 wk post-transplant, showed a significant association with a failure to maintain chimerism by 6 wk. A lack of chimerism at this time point was seen only in mice with host CD8 cell recovery to at least 5% by 6 wk post-BMT (Fig. 2). At earlier time points, the recovery of CD8 cells was minimal in all animals, and did not predict the achievement or failure to achieve durable chimerism.

FIGURE 2.

The relationship between the degree of donor cell chimerism and host CD8 cell recovery. A lack of chimerism in B cells, monocytes, and granulocytes is only seen in mice with early recovery to ≥5% host CD8 cells in PBL (6 wk post-BMT). Significant numbers of circulating CD8 cells were not measurable at earlier time points. The mice in this study received various doses (1/32 to 1/512 of the standard dose of TCD mAbs) on day −1 plus MR1 (0.5 mg) and 3 Gy TBI on day 0. All animals showing chimerism at 6 wk were chimeric in T cells, B cells, monocytes, and granulocytes for the duration of follow-up (23 wk).

FIGURE 2.

The relationship between the degree of donor cell chimerism and host CD8 cell recovery. A lack of chimerism in B cells, monocytes, and granulocytes is only seen in mice with early recovery to ≥5% host CD8 cells in PBL (6 wk post-BMT). Significant numbers of circulating CD8 cells were not measurable at earlier time points. The mice in this study received various doses (1/32 to 1/512 of the standard dose of TCD mAbs) on day −1 plus MR1 (0.5 mg) and 3 Gy TBI on day 0. All animals showing chimerism at 6 wk were chimeric in T cells, B cells, monocytes, and granulocytes for the duration of follow-up (23 wk).

Close modal

The association of early host CD8 recovery with failure of durable engraftment in mice receiving incompletely depleting doses of TCD mAbs along with MR1 and 3 Gy TBI led us to hypothesize that recipient CD8+ cells were responsible for donor marrow rejection in these mice, and to speculate that CD8 depletion alone might be sufficient to ensure the reliable achievement of chimerism and tolerance in mice receiving MR1 and 3 Gy TBI. To address this hypothesis, we evaluated marrow engraftment in mice treated with CD8 TCD mAb alone, along with MR1 and 3 Gy TBI. As is shown in Fig. 3, six of six mice treated with a depleting dose of anti-CD8 mAb (0.35 mg) plus MR1 developed high levels of durable multilineage chimerism. These mice were specifically tolerant to donor Ags, as they accepted donor skin grafted on day 1 post-BMT, while rejecting third-party skin grafted at the same time (Fig. 4). This regimen has produced similar results in many repeated (>7) experiments. In contrast, mice (n = 5) treated with anti-CD8 mAb plus MR1 and 3 Gy TBI without BMT rejected both B10.A and B10.RIII skin within 14 days (not shown). The ability of the mice that did not receive BMT to reject skin grafted on day 1 and of mice that did receive BMT to reject third-party skin illustrates the requirement for BMT for tolerance induction in this model. Skin grafted on day 1 as the only source of Ag did not induce tolerance in mice receiving this regimen.

FIGURE 3.

Long-term multilineage chimerism in peripheral blood of mice receiving CD8-depleting mAbs plus MR1. B6 mice were treated with 3 Gy TBI and received 20 × 106 unseparated BM cells from fully MHC-mismatched B10.A donors. All the groups are from one experiment. a, Percentages of long-term (>20 wk) chimeras among recipients of the various mAb treatments are presented. CD4 and/or CD8-depleting mAbs (0.44 mg GK1.5 and 0.35 mg 2.43) were given on day −1, and MR1 (0.5 mg) was given on day 0. Treatment with anti-CD4 and CD8 mAbs plus MR1 (n = 7), and treatment with anti-CD8 mAb plus MR1 (n = 6) successfully induced long-term chimerism in every mouse. Treatment with anti-CD4 mAb plus MR1 (n = 7) did not induce long-term chimerism in any mice. A single injection of MR1 (n = 7) allowed induction of lasting mixed chimerism in only two of seven mice. Anti-CD4, CD8, or combination treatment (without MR1) did not induce lasting mixed chimerism in any mice. b, The mean percentage of donor cells among peripheral blood granulocytes as determined by two-color FCM at various times post-BMT. The mice treated with anti-CD8 plus MR1 or anti-CD4, anti-CD8 plus MR1 showed high levels of lasting chimerism. Two of seven mice treated with MR1 alone became long lasting chimeras (MR1 chimeras), whereas five mice failed to achieve lasting mixed chimerism (MR1-non chimeras). Anti-CD4 or CD8, or a combination of anti-CD4 plus anti-CD8 mAbs treatment (without MR1) failed to induce lasting chimerism. Results are shown as mean ± SEM in each group. Similar results were obtained in all of the lineages tested (CD4 and CD8 T cells, monocytes, B cells), and granulocytes are shown as being representative of all lineages.

FIGURE 3.

