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The Journal of Immunology, 2000, 164: 4452-4457.
Copyright © 2000 by The American Association of Immunologists

Donor MHC Class II Antigen Is Essential for Induction of Transplantation Tolerance by Bone Marrow Cells1

Akihisa Umemura, Anthony P. Monaco and Takashi Maki2

Transplant Center, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Posttransplant infusion of donor bone marrow cells (BMC) induces tolerance to allografts in adult mice, dogs, nonhuman primates, and probably humans. Here we used a mouse skin allograft model and an allogeneic radiation chimera model to examine the role of MHC Ags in tolerance induction. Infusion of MHC class II Ag-deficient (CIID) BMC failed to prolong C57BL/6 (B6) skin grafts in ALS- and rapamycin-treated B10.A mice, whereas wild-type B6 or MHC class I Ag-deficient BMC induced prolongation. Removal of class II Ag-bearing cells from donor BMC markedly reduced the tolerogenic effect compared with untreated BMC, although graft survival was significantly longer in mice given depleted BMC than that in control mice given no BMC. Infusion of CIID BMC into irradiated syngeneic B6 or allogeneic B10.A mice produced normal lymphoid cell reconstitution including CD4+ T cells except for the absence of class II Ag-positive cells. However, irradiated B10.A mice reconstituted with CIID BMC rejected all B6 and a majority of CIID skin grafts despite continued maintenance of high degree chimerism. B10.A mice reconstituted with B6 BMC maintained chimerism and accepted both B6 and CIID skin grafts. Thus, expression of MHC class II Ag on BMC is essential for allograft tolerance induction and peripheral chimerism with cells deficient in class II Ag does not guarantee allograft acceptance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monaco and colleagues (1, 2) first described the use of posttransplant donor bone marrow cells (BMC)3 in conjunction with antilymphocyte serum (ALS) to induce tolerance to skin allografts. Tolerant mice showed evidence of low degree (up to 4%) chimerism (microchimerism) (3). Subsequent studies in mongrel dogs (4, 5) and nonhuman primates (6, 7) demonstrated that the use of polyclonal ALS and donor BMC is also very effective in producing tolerance to kidney allografts. Posttransplant donor BMC infusion with induction therapy by polyclonal antilymphocyte globulin was clinically applied in living-related and cadaveric kidney transplantation with a standard multidrug immunosuppressive protocol (8, 9, 10). Some BMC-infused patients had a decreased incidence of early rejection episodes and early withdrawal of steroids from immunosuppressive medication. Since Starzl reported that patients with long-surviving kidney and liver allografts had evidence of circulating donor cells (11), induction of posttransplant chimerism was assumed to be essential for long term survival of organ allografts. Consequently, infusion of donor BMC has been used without induction immunosuppression in an attempt to augment graft-derived microchimerism after organ transplantation (12, 13, 14, 15).

We have recently developed a mouse skin allograft model in which tolerance was induced in a fully MHC-mismatched mouse strain combination with addition of posttransplant rapamycin treatment to peritransplant ALS and posttransplant donor BMC infusion (16, 17). In tolerant mice, a low to moderate degree of chimerism developed in association with clonal deletion, anergy, and suppressor cells participating at different phases of tolerance (D. Hale, unpublished observation). In the present study, we used gene knockout mice to examine the role of cell surface molecules in donor BMC-induced allograft tolerance. In addition, we used a radiation chimera model to examine the role of chimerism in tolerance induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice were purchased from Charles River Laboratories, Kingston, NY (B10.A mice), Taconic Farms, Germantown, NY (C57BL/6, MHC class I or class II-deficient mice), and The Jackson Laboratory, Bar Harbor, ME (CD4 Ag-deficient mice). All care and handling of animals were conducted in accordance with guidelines provided in the Guide for the Care and Use of Laboratory Animals published by the U.S. Department of Health and Human Services.

Tolerance induction

Full-thickness skin grafts were transplanted onto the lateral thoracic area of the recipients using standard techniques described previously (18). Skin grafts were scored as described by Billingham et al. (19). Rabbit anti-mouse ALS was produced as previously described (20). Rapamycin (generously provided by Dr. S. Sehgal, Wyeth Ayerst, Princeton, NJ) was suspended in a carboxymethylcellulose and polysorbate vehicle. The tolerance induction protocol consisted of ALS (0.5 ml) i.p. on days -1 and +2, skin grafting on day 0, rapamycin (6 mg/kg) i.p., and donor BMC (25 x 106 cells) i.v. on day 7. BMC were prepared from the femurs and humeri by flushing with HBSS. Mice that received ALS, C57BL/6 (B6) skin grafts, and rapamycin without BMC infusion were included as controls.

