CD4+FoxP3+ Regulatory T Cells Promote B Cell Differentiation and Induce Tolerance to Bone Marrow Grafts

CD4+FoxP3+ regulatory T cells (Treg) adoptive transfer has proved to be an effective treatment for GVHD prevention in several studies. Treg impact on immune reconstitution and bone marrow engraftment after transplantation has been less well studied.

We treated C57BL/6 FoxP3^DTR mice with diphtheria toxin (DT) resulting in a complete Treg ablation. Transplantation of lethally irradiated (TBI 10 Gy) Treg depleted mice with allogeneic (BALB/C or FVB/N) T-cell depleted bone marrow (TCD BM) or purified hematopoietic stem cells (HSCs) resulted in donor rejection or mixed chimerism (p < 0.01). Treg depletion favored host CD4+ (p < 0.001), CD8+ (p < 0.01) and GR1+ cell persistence (p < 0.01) and delayed B cell reconstitution (p < 0.001). Transfer of in vitro activated with anti-CD3/CD28 beads and IL-2 Treg to DT treated mice rescued engraftment (p < 0.01) and boosted B cell reconstitution (p < 0.001).

Syngeneic transplanted Treg depleted mice engrafted but had markedly delayed B cell reconstitution (p < 0.01) thus Treg promote donor B cell differentiation in manner not dependent upon alloreactivity. FACS analysis of bone marrow cells of these mice showed higher numbers of donor Lin-Sca1+cKit+ HSCs (p < 0.05) and donor Lin-Sca1+cKit+Flt3+ lymphoid progenitors (p < 0.05) while B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.01) were reduced demonstrating a block of maturation in the early phases of B cell differentiation. IL-7 production was reduced after Treg depletion (p < 0.05) suggesting that Treg control B cell differentiation through an IL-7 dependent mechanism. Confocal microscopic analysis of femurs after transplantation demonstrated that Treg localize near the endosteum (p < 0.0001) co-located with donor B220+ B cell clusters and gather in the epiphyseal areas where donor HSCs were mainly detectable suggesting that Treg act as an immunological barrier for HSCs and B cell progenitors, providing a protective niche.

Adoptive transfer of in vitro activated Treg induced B cell reconstitution (p < 0.01)  in non irradiated immune deficient BALB/C rag2^-/-γc^-/- mice that received allogeneic TCD BM. Higher numbers of Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.05) were found in the bone marrow of these mice with reduced levels of inflammatory cytokines such as INFγ, IL-12, IL-13, IL-1b, CXCR-9 and CXCR-10 and increased levels of IL-7 and VEGF.

In conclusion, our findings indicate that Treg act as a key regulator of B cell differentiation promoting production of mature B cells through an IL-7 mediated mechanism that is not dependent on alloantigen recognition. Our data suggest that Treg play a role in building the donor HSC and B cell precursor niche. Finally, Treg adoptive transfer enhances B cell reconstitution and induces tolerance to bone marrow grafts even in the absence of conditioning providing a new tool for clinical translation especially in children with SCID or hemoglobinopathies.