Long-term multilineage chimerism in peripheral blood of mice receiving CD8-depleting mAbs plus MR1. B6 mice were treated with 3 Gy TBI and received 20 × 106 unseparated BM cells from fully MHC-mismatched B10.A donors. All the groups are from one experiment. a, Percentages of long-term (>20 wk) chimeras among recipients of the various mAb treatments are presented. CD4 and/or CD8-depleting mAbs (0.44 mg GK1.5 and 0.35 mg 2.43) were given on day −1, and MR1 (0.5 mg) was given on day 0. Treatment with anti-CD4 and CD8 mAbs plus MR1 (n = 7), and treatment with anti-CD8 mAb plus MR1 (n = 6) successfully induced long-term chimerism in every mouse. Treatment with anti-CD4 mAb plus MR1 (n = 7) did not induce long-term chimerism in any mice. A single injection of MR1 (n = 7) allowed induction of lasting mixed chimerism in only two of seven mice. Anti-CD4, CD8, or combination treatment (without MR1) did not induce lasting mixed chimerism in any mice. b, The mean percentage of donor cells among peripheral blood granulocytes as determined by two-color FCM at various times post-BMT. The mice treated with anti-CD8 plus MR1 or anti-CD4, anti-CD8 plus MR1 showed high levels of lasting chimerism. Two of seven mice treated with MR1 alone became long lasting chimeras (MR1 chimeras), whereas five mice failed to achieve lasting mixed chimerism (MR1-non chimeras). Anti-CD4 or CD8, or a combination of anti-CD4 plus anti-CD8 mAbs treatment (without MR1) failed to induce lasting chimerism. Results are shown as mean ± SEM in each group. Similar results were obtained in all of the lineages tested (CD4 and CD8 T cells, monocytes, B cells), and granulocytes are shown as being representative of all lineages.

Close modal
FIGURE 4.

Skin graft tolerance in mixed chimeras prepared with 3 Gy TBI, 20 × 106 BMT, anti-CD8 mAb plus MR1. BMT recipients prepared with anti-CD8 mAb plus MR1 (–•–; n = 6), and both anti-CD4-and CD8 mAbs plus MR1 (▪; n = 7) accepted donor grafts permanently, while third-party grafts were rejected by 47 days (median survival time (MST) 14 days in anti-CD8 mAb plus MR1 group, and MST 39 days in anti-CD4 and CD8 mAbs plus MR1 groups) postgrafting. BMT with anti-CD4 and CD8 mAb treatment alone induced slight donor skin graft prolongation (▴; MST 39 days, n = 7), but all grafts rejected by 47 days postgrafting. BMT recipients treated with anti-CD4 mAb plus MR1 rejected donor skin within 15 days (□; MST 13 days, n = 7). Among BMT recipients treated with MR1 alone, two of seven became chimeras and accepted donor skin grafts permanently(▵; n = 2), and rejected third-party skin by 32 days, but nonchimeras rejected donor skin (▿; MST 25 days, n = 5). BMT recipients treated with anti-CD4 mAb (○; MST 13 days, n = 7) or anti-CD8 mAb alone (∗; MST 13 days, n = 7) did not accept donor skin. Donor (B10.A) and third-party (B10.RIII) skin was grafted 1 day post-BMT.

FIGURE 4.

Skin graft tolerance in mixed chimeras prepared with 3 Gy TBI, 20 × 106 BMT, anti-CD8 mAb plus MR1. BMT recipients prepared with anti-CD8 mAb plus MR1 (–•–; n = 6), and both anti-CD4-and CD8 mAbs plus MR1 (▪; n = 7) accepted donor grafts permanently, while third-party grafts were rejected by 47 days (median survival time (MST) 14 days in anti-CD8 mAb plus MR1 group, and MST 39 days in anti-CD4 and CD8 mAbs plus MR1 groups) postgrafting. BMT with anti-CD4 and CD8 mAb treatment alone induced slight donor skin graft prolongation (▴; MST 39 days, n = 7), but all grafts rejected by 47 days postgrafting. BMT recipients treated with anti-CD4 mAb plus MR1 rejected donor skin within 15 days (□; MST 13 days, n = 7). Among BMT recipients treated with MR1 alone, two of seven became chimeras and accepted donor skin grafts permanently(▵; n = 2), and rejected third-party skin by 32 days, but nonchimeras rejected donor skin (▿; MST 25 days, n = 5). BMT recipients treated with anti-CD4 mAb (○; MST 13 days, n = 7) or anti-CD8 mAb alone (∗; MST 13 days, n = 7) did not accept donor skin. Donor (B10.A) and third-party (B10.RIII) skin was grafted 1 day post-BMT.

Close modal

In contrast to recipients of anti-CD8 mAb, all seven BMT mice treated with anti-CD4 mAb, MR1 and 3 Gy TBI failed to develop chimerism and rejected donor skin grafts within 15 days following BMT (Figs. 3 and 4). As expected from previous results (5), 3 Gy-irradiated mice treated with both anti-CD4 and anti-CD8 mAbs, or with either mAb alone without MR1, also failed to achieve durable chimerism or tolerance (Figs. 3 and 4). Among animals treated with MR1 and 3 Gy TBI without any TCD mAb, only two of seven developed durable chimerism (Fig. 3) and accepted donor skin grafts (Fig. 4). Thus, anti-CD8 mAb, but not anti-CD4 mAb, greatly augmented the capacity of MR1 to overcome resistance to allogeneic marrow engraftment and permit the induction of lasting chimerism and donor-specific tolerance. These results indicate an obligate role for CD40-CD40L interactions in inducing CD4 cell-mediated rejection of allogeneic marrow, and a variable requirement for this pathway in permitting CD8 cell-mediated marrow rejection to occur.

