Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning

Abstract

Background: A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC) engraftment with minimal myelosuppressive treatments.

Results: We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154) at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells.

Conclusion: We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains.

Figure 1

Induction of BM engraftment with CD4, CD8 and CD40L mAbs. (A) CBA mice were treated with three doses of 1 mg of non-depleting CD4, CD8 and CD40L mAbs on days -28, -26, -24, 0, 2 and 4 in relation to the day of BMT (day 0). (B) The level of hematopoietic chimerism was determined among peripheral blood mononuclear cells of CBA mice, transplanted with different numbers of B10 BM, by flow cytometry. CBA mice not subjected to BMT were used as a control group. Results are from day 120 following BMT. Difference from the control group is statistically significant in animals transplanted with 1×10⁷ cells or more (p < 0.02). (C) The mice were transplanted with donor type (B10) skin grafts 50 days following BMT. Grafts survived indefinitely in animals where mixed chimerism had been established, being the difference between animals transplanted with 1×10⁷ BM cells or more, and animals transplanted with 1×10⁶ or 5×10⁶ BM cells statistically significant (p < 0.02).

Figure 2

The requirement for first-stage antibody treatmentin advance of BMT. Except for control mice which received no treatment, all mice represented in this figure received CD4, CD8 and CD40L antibodies at the time of BMT (1 mg each ip on days 0, 2 and 4, relative to BMT on day 0). The first-stage antibody treatment was varied as described. CBA mice (A) and BALB/c mice (B) were transplanted with different doses of T cell depleted B10 BM, under the cover non-depleting CD4, CD8 and CD40L mAbs. Only one group of mice from each strain received the mAb treatment 4 weeks prior to BMT. Hematopoietic chimerism determined by flow cytometry 120 days following BMT is shown. In both strains the difference between untransplanted controls and animals not treated in advance of BMT is not statistically significant. Survival of B10 skin grafts, transplanted 50 days following BMT is represented. Only animals where mixed chimerism could be detected accepted the skin grafts indefinitely (p < 0.01 to any other group). (C) CBA mice were treated with 3 × 1 mg of CD4 and CD8 mAbs alone, or combined with the same dose of CD40L mAbs, 4 weeks before the transplantation of 4×10⁷ T cell depleted B10 BM. One group was not treated at that time. Together with the BMT all animals were treated with CD4, CD8 and CD40L mAbs as described in Figure 1. Animals that did not receive BMT were used as negative controls. (D) Experiment identical to the one described in (C), using different combinations of mAb 4 weeks before BMT. No statistically significant difference was observed between the transplanted groups in the levels of hematopoietic chimerism 120 days following BMT.

Figure 3

MAb requirements at the time of BMT (second-stagetreatment). CBA mice were treated with non-depleting CD4 and CD8 mAbs alone 4 weeks before transplantation of 2 × 10⁷ T cell depleted B10 BM. At the time of BMT the mice were treated with CD4 and CD8 mAb alone, or combined with CD40L mAb. Chimerism was only detected in the mice treated with both co-receptor and co-stimulation blockade at the time of BMT. Data is representative of 2 independent experiments.

Figure 4

BM engraftment in congenic mice. A dose of 2×10⁷ T cell depleted bone marrow cells from B6.CD45.1 mice was transplanted into CBA or congenic B6 mice treated with mAbs as described in figure 1. (A) One group of B6 mice was transplanted in the absence of any mAb treatment, and a group of CBA and B6 mice not subjected to BMT was used as a negative control. Hematopoietic chimerism was determined by quantification of peripheral blood mononuclear cells 120 days following BMT by flow cytometry. The results from two independent experiments are represented. The difference between the groups treated with mAb and any other group is statistically significant (p < 0.001). The difference between CBA and B6 recipients of B6.CD45.1 BM under the cover of mAbs is also significant in both experiments (p = 0.04 and p = 0.0006). (B) B6 mice were treated with 1 mg of the mAb PK136 administered 5 days prior to transplantation of B6.CD45.1 BM, to deplete their NK1.1 cells, while another group was treated with the control mAb YCATE55. Additional animals were subjected to the treatment described in Figure 1. NK cell depletion failed to achieve chimerism (p < 0.001).