Rapid deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells

Abstract

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II(+) donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c(+) dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b(+) monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b(+) cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.

Figure 1. Requirement for direct CD4 alloreactivity for CD8 tolerance

(A) B10.S mice received a wild-type B6 or I-Aβ^−/− BM graft after conditioning with TBI and anti-CD154. The incidence of mixed chimerism for the B cell lineage at indicated time points is shown. Chimerism was defined as ≥5% donor MHC class I positive cells among B220⁺ B cells. One representative experiment out of two is shown (n = 7 animals/group per experiment). (B) The percentage of donor chimerism ±SEM over time is shown for B cells and CD4 T cells in peripheral blood of both groups shown in panel A. (C) Mice were treated as described for panel A. In addition, they received one dose of anti-CD8 mAb. CD8 T cells were <0.5% of lymphocytes in white blood cells by 2 weeks after BMT. The incidence of chimerism for the B cell lineage is shown at indicated time points. One representative experiment out of two is shown (n = 6–7 animals/group per experiment). (D) The percentage of donor chimerism ±SEM over time is shown for B cells and CD4 T cells in peripheral blood of the groups shown in panel C. In all animals, B cell chimerism is similar to chimerism of myeloid cell lineages, and CD4 T cell chimerism is similar to CD8 chimerism (unless CD8s were depleted).

Figure 2. Donor CD11c+ DCs are not necessary for induction of chimerism

(A) Experimental design: B10.S mice received conditioning with 3Gy TBI and anti-CD154. In addition, they were or were not treated with diphtheria toxin (DT). On day 0 they received 20x10⁶ BMCs from diphtheria toxin receptor-transgenic (DTR tg) or wild-type B6 mice, which were either treated or not treated with DT on the day before BM harvest. Panel (B) shows the percentage of donor-derived cells among gated CD11c+ splenocytes one day after the last DT injection for two individual mice receiving DTR tg BM either without (left panel) or with DT treatment (right panel). Both of these mice had similar levels of multilineage chimerism (around 15% for B cells and 30% for granulocytes). (C) Incidence of donor chimerism in the B cell lineage, which is representative also for the granulocyte lineage, is shown 3 and 9 weeks after BMT for mice of the 4 groups mentioned above (n = 7–8/group; one of 2 similar experiments is shown). (D, E) Levels of donor chimerism in the 4 groups mentioned above is shown for B cells (D) and CD4 T cells (E).

Figure 3. Donor B cells are not required for induction of stable chimerism and tolerance

(A) B10.S mice received conditioning as in Methods followed by transplantation with BMCs from wt B6 or B cell-deficient μ^MT mice. Incidence of donor chimerism in the granulocyte lineage is shown (n = 7–8/group; one representative experiment of 2 is shown). (B) Levels of chimerism (mean ± SEM) in granulocytes and CD4 T cells are shown for groups in panel A. (C) The groups in panel A were grafted with donor B6 or 3^rd party B10.RIII skin 2 weeks after BMT, and graft survival is shown over 100 days.

Figure 4. Donor T cells are not necessary for induction of chimerism and tolerance

B10.S mice received the conditioning regimen described under Methods followed by allogeneic BMT. (A) Incidence of donor chimerism in the B cell lineage, which is similar to chimerism in the granulocyte lineage (not shown), is shown for mice that were transplanted with either whole BMCs from wt B6 mice (open bars), with wt B6 BMCs depleted ex vivo of CD4 and CD8 T cells (hatched bars), or with whole BMCs from TCRβ^−/− mice (closed bars; n = 7–8 for each group). (B) Levels of donor chimerism (mean ± SEM) are shown for the B cell and CD4 T cell lineages in the three groups shown in panel A. (C) The groups transplanted with whole BMCs from wild-type B6 or TCRβ^−/− mice were grafted with donor B6 or 3^rd party B10.RIII skin 12 weeks after BMT, and graft survival was followed over 100 days (n=7–8/group).