To assess the robustness of the tolerance induced by BMT in mice treated with anti-CD8 mAb, MR1, and 3 Gy TBI, repeat skin grafting was performed ∼140 days following BMT and primary skin grafting (n = 5). Every mouse that had accepted the primary donor skin graft (i.e., all durable mixed chimeras) also accepted the secondary donor-type skin grafts. Nonchimeric mice that did not accept primary donor skin grafts, also rejected secondary donor skin grafts within 14 days after grafting (data not shown). All mice in all groups rejected secondary third-party (B10.RIII) grafts within 14 days (data not shown). Thus, MR1 is able to reliably prevent CD4 cell-mediated rejection of donor bone marrow cells when CD8 cells are depleted, allowing the establishment of permanent donor-specific tolerance thorough induction of mixed chimerism.

The chimeras prepared with anti-CD8 mAb plus MR1 and 3 Gy TBI also demonstrated high levels of donor chimerism among bone marrow cells, splenic T and B cells and thymocytes when they were sacrificed 28 wk post-BMT (Table I). In contrast, the mice receiving allogeneic BMT after treatment with 3 Gy TBI and MR1 alone in which long-term peripheral blood chimerism was not observed did not show measurable chimerism in the marrow, spleen, or thymus (Table I).

Table I.

Tissue chimerism in chimeras prepared with anti-CD8 mAb plus MR1a

Mean Percentage of Donor Cells ± SD
BMThymusSpleen
CD4+CD8+B220+
NLB6 0.46 0.18 1.13 1.17 
NLB10.A 100 100 99.55 100 100 
Chimeras (n = 4) (anti-CD8 plus MR1) 33.94 ± 15.02b 22.88 ± 8.61b 21.81 ± 3.87b 27.55 ± 3.65b 52.10 ± 15.18b 
Nonchimeras (n = 3) (MR1 alone) 0.28 ± 0.13 0.2 ± 0.29 0.11 ± 0.09 0.07 ± 0.12 0.56 ± 0.12 
Mean Percentage of Donor Cells ± SD
BMThymusSpleen
CD4+CD8+B220+
NLB6 0.46 0.18 1.13 1.17 
NLB10.A 100 100 99.55 100 100 
Chimeras (n = 4) (anti-CD8 plus MR1) 33.94 ± 15.02b 22.88 ± 8.61b 21.81 ± 3.87b 27.55 ± 3.65b 52.10 ± 15.18b 
Nonchimeras (n = 3) (MR1 alone) 0.28 ± 0.13 0.2 ± 0.29 0.11 ± 0.09 0.07 ± 0.12 0.56 ± 0.12 
a

The percentage of donor MHC class I (34-2-12)-positive cells among bone marrow cells, thymocytes, CD4, CD8, and B220-positive splenocytes was determined by two-color FCM 28 wk post-BMT. The mice treated with 0.5 mg MR1 (day 0) alone did not show long-term chimerism in peripheral blood, and none of these mice showed donor chimerism in any of these tissues examined by FCM. Mice treated with CD8-depleting mAb (day 1) plus 0.5 mg MR1 (day 0) showed long-term peripheral blood chimerism, and showed high levels of donor chimerism in these tissues.

b

, p < 0.05; statistically significant differences between CD8-depleting mAb plus MR1 group and MR1-treated group.

To further evaluate the establishment of tolerance, CML and MLR assays were performed in mice sacrificed 13 wk post-BMT. Chimeras prepared with anti-CD8 mAb plus MR1 and 3 Gy TBI were unresponsive toward donor and host Ags, while retaining reactivity against third-party (B10.RIII) alloantigens in both CML and MLR assays (Fig. 5, a and b). In contrast, control mice treated with anti-CD8 mAb plus MR1 and 3 Gy TBI without BMT showed reactivity against B10.A and B10.RIII alloantigens in both CML and MLR assays (Fig. 5, a and b).

FIGURE 5.

Specific MLR and CML tolerance in B6 mice receiving day −1 anti-CD8 mAb (0.35 mg) plus day 0 MRI (0.5 mg), 3 Gy TBI and 20 × 106 B10.A BMC. a, Chimeric mice (n = 4) uniformly failed to generate MLR responses to donor Ags (B10.A), while effectively responding to third-party Ags (B10.RIII). Non-BMT control mice treated with day −1 anti-CD8 mAb plus day 0 MR1 and 3 Gy TBI without BMT (n = 3) responded to donor and third-party Ags effectively. Assays were performed 13 wk post-BMT. Results are shown as mean ± SD for each group. b, Chimeric mice (n = 4) uniformly failed to generate CTL responses to donor Ags (B10.A), while effectively responding to third-party Ags (B10.RIII). Non-BMT control mice treated with day −1 anti-CD8 mAb plus day 0 MR1 and 3 Gy TBI, without BMT (n = 3) responded to donor and third-party Ags effectively. CML assays were performed 13 wk post-BMT. Results are shown as mean ± SD for each group.

