CD4+FoxP3+ Regulatory T Cells Regulate B Cell Differentiation and Induce Tolerance to Bone Marrow Grafts
Authors: Pierini Antonio , Nishikii Hidekazu , Pan Yuqiong , Baker Jeanette , Leveson-Gower Dennis B , Florek Mareike , Negrin Robert 
Published: October 2019

Abstract
CD4+FoxP3+ regulatory T cells (Treg) are a rare cell population that is responsible for peripheral immune tolerance. Treg adoptive transfer has proved to be an effective treatment for graft versus host disease prevention in several preclinical and clinical studies. The impact of Treg on immune reconstitution and bone marrow engraftment after transplantation has been less well studied.
We treated C57BL/6 FoxP3-DTR mice that carry the diphtheria toxin (DT) receptor in the promoter of the FoxP3 gene with DT resulting in a complete ablation of Treg (percentage of FoxP3+CD4+ cells over CD4+ cells < 0.2% in peripheral blood, spleen, lymph nodes, thymus and bone marrow). Transplantation of lethally irradiated (TBI 10 Gy) Treg depleted mice with allogeneic (BALB/C or FVB/N) T-cell depleted bone marrow (TCD BM) resulted in rejection or reduced donor 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). Adoptive transfer of purified host type Treg in vitro activated with Interleukin-2 (IL-2) and anti-CD3/CD28 beads to DT treated mice rescued engraftment (p < 0.01) and boosted B cell reconstitution (DT treated mice that received Treg vs DT treated mice only p < 0.001; DT treated mice that received Treg vs untreated mice p < 0.01). Moreover Treg ablation resulted in rejection or reduced donor chimerism (p < 0.01) in mice transplanted with allogeneic (FVB/N) purified Lin-Sca1+cKit+ hematopoietic stem cells (HSCs) demonstrating that Treg promote donor HSC engraftment without interacting with donor derived facilitating cells.
We explored the mechanism through which Treg impact donor engraftment and immune reconstitution and observed that Treg depleted mice transplanted with syngeneic (C57BL/6 CD45.1) TCD BM engrafted (p > 0.05) 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 syngeneic transplanted 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 numbers of B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.01) were reduced after Treg depletion demonstrating a block of maturation in the early phases of B cell differentiation. Confocal microscopic analysis of femurs after transplantation with TCD BM were used to determine the spatial relationship between Treg, B cells and HSCs. Treg localize near the endosteum (p < 0.05) and cluster in the epiphyseal areas where donor HSCs and B220+ B cells were mainly detectable suggesting that Treg act as an immunological barrier for HSCs and B cell progenitors, providing a protective immunological niche. Further, adoptive transfer of IL-2 and CD3/CD28 bead activated Treg induced B cell reconstitution in non irradiated immune deficient BALB/C rag2-/-γc-/- mice that received an infusion of allogeneic (C57BL/6) TCD BM. Bone marrow analysis of these mice revealed higher numbers of B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.05) in mice that received Treg. Analysis of the sera after Treg adoptive transfer showed reduced production of inflammatory cytokines and chemokines such as Interferon-γ, Interleukin-12, Interleukin-13, Interleukin-1b, CXCR-9 and CXCR-10 and increased production of VEGF.
In conclusion, our findings clearly indicate that Treg act as a key regulator of B cell differentiation promoting production of mature B cells. This effect is not dependent on alloantigen recognition and Treg localization after transplantation suggests that Treg play a role in building the donor HSC and B cell precursor niche. Finally, adoptive transfer of Treg enhances B cell immune reconstitution and induces tolerance to allogeneic bone marrow grafts even in the absence of conditioning providing a new tool for clinical translation in children with severe combined immune deficiencies or hemoglobinopathies and in patients undergoing organ transplantation.

Disclosures
No relevant conflicts of interest to declare.