Rabbit ATG but Not Horse ATG Promotes Expansion of Functional CD4+CD25highFoxP3 Regulatory T Cells In Vitro.
Authors: Feng Xingmin , Solomou Elena E. , Keyvanfar Keyvan , Herndon Thomas , Chen Jichun , Kajigaya Sachiko , Young Neal S. 
Published: April 2020

Abstract
CD4+CD25+ regulatory T cells (Treg) are believed to play important roles in suppressing immune responses and maintaining tolerance. Treg have the ability to prevent the development of autoimmune diseases, graft rejection and graft versus host disease (GVHD) in mice and perhaps also in humans. Immunosuppressive drugs such as rabbit ATG (rATG), horse ATG (hATG) and cyclosporine A (CsA) are widely used in conditioning for transplantation and for the treatment of autoimmune diseases and GVHD, but their effects on Treg remain to be fully elucidated. Lopez et al. (J Am Soc Nephrol. 2006;17:2844–2853.) first reported that in vitro culture of human peripheral blood mononuclear cells (PBMC) with rATG resulted in expansion of CD4+CD25+ T cells. In the current study, we show that in vitro culture of normal human PBMC with low dose rATG (10 μg/ml) resulted in marked expansion of CD4+CD25high T cells (rATG 8.00±0.95% versus untreated 0.99±0.11%, n=10, p&lt;0.0001) and CD4+CD25high FoxP3 T cells (rATG 2.24±0.11% versus untreated 0.90±0.10%, n=10, p&lt;0.0001). rATG exposure converted CD4+CD25− T cells into CD4+CD25+ T cells, which proliferated better in comparison to CD4+CD25− T cells. In immunoblots of protein extracted from PBMC treated with rATG, there was increased expression of FoxP3 and nuclear factor of activated T cells (NFAT1) in CD4+CD25− and CD4+CD25+ T cells; rATG-induced NFAT1 expression correlated with FoxP3 expression. Expanded Treg suppressed autologous T-cell proliferation after T-cell receptor (TCR) stimulation by 64% when cultured with autologous PBMC at a 1:1 ratio, consistent with functional activity. Culture supernatants of PBMC treated with rATG showed increased levels of IL-10, compared with supernatants of PBMC treated with hATG or CsA, but no differences in INF-γ, IL-2, and IL-4. Unexpectedly, hATG did not expand but rather decreased Treg [For CD4+CD25high T cells (n=10): hATG 0.67±0.10% versus untreated 0.99±0.11%, p=0.0386; For CD4+CD25high FoxP3 T cells (n=10): hATG 0.62±0.08% versus untreated 0.90±0.10%, p=0.0435]. Furthermore, rATG and hATG showed differences in binding to lymphocytes, they contained different amounts of CD3 and TCRαβ antibodies, and they induced different activation states (expression of glucocorticoid-induced tumor necrosis factor receptor, cytotoxic T lymphocyte-associated antigen-4, and CD62L) for CD4+ T cells. In vitro, Treg expansion mediated by rATG occurred at submitogenic concentrations (&lt; 50 μg/ml) rather than at lymphocyte depletion levels (50–100 μg/ml). Our findings suggest that rATG expanded Treg by converting CD4+CD25− T cells into CD4+CD25+ T cells, probably through a mechanism of transcription regulation, and enhanced NFAT1 expression, in turn conferring on CD4+CD25− T cells FoxP3 expression and regulatory activity. The therapeutic effects of rATG in the treatment of autoimmune diseases and GVHD may occur due to not only lymphocyte depletion but also enhanced Treg cell number and function. Our observation may provide a useful method for expansion of Treg in cellular treatment in transplantation and autoimmune diseases.