FIGURE 5.

Specific MLR and CML tolerance in B6 mice receiving day −1 anti-CD8 mAb (0.35 mg) plus day 0 MRI (0.5 mg), 3 Gy TBI and 20 × 106 B10.A BMC. a, Chimeric mice (n = 4) uniformly failed to generate MLR responses to donor Ags (B10.A), while effectively responding to third-party Ags (B10.RIII). Non-BMT control mice treated with day −1 anti-CD8 mAb plus day 0 MR1 and 3 Gy TBI without BMT (n = 3) responded to donor and third-party Ags effectively. Assays were performed 13 wk post-BMT. Results are shown as mean ± SD for each group. b, Chimeric mice (n = 4) uniformly failed to generate CTL responses to donor Ags (B10.A), while effectively responding to third-party Ags (B10.RIII). Non-BMT control mice treated with day −1 anti-CD8 mAb plus day 0 MR1 and 3 Gy TBI, without BMT (n = 3) responded to donor and third-party Ags effectively. CML assays were performed 13 wk post-BMT. Results are shown as mean ± SD for each group.

Close modal

We have previously demonstrated that B6 mice receiving allogeneic B10.A BMT with costimulatory blockade alone (MR1 plus CTLA4Ig) show partial deletion of Vβ5+ and Vβ11+ peripheral blood CD4 cells as early as 1 wk after BMT (11). To examine whether deletion of donor-reactive T cells occurs in mice treated with CD8 depletion plus MR1, PBLs were analyzed for the presence of these Vβ subunits on their TCRs. Mice receiving BMT with CD8 depletion plus MR1 and 3 Gy TBI did not show deletion of donor-reactive Vβ5+ (not shown) or Vβ11+ CD4 cells at 1 wk post-BMT (Fig. 6). However, deletion of these Vβ was observed at subsequent time points, and progressed over time (Fig. 6). The percentages of Vβ8.1/2-bearing CD4 cells, which do not recognize superantigens on the donor or host, were not reduced at any time point, ruling out a nonspecific deletional process. The slight increase in mean percentage of Vβ8.1/2-bearing CD4+ cells in the mice receiving BMT with CD8 depletion plus MR1 and 3 Gy TBI may be a compensatory effect reflecting the deletion of Vβ5+ and Vβ11+ populations.

FIGURE 6.

Delayed deletion of donor-reactive peripheral blood CD4 T cells in chimeras prepared with CD8 depletion plus MR1. The mean percentage (±SD) of CD4+ PBL expressing Vβ8.1/2 or Vβ11 is shown at various times post-BMT. Mice that received anti-CD8 mAb plus MR1 showed progressive deletion of CD4+ Vβ11+ peripheral T cells over time (p < 0.05 vs normal B6 mice at 3 wk post-BMT and all subsequent time points). These mice showed stable multilineage chimerism and tolerance. The percentage of Vβ8.1/2-bearing CD4 cells was not reduced at any time point, ruling out a nonspecific deletional process.

FIGURE 6.

Delayed deletion of donor-reactive peripheral blood CD4 T cells in chimeras prepared with CD8 depletion plus MR1. The mean percentage (±SD) of CD4+ PBL expressing Vβ8.1/2 or Vβ11 is shown at various times post-BMT. Mice that received anti-CD8 mAb plus MR1 showed progressive deletion of CD4+ Vβ11+ peripheral T cells over time (p < 0.05 vs normal B6 mice at 3 wk post-BMT and all subsequent time points). These mice showed stable multilineage chimerism and tolerance. The percentage of Vβ8.1/2-bearing CD4 cells was not reduced at any time point, ruling out a nonspecific deletional process.

Close modal

The chimeras that received BMT with CD8 depletion plus MR1 and 3 Gy TBI were sacrificed 28 wk post-BMT and intrathymic deletion was examined. As is shown in Table II, these chimeras showed profound deletion of Vβ5+ and Vβ11+ host-type CD4 single-positive thymocytes. In contrast, nonchimeric controls that received BMT with MR1 alone did not show marked deletion of these donor-reactive thymocyte subpopulations.

Table II.

Intrathymic deletion in long-term chimeras prepared with anti-CD8 mAb, MR1, and 3 Gy TBIa

CD4 single positive thymocytes
Vβ8.1/2Vβ5.1/2Vβ11
NLB6 14.74 3.37 3.36 
NLB10.A 17.54 0.60 0.37 
Chimera (n = 4)(anti-CD8 plus MR1) 17.98 ± 1.72 0.97 ± 0.77b 0.40 ± 0.15b 
Nonchimera (n = 3) (MR1 alone) 16.51 ± 1.63 2.4 ± 0.37 2.97 ± 0.27 
CD4 single positive thymocytes
Vβ8.1/2Vβ5.1/2Vβ11
NLB6 14.74 3.37 3.36 
NLB10.A 17.54 0.60 0.37 
Chimera (n = 4)(anti-CD8 plus MR1) 17.98 ± 1.72 0.97 ± 0.77b 0.40 ± 0.15b 
Nonchimera (n = 3) (MR1 alone) 16.51 ± 1.63 2.4 ± 0.37 2.97 ± 0.27 
a

The percentage of Vβ8+, Vβ5+, and Vβ11+ cells among host MHC class I high and CD8 negative (CD4 single-positive) thymocytes, respectively, was determined by three-color FCM 28 wk post-BMT. The data are presented as mean percentage ± SD.

b

, p < 0.05; statistically significant differences between chimeras prepared with anti-CD8 plus MR1 vs nonchimeric recipients of MR1 alone.