For bone marrow transplantation, lethally irradiated (950 rad) syngeneic B6 mice or sublethally irradiated (750 rad) allogeneic B10.A mice were infused with 25 x 106 BMC derived from wild-type B6 or CIID mice within 6 h after irradiation. Chimeric B10.A mice were grafted with either wild-type B6 or CIID skin on day 30.

Flow cytometry

The cells were first incubated with an anti-CD16/32 mAb for 10 min to block nonspecific binding of labeled Abs. Splenocytes and PBL were stained with the FITC-, PE-, or CyChrome-conjugated mAbs directed to CD4, CD8a, CD11b, CD11c, CD45R, CD34, CD40, CD80, and CD 86, all of which were purchased from PharMingen (San Diego, CA). FITC-, PE- or CyChrome-conjugated isotype Abs were used as controls. Stained cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA).

Determination of chimerism

Donor cell chimerism was determined by flow cytometric analysis of recipient peripheral and/or splenic lymphoid cells with normal donor-type (B6) and recipient-type (B10.A) cells as positive and negative controls. The percentage of chimeric H-2Kb-positive cells was calculated by the formula: 100 x [(net % in the test samples - net % in the negative control samples)]/(net % in the positive control samples - net % in the negative control samples]. Net percentage refers to the percentage obtained after subtraction of staining with the appropriate isotype controls.

Depletion of I-Ab+ cells from B6 BMC

B6 BMC were incubated with anti-I-Ab mAb (clone M5/114.15.2, Phar-Mingen) or purified isotype control Ab (clone A95–1, PharMingen) at 4°C for 60 min. The cells were washed twice in HBSS, resuspended in RPMI 1640, and incubated with immunomagnetic beads conjugated with anti-rat IgG (Dynabeads M-450, Dynal, Lake Success, NY) at 4°C for 30 min. After incubation, I-Ab+ cells were removed by applying the magnet. The unbound cells were collected, washed, and resuspended in HBSS. For each experiment, adequacy of depletion of I-Ab+ cells was confirmed by flow cytometric analysis.

Statistics

Graft survival was analyzed by the Kaplan-Meier estimate using Mac SYSTAT 5.1 and SURVIVAL programs (Systat, Evanston, IL). The significance level (p value) was obtained by Mantel’s log rank test in the SURVIVAL program. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of MHC class I and class II Ag in BMC-induced allograft tolerance

Mice deficient in MHC class I Ag (CID mice) (21) or class II Ag (CIID mice) (22), both of which were derived from mice of the H-2b haplotype (C57BL/6; B6), were used as donors of BMC and skin grafts. B10.A recipient mice (H-2a) were given ALS (days -1 and 2), B6 skin (day 0), rapamycin (day 7), and B6, CID, or CIID BMC (day 7). B10.A mice that received ALS, B6 skin grafts, and rapamycin without BMC infusion were included as controls. As shown in Fig. 1GoA, infusion of B6 BMC markedly prolonged B6 skin graft survival with a median survival time (MST) of 80 days compared with graft survival in control mice (MST = 18 days) (p < 0.001). CID BMC were as effective as wild-type B6 BMC in B6 skin graft survival (p < 0.001 vs controls). In contrast, BMC prepared from CIID mice had no effect in prolonging B6 skin graft survival (MST = 21 days) (p = 0.496 vs controls). Although CIID donors are deficient in CD4+ T cells (22), CD4+ T cells were unlikely to play a role in BMC-induced tolerance because BMC obtained from CD4-deficient mice (23) were also effective in inducing prolongation of B6 skin allografts in B10.A recipients (p = 0.004 vs controls) (Fig. 1GoB).



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  		FIGURE 1. Effect of BMC infusion on skin allograft survival. A, B10.A mice were treated with ALS on days -1 and +2 relative to B6 skin grafting on day 0. On day 7, recipient mice were treated with 6 mg/kg rapamycin i.p. and infused with 25 x 106 BMC derived from B6 (n = 10), CIID (n = 10), or CID (n = 10) mice. Control mice were treated with ALS and rapamycin without BMC infusion (n = 9). B, B10.A mice were treated with ALS, B6 skin grafting, and rapamycin and infused with BMC of B6 (n = 7) or CD4 Ag-deficient B6 (n = 8) mice. Control mice were not given BMC infusion (n = 7). C, B10.A mice were treated with ALS, CIID skin grafting, and rapamycin and infused with BMC of B6 (n = 16) or CIID (n = 14) mice. Control mice were not given BMC infusion (n = 15).

