CD8+/TCR− Facilitating Cells Induce Generation of Regulatory T cells in Vivo: Chimeric Regulatory T Cells are Antigen-Specific and Enhance Engraftment of Allogeneic HSC in Non-Obese Diabetic (NOD) Mice.
Authors: Huang Yiming , Bozulic Larry D , Miller Thomas , Xu Hong , Hussain Lala-Rukh , Ildstad Suzanne T. 
Published: April 2020

Abstract
CD8+/TCR− graft facilitating cells (FC) are a novel tolerogenic cell population in bone marrow that potently enhance engraftment of hematopoietic stem cells (HSC) in allogeneic and syngeneic recipients. The CD11c+/CD11b−/B220+ plasmacytoid precursor dendritic cell (p-preDC) subpopulation of FC (p-preDC FC) comprises over 60% of FC total and plays a critical and nonredundant role in facilitation. FC prevent graft-versus-host disease and remain tolerogenic after in vivo infusion. Regulatory T cells (Treg) are immunomodulatory cells that maintain tolerance in vivo. They can be generated in vitro via co-culture with p-preDC. There is great interest regarding the use of Treg as a cell-based therapy to induce graft/host tolerance in vivo. However, a major challenge to the clinical use of Treg has been to obtain sufficient numbers of cells for in vivo use and maintain their tolerogenic properties in vivo after in vitro expansion. Here, we evaluated whether FC function by inducing the production of Tregin vivo and examined the function of these chimeric Tregin vivo and in vitro. HSC (c-Kit+Sca-1+Lin−; KSL) were sorted from donor B6 and NOD mice. 10,000 B6 HSC and 1,000 NOD HSC were transplanted by tail-vein injection into recipient NOD mice conditioned with 950 cGy of total body irradiation (TBI). Spleen, thymus, and bone marrow were harvested from recipient NOD mice 5 weeks after transplantation. CD4+CD25+Foxp3+ Treg were analyzed by flow cytometry. FC induced the generation of both donor and recipient CD4+CD25+Foxp3+ Tregin vivo; the majority of Treg were recipient-derived (89% to 97%). To test the function of Treg from HSC + FC chimeras (chimeric Treg), CD8− CD4+CD25+ Treg were sorted from the spleen of chimeras 5 weeks after transplantation. 50,000 chimeric Treg plus 10,000 B6 HSC were transplanted into NOD recipients conditioned with 950 cGy TBI. Recipients of 50,000 Treg from naïve B6 spleens (B6 Treg) + HSC or HSC alone served as controls. Five of 26 recipients of HSC alone engrafted and survived up to 100 days. Only 2 of 5 recipients of HSC plus 50,000 B6 Treg engrafted and none of the recipients exhibited durable engraftment beyond 100 days. In striking contrast, 100% (4 of 4) recipients of HSC + 50,000 chimeric Treg engrafted durably, with survival ≥ 100 days. Chimeric Treg function was confirmed in vitro by MLR suppressor assays, as evidenced by strong suppression of T cell proliferation. Sorted chimeric Treg demonstrated an 87.2% suppression of cell proliferation when plated in a 1:1 ratio with naïve NOD responder cells and B6 stimulator cells. Moreover, when plated at a 1:4 and 1:8 ratio with naïve NOD responders, Treg suppressive function titrated to 62.7% and 43.3%, respectively. In contrast, sorted Treg from naïve B6 animals showed 75.8%, 35.4, and 29.4% suppression when plated in ratios of 1:1, 1:4, and 1:8, respectively. Taken together, these data suggest that FC induce the production of antigen-specific Tregin vivo and chimeric Treg are superior to naïve Treg in suppressing the proliferation of effector T cells and potently enhance engraftment of allogeneic HSC.