To determine whether the above results could be generalized to include additional strain combinations and genetic backgrounds, we evaluated a similar treatment strategy in BALB/c (H-2d) recipients of fully MHC-mismatched plus multiple minor Ag-mismatched B10.BR (H-2k) marrow. Because BALB/c mice tend to have higher percentages of CD8 cells in the PBL than B6 mice, we treated these animals with a full standard dose of anti-CD8 mAb (1.4 mg on day −1) in combination with MR1 (2 mg on day 0). All animals receiving BMT following treatment with anti-CD8 mAb, MR1, and 3 Gy TBI in this strain combination became durable chimeras (Fig. 7, a and b) and also showed specific acceptance of donor skin grafts (Fig. 7,c). Neither anti-CD8 mAb nor MR1 treatment alone (with 3 Gy TBI) was sufficient to induce durable mixed chimerism or tolerance in this strain combination (Fig. 7, a–c).

FIGURE 7.

Long-term (>10 wk) multilineage chimerism and skin graft tolerance of mice receiving CD8-depleting mAbs plus MR1 in the B10.BR→BALB/c combination. BALB/c mice were treated with 3 Gy TBI and received 20 × 106 unseparated BM cells from fully MHC and multiple minor histocompatibility Ag-mismatched B10.BR donors. a, Percentages of long-term chimeras among groups receiving the various mAb treatments plus 3 Gy TBI are presented. CD8-depleting mAb (1.4 mg 2.43) was given on day −1, and MR1 (2 mg) was given on day 0. Treatment with anti-CD8 mAb plus MR1 (n = 6) successfully induced long-term chimerism in all mice. Neither treatment with anti-CD8 mAb alone (n = 7) nor with MR1 alone (n = 5) induced long-term chimerism. b, The mean percentage of donor peripheral blood granulocytes as determined by two-color FCM analysis. The mice treated with anti-CD8 plus MR1 showed high levels of lasting chimerism. Results are shown as mean ± SEM in each group. Results for granulocytes are representative of results obtained for all lineages among CD4 and CD8 T cells, monocytes and B cells. c, BMT recipients treated with anti-CD8 mAb plus MR1 (□; n = 6) accepted donor grafts (upper) long-term, whereas third-party grafts (lower) were rejected by 44 days (MST 14 days) postgrafting. The mice treated with MR1 alone (○; n = 5) or anti-CD8 mAb alone (▴; n = 7) rejected donor and third-party skin by 17 days postgrafting.

FIGURE 7.

Long-term (>10 wk) multilineage chimerism and skin graft tolerance of mice receiving CD8-depleting mAbs plus MR1 in the B10.BR→BALB/c combination. BALB/c mice were treated with 3 Gy TBI and received 20 × 106 unseparated BM cells from fully MHC and multiple minor histocompatibility Ag-mismatched B10.BR donors. a, Percentages of long-term chimeras among groups receiving the various mAb treatments plus 3 Gy TBI are presented. CD8-depleting mAb (1.4 mg 2.43) was given on day −1, and MR1 (2 mg) was given on day 0. Treatment with anti-CD8 mAb plus MR1 (n = 6) successfully induced long-term chimerism in all mice. Neither treatment with anti-CD8 mAb alone (n = 7) nor with MR1 alone (n = 5) induced long-term chimerism. b, The mean percentage of donor peripheral blood granulocytes as determined by two-color FCM analysis. The mice treated with anti-CD8 plus MR1 showed high levels of lasting chimerism. Results are shown as mean ± SEM in each group. Results for granulocytes are representative of results obtained for all lineages among CD4 and CD8 T cells, monocytes and B cells. c, BMT recipients treated with anti-CD8 mAb plus MR1 (□; n = 6) accepted donor grafts (upper) long-term, whereas third-party grafts (lower) were rejected by 44 days (MST 14 days) postgrafting. The mice treated with MR1 alone (○; n = 5) or anti-CD8 mAb alone (▴; n = 7) rejected donor and third-party skin by 17 days postgrafting.