 
We also investigated the effect of BMC infusion on survival of CIID skin grafts which are rapidly rejected in normal allogeneic hosts (24). As shown in Fig. 1GoC, infusion of B6 BMC effectively prolonged the CIID skin allograft survival in ALS- and rapamycin-treated B10.A mice (MST of 61 days compared with MST of 25 days for ALS and rapamycin controls) (p = 0.002). However, skin donor-specific CIID BMC had no effect (MST = 26 days) (p = 0.255 against controls).

Effect of depleting I-Ab-positive cells from BMC on induction of allograft tolerance

We investigated whether depletion of MHC class II Ag-positive cells from donor BMC before infusion abrogates their tolerogenic effect. I-Ab-positive cells were removed from B6 BMC with the use of anti-I-Ab mAb and immunomagnetic beads. Because ~20% of B6 BMC were I-Ab positive by flow cytometric analysis, 20 x 106 I-Ab+ cell-depleted B6 BMC were infused on day 7 into B10.A recipient mice that received ALS (days -1 and 2), B6 skin grafts (day 0), and rapamycin (day 7). Control B10.A mice received ALS, rapamycin, and either untreated B6 BMC (25 x 106) or B6 BMC treated with Ig isotype (25 x 106). B10.A mice that received ALS, B6 skin grafts, and rapamycin without BMC infusion were also included as controls. As shown in Fig. 2GoA, depletion of I-Ab-positive cells from BMC markedly reduced the capacity of donor BMC to prolong skin allograft survival (p = 0.003 and 0.03 against wild-type B6 BMC and isotype-treated B6 BMC, respectively) although the graft survival in mice given I-Ab+ cell-depleted BMC was significantly greater than that in ALS/rapamycin control mice (p = 0.004).



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  		FIGURE 2. Effect of depleting I-Ab-positive cells from B6 BMC on skin allograft survival. A, B10.A mice were given ALS, B6 skin grafts, and rapamycin and infused on day 7 with 25 x 106 wild-type B6 BMC (n = 15), 20 x 106 I-Ab+ cell-depleted B6 BMC (n = 20), or 25 x 106 isotype-treated B6 BMC (n = 10). Control mice were treated with ALS and rapamycin without BMC infusion (n = 14). B, Flow cytometric analysis of splenocytes after BMC infusion. The presence of lymphoid cell chimerism was assessed 60 days after skin grafting by flow cytometric analyses of splenocytes with mAbs directed to donor MHC class I (Kb; FITC) and B cells (CD45R, CyChrome). In addition, monoclonal anti-CD4 Ab (PE) was used for assessment of CD4+ T cell recovery.

 
Our previous studies (36)4 established that low level donor chimerism (~3%) was detectable for a brief period around day 60 in C57BL/10 (B10) mice given ALS, B10.A skin grafts, rapamycin, and 25 x 106 B10.A BMC. Splenocytes prepared on postgraft day 60 from B10.A mice bearing intact B6 skin grafts following infusion of untreated or I-Ab+ cell-depleted B6 BMC were analyzed for the presence of chimerism. As illustrated in Fig. 2GoB, the same degree of chimerism was found in two groups of mice given either untreated B6 BMC (n = 3) or I-Ab+ cell-depleted B6 BMC (n = 3) (4.2 ± 1.3% vs 5.1 ± 0.6%, respectively). Moreover, significant numbers of CD45R+ cells of donor origin were found in both groups (3.0 ± 1.0% vs 2.5 ± 0.2%, respectively), whereas CD4+ T cells (0.1 ± 0.1% vs 0.2 ± 0.1%, respectively) and CD8a+ T cells (not shown) of donor origin were absent.

Ability of CIID BMC to fully restore peripheral lymphoid cells in lethally irradiated B6 mice

Lethally irradiated (950 rad) B6 mice were reconstituted with 25 x 106 wild-type B6 or CIID BMC. All BMC-reconstituted mice survived >80 days, whereas irradiated mice died within 13 days without BMC reconstitution. The proportion of various lymphoid cell populations within the spleen and lymph nodes (not shown) as determined by flow cytometric analysis were virtually identical for all cell populations tested between the B6 BMC-reconstituted and CIID BMC-reconstituted mice at three time points except that the majority of cells in CIID BMC-reconstituted mice were deficient in class II Ag expression. Results of staining with CD4, CD8a, CD45R, and CD11b are shown in Fig. 3Go. CD4+ T cells recovered not only in B6 BMC-reconstituted mice but also in CIID BMC-reconstituted mice. Recovery was seen 30 days after BMC infusion and stabilized by 60 days after BMC infusion.



