Hematopoietic chimerism and central tolerance created by peripheral-tolerance induction without myeloablative conditioning

Abstract

Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Realization of that potential has been impeded by the need for myeloablative conditioning of the host and development of graft-versus-host disease (GVHD). To surmount these impediments, we have adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines donor-specific transfusion (DST) with anti-CD154 mAb. When applied to stem cell transplantation, administration of DST, anti-CD154 mAb, and allogeneic bone marrow leads to hematopoietic chimerism and central tolerance with no myeloablation and no GVHD. Tolerance in this system results from deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the transfusion that precedes transplantation need not be of donor origin, suggesting that both allospecific and non-allospecific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance impair establishment of chimerism. We conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.

Figure 1

In independent trials, two groups of BALB/c (H2^d) mice (n = 5 in each group) were injected with 50 × 10⁶ C57BL/6 (H2^b) bone marrow cells on day 0. All mice were treated with a DST consisting of 10⁷ C57BL/6 spleen cells on day –7 relative to bone marrow transplantation. They also received anti-CD154 mAb at a dose of 0.5 mg intraperitoneally on days –7, –4, 0, and +3 relative to bone marrow transplantation. The percentage of donor-origin PBMCs was measured by flow microfluorometry in all mice 4 weeks, 8 weeks, 14 or 21 weeks, and 30 weeks after bone marrow transplantation as described in Methods.

Figure 2

Deletion of peripheral host alloreactive CD8⁺ T cells. KB5 CBA synchimeras were randomized into four cohorts. Group 1 was untreated. Group 2 was injected with four doses of anti-CD154 mAb on days 0, +3, +7, and +10 (short arrows), and with 50 × 10⁶ C57BL/6 bone marrow cells on day +7 (long arrow). Group 3 received four doses of 0.5 mg of anti-CD154 mAb at the same intervals (short arrows) plus a transfusion of C57BL/6 spleen cells on day 0. Group 4 received a DST of C57BL/6 spleen cells on day 0 and anti-CD154 mAb on days 0, +3, +7, and +10, in addition to an injection of 50 × 10⁶ C57BL/6 bone marrow cells on day +7. The percentage of DES⁺CD8⁺ cells in the blood was determined on day 0 (before any treatment) and then at the indicated times. Within 2 weeks of treatment, the percentage of DES⁺CD8⁺ peripheral blood cells was significantly lower in all treatment groups compared with controls (P < 0.001). Thereafter, the percentage of DES⁺CD8⁺ peripheral blood cells in groups 2 and 3 tended to rise toward that observed in controls, but even at week 15 the percentage remained less than in controls (P < 0.001). In contrast, the percentage of DES⁺CD8⁺ peripheral blood cells in group 4 remained extremely low throughout the course of the experiment and, at week 15, was significantly lower than in all other groups (P ≤ 0.001 for each comparison). With respect to chimerism, defined as at least 0.5% donor-origin (H2-K^b+) PBMCs 9 weeks after transplantation, all mice in group 4 were chimeric and none in groups 2 or 3 was chimeric.

Figure 3

Intrathymic deletion of host alloreactive DES⁺CD8⁺CD4^– thymocytes. KB5 CBA synchimeras were randomized into two groups. Group 1 (upper panels) was left untreated. Group 2 (lower panels) was injected with a C57BL/6 DST on day –7 and anti-CD154 mAb on days –7, –4, 0, and +3 relative to injection of 50 × 10⁶ C57BL/6 bone marrow cells on day 0. Thymi were recovered 35 weeks after bone marrow transplantation and analyzed by flow microfluorometry for the percentage of DES⁺CD8⁺CD4^– thymocytes as described in Methods. Shown in the left column are representative dot plots; the percentage of cells expressing CD4 and CD8 is indicated in each quadrant. The right column presents histograms; the horizontal bars depict the gates used to determine the number of DES⁺ cells in the CD8⁺CD4^– quadrant. Representative data are shown; the complete data set is given in Table 4.