Upregulation of Molecules Associated With T-Regulatory Function by Thymoglobulin Pretreatment of Human CD4+ Cells

Abstract

This study evaluated the immunologic effects of thymoglobulin in modulating human CD4+ cells. Human CD4+ cells were purified from peripheral blood mononuclear cells by negative selection method. CD4+ cells were pretreated with thymoglobulin and incubated for 72 hr. Cells and culture supernatants were collected and studied by real-time quantitative polymerase chain reaction, fluorescence activated cell scanning, multiplex cytokine assay, and mixed lymphocyte reaction (MLR). Thymoglobulin pretreated CD4+ cells demonstrated up-regulation of gene transcripts for CTLA-4, OX40, forkhead box P3 (Foxp3), CD25, IFN-gamma, IL-10, and IL-2 as determined by real-time quantitative polymerase chain reaction. Fluorescence-activated cell scanning analysis demonstrated that CD4+ cells, pretreated with thymoglobulin, up-regulated CD25 expression on their surface, and the surface expression of CTLA-4 and OX40 and the expression of intracellular Foxp3 were observed in these CD4+CD25+ cells. Additionally, MLR demonstrated that thymoglobulin-pretreated cells partially inhibited proliferation of untreated autologous CD4+ cells in response to allogeneic cells. The high levels of IFN-gamma, IL-10, IL-2, and IL-4 were detected by multiplex cytokine assay in supernatants collected from cultures of thymoglobulin-pretreated CD4+ cells. The lymphocyte proliferation of allogeneic MLR was also partially blocked in the presence of supernatants from cultures of thymoglobulin-pretreated CD4+ cells. This study demonstrates that the unique effects of thymoglobulin in modulating CD4+ cells may be an important mechanism for its action in inducing immunosuppression and transplant tolerance.