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  		FIGURE 3. Flow cytometric analysis of splenocytes after BMC reconstitution. B6 mice were lethally (950 rad) irradiated and reconstituted with 25 x 106 B6 or CIID BMC. Flow cytometric analyses were performed at various days after reconstitution (days 14, 30, and 60). Analysis of na{epsilon}ve B6 and CIID splenocytes is also shown. Cells were doubly stained for I-Ab and multiple cells surface markers. The analyses were repeated with three mice, and the mean values are shown. Cells were also analyzed for CD34, CD11c, CD40, CD80, and CD86 (not shown).

 
Impact of CIID BMC on formation of allogeneic chimerism and induction of tolerance

To study the impact of allogeneic chimerism on tolerance induction, allogeneic radiation chimeras were created by infusing T cell-depleted B6 or CIID BMC (25 x 106 cells) into sublethally irradiated (750 rad) allogeneic B10.A mice. Recipient mice did not receive ALS or rapamycin. Flow cytometric analyses of PBL 28 days after BMC reconstitution confirmed a high degree of chimerism with H-2b-positive donor cells; 91.1 ± 0.7% for B6 BMC-reconstituted mice (n = 12) and 83.8 ± 2.1% for CIID BMC-reconstituted mice (n = 10). I-Ab-positive cells within the chimeric cell population were negligible in CIID BMC-reconstituted mice whereas they were 37.3 ± 3.4% in B6 BMC-reconstituted mice. Recovery of both donor-type (Kb-positive) and recipient-type (Kb-negative) CD4+ T cells was also observed in both B6 BMC-reconstituted mice (4.5 ± 0.4% and 5.0 ± 0.4%, respectively) and CIID BMC-reconstituted mice (5.6 ± 0.4% and 5.4 ± 0.5%, respectively). Similar results were obtained with splenic lymphoid cells (data not shown), indicating that CIID BMC were fully capable of generating high degree chimerism. B10.A mice that were confirmed chimeric with B6 or CIID cells by flow cytometric analyses were grafted with B6 or CIID skin grafts 30 days after BMC reconstitution. As shown in Table IGo, B10.A mice reconstituted with B6 BMC accepted both B6 and CIID skin grafts. In contrast, B10.A mice reconstituted with CIID BMC acutely rejected all B6 skin grafts within 15 days (MST = 10 days) and the majority of CIID skin grafts within 19 days (MST = 15.5 days).


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  		Table I. Effect of allogeneic chimera on skin allograft survival1

 
Flow cytometric analyses revealed that B6 BMC-reconstituted B10.A mice maintained high degrees of chimerism after skin grafting: 92.3 ± 0.7% (n = 11) or 91.9 ± 0.9% (n = 9) at day 50 (20 days after skin grafting) and 95.4% ± 0.8% (n = 5) or 94.7 ± 1.2% (n = 5) at day 60 (30 days after skin grafting) for B6 or CIID skin graft recipients, respectively. Interestingly, B10.A mice reconstituted with CIID BMC also continued to maintain high degrees of chimerism regardless of the conditions (rejection or acceptance) of skin grafts: 91.5 ± 1.7% (n = 10) or 90.8 ± 1.5% (n = 12) at day 50 when most of the grafts were rejected and 94.3 ± 0.9% (n = 5) and 93.8 ± 0.8% (n = 5) at day 60 (all 5 mice tested had rejected skin grafts more than 20 days earlier) for B6 and CIID skin graft recipients, respectively. Representative flow cytometric analyses at day 28 (before grafting) and day 50 (after grafting) are shown in Fig. 4Go.



