Ex Vivo Expanded Human Regulatory T Cells Prevent Acute Graft Versus Host Disease In A Mouse Model

Regulatory T cells (Tregs) play an essential role in modulating the immune system and maintaining peripheral tolerance. Because the number of Tregs that can be obtained from donor peripheral blood is limited, freshly isolated Tregs need to be expanded ex vivo to generate a sufficient number of cells for therapeutic applications. It remains controversial as to whether Tregs from cord blood (CB) possess better suppressive function compared to Tregs from adult peripheral blood (APB). Initial studies have shown that human Tregs can be effectively expanded ex vivo. However, the efficacy of expanded human Tregs in the prevention of acute graft versus host disease (aGVHD) in vivo is poorly understood.In this study, we expanded Tregs ex vivo from CD4+CD25+T cells from CB and CD4+CD25+CD127-T cells from APB and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. The phenotypes, expression of cytokines, and suppression of expanded Tregs were analyzed after two cycles of stimulation. Bone marrow and splenocytes of C57BL/6mice were transferred into lethally irradiated DBA/2mice, which were used as an aGVHD model. In Treg-treated mice, additional ex vivo expanded Tregs from CB or APB were transferred to the mice. Survival, body weight, GVHD scoring, histopathologic specimens, serum cytokines, and Th subsets were analyzed in Treg-treated mice and untreated controls.The average fold expansion of CB and APB Tregs with two cycles of polyclonal stimulation was1.5x103and1.8x103, respectively, whereas the average fold expansion of CB and APB Tregs with an alloantigen stimulus followed by a polyclonal stimulus was5x102and4.7x102, respectively. All of the expanded Tregs expressed high levels of Foxp3, CD39and CTLA-4, low levels of CD127, IL-2and IFN-γ, and retained anergic properties. CB Tregs expanded with two cycles of polyclonal stimulation suppressed not only the proliferation of the polyclonal antigen-driven responder T cells (Tresp) but also the HLA mismatched dendritic cell-driven Tresp. When CB and APB Tregs expanded with two cycles of polyclonal stimulation were transferred to the aGVHD mice,87.5%and16.7%of the mice, respectively, survived at least63days, however, all aGVHD mice died within18days. The production of IFN-γ and TNF-a in mice treated with CB Tregs was significantly lower compared to that in aGVHD mice on Day7. Subsequently, the production of IFN-γ, TNF-a and IL17rapidly dropped in CB-Treg-treated mice and remained at low levels, meanwhile the secretion of TGF-3increased continuously. Consistent with cytokine production in serum, the percentage of Thl cells was reduced from30%on Day7to10%on Day14and remained at low levels. Mouse CD4+Foxp3+cells (Tregs) were continually induced in vivo, in contrast, IL-17-positive cells (Th17) declined from high levels (approximately40%) to undetectable levels (less than2%). CD39expressed on the surface of CB Tregs could inhibit the proliferation of mouse T cells and the TGF-β secreted by them might polarize the Treg/Th17balance towards Tregs in aGVHD mouse model.CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs and could alleviate mouse aGVHD symptoms in allogeneic bone marrow transplant by expression of CD39and secretion of TGF-β, which suggests the potential use of expanded CB Tregs as a therapeutic approach for aGVHD. It is feasible to obtain human functional polyclonal or alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers to meet the needs of clinical application.