Close modal

Recent reports have demonstrated that administration of anti-CD40L, with or without CTLA4Ig or donor-specific blood transfusion, could induce prolonged acceptance of donor heart, skin, and islet allografts in mice (22, 23), as well as kidneys and islets in primates (24, 25, 26). However, none of these models have been associated with systemic donor-specific tolerance across MHC barriers in euthymic recipients, as measured by the stringent tests of primary donor skin graft acceptance and by in vitro assays. Costimulatory blockade has only been shown to lead to systemic tolerance across full MHC barriers in euthymic mice when used in combination with allogeneic BMT. This tolerance can be achieved by giving conventional marrow doses to mice treated with anti-CD40L mAb, CTLA4Ig, and 3 Gy TBI (11), or by giving very high marrow doses to mice receiving similar treatment without any host irradiation (17). In both instances, tolerance is associated with early (by 1 wk) peripheral deletion of donor-reactive CD4 cells and, in the long term, after donor hematopoietic cells have seeded the host thymus, with a central deletional mechanism of tolerance (11, 17). However, a major limitation to the clinical applicability of these strategies is that they are not successful in 100% of mice. Complete reliability in animal models would be an essential requirement before such an approach could be considered for clinical application.

Based on results obtained in the above model, we postulated that a failure of early CTL tolerance might be responsible for the inability to achieve durable chimerism in some mice receiving combined anti-CD40L and CTLA4Ig treatment without T cell depletion. Therefore, we compared the timing of development of MLR (largely CD4 cell dependent) and CTL (largely CD8 cell mediated) unresponsiveness to donor Ags in mice receiving this regimen. B6 mice received MR1 and 3 Gy TBI on day 0, B10.A BMT on day 0, and CTLA4Ig on day 2, as previously described (11). MLR assays were performed using the spleen cells from BMT recipients and controls at the time of sacrifice on days 4, 8, 15, 21, and 35 after BMT. At all time points analyzed, control animals that received the entire conditioning regimen without BMT showed an intact response to both B10.A and third-party (A.SW) stimulators, demonstrating that the regimen was not globally immunosuppressive. However, spleen cells from the mixed chimeras demonstrated a lack of proliferation when cocultured with irradiated donor-type or third-party stimulator cells at the earliest time point tested, day 4 (data not shown). Specific tolerance to the donor became apparent by day 8, when responses to third-party (A.SW) stimulator cells were measurable and comparable to those of similarly treated non-BMT control animals (data not shown). Unresponsiveness to donor Ag in the MLR assay persisted at all subsequent time points tested in all animals receiving BMT with the costimulatory blocking protocol. Most of these animals showed low, but measurable, anti-third-party responses, indicating the presence of donor-specific tolerance in the MLR assay. Despite the presence of donor-specific tolerance in MLR assays in animals receiving allogeneic BMT with this regimen, significant donor chimerism was not always measurable.

When CTL responses were tested in the same animals, a different pattern was observed. Some animals showed donor-specific unresponsiveness, but some did not, even though they showed chimerism by day 25. However, at later time points (day 35 or later), all chimeric animals showed donor-specific unresponsiveness. These results demonstrate that, in contrast to MLR responses, CTL responses were not uniformly tolerant to the donor in this early period, even when substantial donor chimerism was present. These results suggest that CD8 cells were variably tolerized to donor Ags by BMT with costimulatory blockade, and that this cell population may be responsible for the failure to achieve durable chimerism in a proportion of animals receiving BMT with this regimen.

The results presented here are consistent with previous studies, in which donor CD4+ cells exposed in vitro to host alloantigens in the presence of anti-CD40L mAb were tolerized and lost the ability to induce graft-vs-host disease (27). We demonstrate in this study that such tolerization can occur in vivo when CD40/40L interactions are blocked. Mice receiving CD8-depleting mAb along with a single injection of anti-CD40L and 3 Gy TBI consistently showed lasting engraftment of fully MHC-mismatched donor marrow. Other studies indicate that the efficacy of anti-CD40L in this model is due only to blocking of the interaction of CD40L with CD40 on APC, and not to other mechanisms (J. Kurtz, H. Ito, J. Shaffer, and M. Sykes, Submitted for publication). Animals receiving BMT with 3 Gy TBI and combined treatment with anti-CD40L and CTLA4Ig, in which the achievement of long-term chimerism is more variable, showed early induction of tolerance in MLR assays, but far more variable tolerance to the donor in CML assays, suggesting that CD4 cells were more reliably tolerized than CD8 cells by BMT with costimulatory blockade. We speculate that chimeric animals with persistent anti-donor CTL responses were destined to lose chimerism, and that nonchimeric animals lost their chimerism due to these anti-donor CTL responses. Because anti-CD40L mAb is sufficient to overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and allow the induction of tolerance, we surmise that the requirement for CTLA4Ig in non-CD8-depleted mice reflects the need to specifically block the CD28-B7 pathway of CD8 T cell activation, and that this pathway does not need to be independently blocked for tolerance to be induced among CD4 cells that encounter APC in the presence of anti-CD40L. It can be inferred that B7 is expressed on some APC in the presence of CD40 blockade, and that this is sufficient to activate CD8 T cells, but not CD4 T cells, to donor Ags.