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  		FIGURE 4. Induction of allogeneic radiation chimera with B6 and CIID BMC. B10.A mice were irradiated (750 rad) and infused with 25 x 106 B6 or CIID BMC. Lymphoid cell chimerism was examined by flow cytometric analyses of PBL with mAbs directed to donor MHC class I (Kb; FITC) and class II (I-Ab; PE) Ags. In addition, monoclonal anti-CD4 Ab (PE) was used for assessment of CD4+ T cell recovery in chimeric mice. The chimeric state in two representative B10.A mice at day 28 (2 days before skin grafting) and day 50 (20 days after skin grafting) is shown. a, Irradiated, B6 BMC-reconstituted B10.A mouse transplanted with CIID skin grafts. The graft was intact for >80 days. b, Irradiated, CIID BMC-reconstituted B10.A mouse transplanted with CIID skin grafts. The graft was rejected 9 days after grafting (day 39). Chimerism at day 60 was virtually identical with that of day 50 (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We showed that expression of MHC class II Ag by donor BMC is an absolute prerequisite for induction of allograft tolerance in two donor BMC-mediated transplantation tolerance models. In an ALS/rapamycin/donor BMC model, wild-type B6 BMC that express class II (I-A but not I-E) Ag induced significant prolongation of both donor-specific B6 and class II Ag-deficient (CIID) skin graft survival, whereas CIID BMC failed to prolong survival of not only B6 but also donor-specific CIID skin allografts. Lack of tolerogenicity by CIID BMC is due to loss of class II Ag and not to CD4+ T cell deficiency because CD4 Ag-deficient BMC are as effective as normal BMC. Similarly, CD8+ T cells do not participate in donor BMC-induced transplantation tolerance because CID BMC that are deficient in CD8+ T cells are effective in tolerance induction. This result rules out the involvement of CD8+ veto T cells (25, 26) in induction of allograft tolerance by donor BMC, at least in the MHC fully mismatched mouse skin allograft model.

B10.A mice tolerant to B6 skin grafts after ALS, rapamycin and donor-specific BMC infusion showed low degrees of chimerism (4%) at 60 days after skin grafting. The chimeric donor cells were dominated by B cells with no detectable T cells. These results are in agreement with our previous finding. In a reverse strain combination, B10.A skin to B10 recipients, and using an ALS/rapamycin/donor BMC infusion model, we showed that low to moderate chimerism (depending on the dose of BMC) was present in tolerant mice (36). In all BMC doses, chimeric donor-derived cells were predominantly class II Ag positive, including B cells (~80%) and monocytes/macrophages (20%). No T cells of donor origin were detected by flow cytometric analyses. This is in contrast to irradiation-induced chimerism where all lymphoid cell types of donor origin including T cells were identified. The reason for the absence of donor T cell chimerism in ALS- and rapamycin-treated (no irradiation) mice is not known, although it is possible that donor T cells were not detected by flow cytometric analyses because of their low numbers. The presence of chimerism in B10.A mice given CIID BMC was not tested due to early skin graft rejection.

It has been shown that skin grafts from MHC class II Ag-deficient (CIID) mice were rapidly rejected by normal recipients through the indirect recognition of alloantigen in which recipient CD4+ T cells recognized donor Ags presented in association with recipient class II molecules (24). Both CD4+ and CD8+ T cells were involved in rejection of skin grafts when there was a class I Ag disparity between the donors (CIID mice) and recipients. Although we have not investigated the involvement of the direct vs indirect Ag recognition pathway in the B6 to B10.A combination using either the ALS/rapamycin/BMC model or the irradiation/BMC reconstitution model, it is reasonable to assume that B10.A mice rejected class I Ag disparate CIID skin grafts through the indirect Ag recognition pathway. The significance of both CD4+ and CD8+ T cells in rejection of CIID skin grafts in irradiated, CIID BMC-reconstituted B10.A mice was suggested by activation of CD44+ cells in both recipient CD4+ and CD8+ T cell populations after skin graft rejection (A. Umemura, unpublished data).

We do not know at present the identity of class II Ag-positive cells in BMC responsible for induction of transplantation tolerance or where the event takes place in the ALS/rapamycin/donor BMC model. Because removal of I-A-positive cells before injection markedly reduced the tolerogeneic effect of donor BMC, we postulate that mature class II Ag-positive cells, such as B cells, macrophages, and dendritic cells, are responsible for early stage of tolerance by migrating into the host thymus and inducing deletion of donor alloantigen-reactive T cells. We also postulate that hemopoietic stem cells within donor BMC are important in maintaining the peripheral chimerism with class II Ag-positive cells, which trigger the peripheral tolerance mechanisms such as activation-induced cell death (27) or production of immunosuppressive cytokines by activated CD4+ T cells (28, 29) at the maintenance stage of transplantation tolerance. This possibility is suggested by the different tolerogenic effect mediated by genetically class II Ag-deficient BMC and wild-type B6 BMC depleted of class II Ag-positive cells. With the use of genetically class II Ag-deficient (CIID) BMC, recovery of donor class II-positive cells never occurred, and the tolerogenic effect was completely abrogated with allograft survival virtually identical with that of ALS/rapamycin controls. In contrast, I-Ab+ cell-depleted BMC, although much less effective than untreated BMC, still induced significant prolongation of graft survival over that in mice given ALS and rapamycin alone (no BMC). Because donor class II Ag-positive cells eventually recovered in the recipients by 60 days to the same degree as those seen in mice given untreated BMC, we postulate that later resurgence of I-Ab+ cells led to partial tolerance through the late peripheral event(s).