Figure 5. Donor-derived IL-10 is not required for induction of chimerism

B10.S mice received the conditioning regimen described under Methods followed by allogeneic BMT. (A) Incidence of donor chimerism in the B cell lineage, which is similar to chimerism in the granulocyte lineage (not shown), is shown for mice that were transplanted with either T cell-depleted BMCs from wt B6 mice (open bars) or from IL-10 KO mice (closed bars; n = 7–8/group). (B) Levels of donor chimerism (mean ± SEM) are shown for the B cell and CD4 T cell lineages in the two groups shown in panel A.

Figure 6. Tolerance can be assessed by early chimerism and cytotoxicity assays 7–8 days after BMT

(A) B6 mice received either conditioning with TBI and anti-CD154 mAb or with TBI only without anti-CD154. They were then transplanted with BMCs from fully MHC-mismatched B10.A mice. The percentage of donor B cells and granulocytes in the spleen is shown on day 7 after BMT. (B) B6 mice received conditioning with TBI and anti-CD154 and were then transplanted with BMCs from fully MHC-mismatched B10.A mice. Cytotoxic activity of whole splenocytes on day 8 after BMT was measured in a ⁵¹Cr release assay against donor B10.A (closed symbols) or 3^rd party B10.RIII targets (open symbols) after a 5 day restimulation culture with the respective stimulators (mean ± SEM, 4 mice/group). One representative experiment out of three is shown.

Figure 7. Donor B cells but not Mac1+ cells promote early chimerism

Recipient mice were conditioned as in Methods, then transplanted with 25x10⁶ whole BMCs (WBM), MACS-selected CD19+ or Mac1+ cells or MACS depleted CD19− or Mac1− cells from a fully allogeneic donor strain. Percentage of donor B cells and granulocytes in spleen on day 7 after BMT is shown. Pooled data from 2–3 experiments per strain combination are shown. (A) Recipient mice were 10.S, donor mice were B6 (n=6/group from 2 experiments). (B) Recipient mice were B6 and donor mice were B10.A (n=10–12/group from 3 experiments).

Figure 8. Donor B cells induce donor-specific unresponsiveness in CML assay seven days after BMT

Recipient B10.S or B6 mice were conditioned as in Methods, then transplanted with 25x10⁶ whole BMCs (WBM), MACS-selected CD19+ or Mac1+ cells or MACS-depleted CD19− from fully MHC-mismatched B6 or B10.A donor mice as indicated. Cytotoxic activity in whole splenocytes on day 7 after BMT was measured in a ⁵¹Cr release assay against donor B6 (closed symbols) or 3^rd party B10.RIII targets (open symbols) after a 5 day restimulation culture with the respective stimulators. Each line indicates mean ± SEM of % specific lysis for pooled mice from 2–3 different experiments. Upper row: recipient mice were B10.S, donor mice were B6 (n=6/group from 2 experiments); lower row; recipient mice were B6 and donor mice were B10.A (n=8–12/group from 3 experiments).

Figure 9. B cells induce early deletional CD8 T cell tolerance

Recipient B6 mice expressing a transgenic donor-specific alloreactive TCR on a fraction of recipient CD8 T cells were generated (“2C/B6 chimeras”, for details see Methods section). Eight weeks later, these 2C/B6 chimeras received conditioning with TBI and anti-CD154 and were then transplanted with 25x10⁶ whole BMCs (WBM), MACS-selected CD19+ or Mac1+ cells or MACS-depleted CD19− cells from fully MHC-mismatched ligand-bearing B10.A donor mice (L^d positive; black columns indicating B cell-containing and white columns indicating B cell-depleted grafts) or whole BMCs from non-ligand bearing B10.S donor mice (L^d negative; hatched columns). The percentage of 2C+ cells among splenic CD8 T cells was determined by FACS on day 7 after BMT and then normalized to the percentage of 2C TCR expression among peripheral blood CD8 T cells 1 week before BMT. Mean + SEM are shown for 5 animals/group.