Because Th appeared to be unresponsive to the donor at very early times post-BMT, the anti-donor CTL that persist in some animals probably differentiate via CD4 cell-independent pathways. CD40L is expressed mainly on activated CD4+ T cells, and not on CD8 cells (28, 29, 30). CD40-dependent activation of APC by CD40L on activated Th is a major pathway by which help is provided for CTL generation against minor histocompatibility Ags and other peptide Ags (31, 32, 33). However, anti-viral CTL responses can occur in CD40L-deficient mice (34). CD8+ CTL activation that is independent of CD40L/CD40 interactions occurs by both CD4 Th-dependent (35) and CD4 cell-independent pathways (34, 36, 37, 38) and in the absence of CD4-mediated APC conditioning (31, 32, 33, 34, 39, 40, 41). CD4 cell-independent APC activation can occur via LPR (42, 43, 44), C3R (45), FcγR (46), and CpG oligodinucleotides (45, 47, 48, 49), and these pathways are associated with the production of mediators of CD8 T cell activation, such as type I IFNs (44, 46, 50), TNF-α (51), IL-12 (52, 53, 54, 55, 56, 57), and IL-15 (58). Thus, we hypothesize that the failure to achieve engraftment in a proportion of animals receiving BMT with costimulatory blockade as the only immunosuppression may reflect such “bypass activation” of APCs due to exposure to microorganisms that cannot be controlled, and that this activation leads to the Th-independent activation of alloreactive recipient CD8 cells that then reject the donor marrow and prevent tolerance induction. Such a pathway could be important, because humans are frequently exposed to microorganisms that might thereby preclude the ability to reliably use this approach to tolerance induction.

Although the CD28 pathway of costimulation both stimulates high-level IL-2 production and may provide an essential survival signal, CD28-independent T cell activation clearly occurs and is capable of causing graft rejection in mice (59, 60). Several additional costimulatory pathways have been described (reviewed in Ref. 61) that may have the capacity to compensate for the absence or blockade of CD28 signaling. Furthermore, naive CD8+ T cells with high affinity for their ligands can differentiate into cytolytic effector cells with “signal 1” without the apparent involvement of costimulatory molecules (62). Thus, there are several possibleexplanations for the inability of CTLA4Ig to reliably prevent CD8+ T cell activation, and further studies will be needed to determine the role of additional costimulatory pathways in CD8+ T cell responsiveness to alloantigens in the absence of CD4 cell help.

In most of our experiments (e.g., Figs. 1 and 3), a minority of animals receiving BMT with anti-CD40L alone (plus 3 Gy TBI) developed lasting mixed chimerism and donor-specific tolerance (Fig. 4). Therefore, blockade of APC activation via the CD40 pathway is sometimes sufficient to allow tolerization of allo-reactive CD8 cells in addition to CD4 cells, even without specific blockade of the CD28-B7 pathway. We hypothesize that this occurs in animals in which CD40-independent APC activation has not occurred, and in which B7 expression on APC presenting alloantigens is markedly down-regulated due to CD40 blockade. We have attempted to improve the reliability of tolerance induction with MR1 alone, 3 Gy TBI, and BMT by increasing the MR1 dose. The highest dose evaluated, 4 mg, increased the induction of chimerism, but was still much less than 100% reliable. Thus, if MR1 alone has the potential to reliably overcome CD8-mediated in addition to CD4-mediated resistance, exceedingly high MR1 doses would be required.

It has recently been demonstrated that asialo GM1+CD8+ T cells are responsible for costimulatory blockade-resistant mouse skin graft rejection (63), and CD8+ T cells have been shown to be responsible for costimulatory blockade-resistant rejection in intestinal (64) and skin allograft models (65). Although these studies demonstrated prolonged allograft acceptance in the presence of costimulatory blockade with CD8 cell depletion, the allografts were ultimately rejected, and donor-specific tolerance was not achieved. In a cardiac allograft model, treatment with anti-CD8 mAb plus anti-CD40L induced long-term heart graft acceptance and operational tolerance (66). However, systemic tolerance was not demonstrated in those models, and our results suggest that tolerance for MHC-mismatched skin grafts, a more stringent test of tolerance, is not achieved with anti-CD8 and MR1 without BMT. Although anti-CD8 plus anti-CD40L induced skin graft tolerance across minor Ag barriers (67), our data using fully MHC-mismatched donors suggest that it is unlikely that tolerance could be achieved for fully MHC-mismatched skin grafts without BMT. In our studies, mice receiving CD8 depletion plus anti-CD40L and 3 Gy TBI without BMT rejected fully MHC-mismatched allogeneic skin grafts within 14 days (not shown) and did not develop tolerance in MLR and CML assays (Fig. 5). In contrast, the addition of BMT at the time of skin grafting allows skin graft tolerance (primary and secondary grafts) and MLR and CML tolerance to be observed, indicating that systemic tolerance is achieved. Unlike responses to MHC-mismatched skin allografts given without BMT, and unlike some antiviral responses (39, 65, 68), CD4 cell-mediated resistance to MHC-mismatched marrow engraftment is completely dependent on the CD40-CD40L pathway. Because CD4 cells become rapidly tolerized by donor marrow given in the presence of anti-CD40L, it is possible that their early tolerant state makes them resistant to activation by APC activated by CD40-independent pathways. Additionally, interactions between the rapidly tolerized CD4 cells and APC may render the APC tolerogenic for CD8 cells, and perhaps naive CD4 cells, that subsequently encounter donor Ag on those APC. Such transfer of tolerance to CD8 cells via an APC encountered by a tolerant CD4 cell may account for the donor-reactive CD8 cell deletion that has been seen in mice receiving donor-specific transfusions and anti-CD40L mAb (65).