CIID BMC were capable of rescuing radiation injury and achieving high degrees of donor cell chimerism (macrochimerism) on infusion into irradiated syngeneic B6 or allogeneic B10.A mice. In chimeric mice, the proportion of various donor-derived lymphoid cell populations was almost identical, except for the absence of class II Ag-positive cells, with that seen in mice reconstituted with wild-type B6 BMC. CD4+ T cells, which are absent in CIID mice, developed in both syngeneic and allogeneic hosts by 30 days after CIID BMC infusion. Although Markowitz et al. (30) have previously reported that CD4+ T cells developed from CIID BMC when syngeneic class II molecules were expressed on radioresistant thymic cells, our data clearly show that positive selection of CD4+ T cells also occurs in the presence of allogeneic class II MHC molecule in the thymus. This is in agreement with the previous finding that functional murine CD4+ T cells develop even in xenogeneic pig thymus transplanted into T cell-depleted, thymectomized mice (31).

Despite the presence of macrochimerism immediately before grafting, B10.A mice reconstituted with CIID BMC acutely rejected both wild-type B6 and donor CIID skin grafts. More surprisingly, these mice maintained the state of macrochimerism even after graft rejection. In contrast, B10.A mice reconstituted with wild-type B6 BMC maintained both skin graft survival and macrochimerism. Thus, creation of macrochimerism with cells deficient in MHC class II Ag expression did not lead to acceptance of donor-type skin grafts, and conversely, rejection of donor skin grafts or class II Ag-expressing skin grafts did not cause loss of macrochimerism. The dissociation of macrochimerism and transplantation tolerance observed in CIID BMC-reconstituted mice contradicts the reports showing a strong correlation between the presence of macrochimerism and specific transplantation tolerance (32, 33).

Because cells of CIID mice lack class II Ag, it is reasonable to assume that the tolerance to circulating CIID cells does not protect wild-type B6 skin allografts that express class II Ag. But why did conditions that allowed continued macrochimerism with CIID cells fail to achieve tolerance to CIID skin allografts? Similarly, why did CIID BMC fail to induce tolerance to CIID skin grafts in the ALS/rapamycin/donor BMC model? We do not know the answer at present. One possibility is that in a fully MHC-mismatched strain combination, induction of tolerance to allografts (but not to hemopoietic cells) may require interaction between recipient CD4+ T cells with donor class II Ag. Class II Ag may be presented by either donor APC (direct Ag recognition pathway) or recipient APC (indirect recognition), given that involvement of both direct and indirect pathways in inhibitory response has been proposed (34, 35). When there is no donor class II Ag, indirect recognition of donor class I Ag presented by recipient APC evokes rejection responses. On the other hand, tolerance to hemopoietic cells may not require interaction between donor class II Ag and recipient CD4+ T cells; presentation of class I Ag to CD4+ T cells through recipient APC in a tolerizing environment (for example, myeloablative immunosuppression and intrathymic migration of alloantigen) may be sufficient. Rejection of skin grafts but sparing of chimeric cells from rejection in the CIID BMC radiation chimera model may also suggest the fundamental differences between hemopoietic cells and tissue grafts in susceptibility to tolerance and rejection processes. Answers to these questions await future investigation.


   Acknowledgments
 
We thank Dr. Xian C. Li for critical review and Rita Gottschalk for technical assistance.


   Footnotes
 
1 This work was in part supported by National Institutes of Health Grant 2RO1AI14551. Back

2 Address correspondence and reprint requests to Dr. Takashi Maki, Research North, Beth Israel Deaconess Medical Center, P.O. Box 15707, Boston, MA 02215. Back

3 Abbreviations used in this paper: BMC, bone marrow cells; CIID, class II deficient; CID, class I deficient; B6, C57BL/6; ALS, antilymphocyte serum; MST, median survival time. Back

4 D. A. Hale, R. Gottschalk, A. Umemura, T. Maki, and A. P. Monaco. Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation: a unique role for sirolimus. Transplantation (in press). Back

Received for publication November 3, 1999. Accepted for publication February 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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