Host CD4+CD25+FoxP3+ Regulatory T Cells Facilitate Donor Engraftment Without Impacting Graft Versus Host Disease Onset After Major-Mismatched Hematopoietic Stem Cell Transplantation
Authors: Pierini Antonio , Nishikii Hidekazu , Florek Mareike , Leveson-Gower Dennis B , Pan Yuqiong , Kim Byung-Su , Baker Jeanette , Negrin Robert 
Published: October 2019

Abstract
A major challenge following allogeneic hematopoietic stem cell transplantation (HCT) is to establish persistent engraftment of donor hematopoietic cells. Many strategies have been developed to permit engraftment involving high dose chemotherapy, serotherapy with anti-lymphocyte drugs or myeloablative irradiation resulting in highly toxic conditioning regimens. The introduction of less harmful therapies could result in less toxicity especially in the major mismatched setting and when reduced intensity conditioning is required. While recent studies have explored the mechanisms through which donor-type CD4+CD25+FoxP3+ regulatory T cells (Tregs) restrict the development of graft versus host and host versus graft reactions, less is known about the role of host-type Treg in the transplant setting. In syngeneic and minor mismatched HCT host Tregs comprise a major component of the Treg compartment in the first weeks after transplant. Moreover the transplant of in vitro primed host Tregs can improve donor engraftment in major mismatched models of HCT; therefore host Tregs could be one of the key controllers of the host versus graft reaction mediated by residual host CD4+ and CD8+ conventional T cells (Tcons), possibly influencing graft versus host disease (GvHD) onset and severity. In this study we investigated the role of host Treg after major mismatched HCT to understand their impact in graft facilitation and rejection and in GvHD induction and prevention. We investigated the mechanism through which this cell population works and we explored the feasibility and the effectiveness of host Treg adoptive transfer for cellular therapy in HCT animal models.

Results
CD4+CD25+FoxP3+ host Tregs persist for at least 28 days after total body irradiation (8 Gy) and transplantation of C57BL/6 (H-2b) T cell depleted bone marrow (TCD BM) into BALB/C (H-2d) mice. Host Treg could be found in spleen, lymph nodes and bone marrow with an increase in the Treg/CD4+ cell ratio. Moreover we observed that these residual host Tregs maintain their suppressive function in vitro if harvested 14 days after transplant and incubated with healthy mouse derived Tcons in a MLR. These results are even more relevant as transplanted mouse derived host Tcons lose their ability to proliferate confirming that host Tregs possess a numeric and functional advantage compared to residual host Tcons.
Using FOXP3-DTR mice as hosts we observed that host Treg ablation results in reduced donor chimerism after major mismatched TCD BM transplant (p < 0.01, analysis performed 2 months after transplant). At the same time, the absence of host Tregs favors host CD4+ T cell persistence (p < 0.001) and delays B cell reconstitution (p < 0.001). Furthermore, we hypothesized that host Treg act as an immunological barrier for HSCs, providing a protective immunological niche. Confocal microscopic analysis of femurs performed at day 7 after TCD BM transplant confirmed that hypothesis showing host Tregs clustering in the epiphysis where donor hematopoietic stem cell (HSC) engraftment is mainly detectable.
To strengthen these results and to provide a clinical translatable tool, we adoptively transferred 5x105/mouse highly purified unmanipulated host Tregs in a non myeloablative (TBI 5.5 Gy) major mismatched model of rejection. We found that the transferred host Tregs induce persistent full donor chimerism if injected together with a sublethal dose of donor Tcons (5x105/mouse, p=0.016) and transiently enhance donor chimerism in the first three weeks after transplant if injected with low dose interleukin-2 (IL-2, 50,000 IU bid for 7 days; p < 0.001) without impacting on GvHD incidence and lethality. The relatively low dose of injected Tregs, the possibility to stimulate and expand them in vivo with IL-2 and the safety of this model provide the first evidence of the feasibility of this clinical approach.

Conclusion
Our findings indicate that host Tregs facilitate bone marrow engraftment in major mismatched HCT models without impacting GvHD. Notably, our observations on the bone marrow environment after transplant strongly suggest that host Tregs can play a role in building the donor HSC cell niche. Finally host Treg adoptive transfer proved to be feasible and effective in animal models providing a new tool for cellular therapy and clinical translation.

Disclosures:
No relevant conflicts of interest to declare.