Central deletion of donor-reactive thymocytes is the major mechanism maintaining long-term tolerance in mixed allogeneic chimeras prepared with anti-CD40L and CTLA4Ig (11, 17). However, evidence has been obtained for early (by 1 wk post-BMT) peripheral deletion of donor-reactive CD4+ T cells in both of these models (11, 17). B6 mice receiving B10.A BMT with the new regimen described here (i.e., CD8 cell depletion plus MR1 with 3 Gy WBI) also showed long-term central deletion of donor-reactive T cells. In the periphery, these mice showed complete deletion of donor-reactive Vβ11+ CD4+ T cells by 5–8 wk post-BMT, but showed no evidence of such deletion at 1 wk post-BMT. In the same experiments, mice receiving BMT with CTLA4Ig plus MR1, without CD8 depletion, showed statistically significant deletion of this Vβ at 1 wk (data not shown), consistent with our previous results (11). The lack of Vβ11 deletion by day 7 in the mice treated with CD8 cell-depleting mAb is consistent with published data suggesting that CD8+ T cells, especially when activated, are the most efficient producers of endogenous superantigens that delete CD4 cells using this Vβ, and may transfer these superantigens to the class II+ cells that present them (69, 70, 71). The chimeric mice in our studies that were depleted of CD8+ cells showed marked deletion of Vβ11+ CD4+ cells before the recovery of CD8+ cells, perhaps due to viral superantigens produced by other organs, such as lung, brain, gonadal tissue (70), and intestine (72). Because endogenous superantigens do not necessarily behave as transplantation Ags (73), definitive data on peripheral deletion will await ongoing studies using TCR transgenic mice.

In previous studies, we showed that costimulatory blockade with one injection of anti-CD40L or of CTLA4Ig obviates the need for thymic irradiation or repeated administration of TCD mAbs to overcome intrathymic alloresistance in mice receiving one injection of depleting anti-CD4 and anti-CD8 mAbs (10). The current demonstration that anti-CD4 mAb is not required to achieve such results (i.e., that anti-CD40L and anti-CD8 mAb alone are sufficient to allow the reliable induction of durable mixed chimerism and transplantation tolerance) is of considerable clinical relevance. The capacity of the adult human thymus to reconstitute T cells declines steadily with increasing age, so that the time to achieve T cell reconstitution after chemotherapy with or without stem cell transplantation increases with advancing age (74). The consequences of delayed thymic reconstitution are much more dramatic for CD4 cells than for CD8 cells in humans, as the latter subset recovers much more readily (75). Thus, a greater concern exists about prolonged CD4 cell than CD8 cell depletion in adult humans receiving T cell ablation in a conditioning protocol. Therefore, the observation that CD4 depletion is not required in a regimen that reliably allows the induction of lasting mixed chimerism and transplantation tolerance is highly encouraging. The low toxicity and reliability of mixed chimerism and tolerance induction across different, full MHC barriers, with or without multiple minor Ag differences, with the nontoxic regimen of CD8-depleting mAb, 3 Gy TBI, and anti-CD40L, suggests that this approach may have considerable potential for clinical application. Evaluation of similar regimens in large animal models is clearly warranted at this point.

In summary, an absence of CD40/40L signaling is not sufficient to reliably allow the induction of mixed chimerism and donor-specific tolerance in 3 Gy-irradiated mice, but does reliably overcome the CD4 cell-mediated barrier to allogeneic engraftment and allows the rapid tolerization of host CD4 cells. CD8 T cell-mediated allogeneic marrow rejection sometimes, but not always, occurs independently of the CD40-CD40L pathway. Recipient CD8 depletion overcomes this variable, but as yet poorly understood and therefore uncontrollable CD8 cell-mediated alloresistance. BMT plays a critical role in inducing long-term systemic tolerance of both CD4 and CD8 cells under blockade of the CD40/40L pathway. This reliable approach (BMT with anti-CD40L mAb and CD8-depleting mAb) to inducing donor-specific skin graft tolerance, which is considered to be the most stringent test of tolerance, warrants evaluation in large animal preclinical models, as it may have considerable clinical potential.

We thank Dr. Henry J. Winn and Dr. Yong Zhao for critical review of the manuscript, Dr. David H. Sachs for his advice, and Julia Lundell for expert secretarial assistance.

1

This work was supported by National Institutes of Health Grant R01HL49915 and through a sponsored research agreement with Biotransplant, Inc. H.I. was supported by the Uehara Memorial Foundation.

4

Abbreviations used in this paper: TBI, total body irradiation; BMT, bone marrow transplantation; CD40L, CD40 ligand; FCM, flow cytometric analysis; MR1, hamster anti-mouse CD40L mAb; MST, median survival time; CML, cell-mediated lympholysis; TCD, T cell-depleting; B6, C57BL/6.

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