Regulatory T cells (Tregs), a subset of CD4+CD25+ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25+ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.
Lymph node or splenic tumor-induced Tregs are isolated and purified by a two-step procedure using CD4+CD25+ regulatory T cell isolation kit from MACS Miltenyi Biotec. First, CD4+ T cells are enriched by negative selection, followed by positive selection of CD25+ T cells. Tumor-induced purified Tregs (CD3+CD4+CD25+FoxP3+) are then co-injected subcutaneously together with tumor cells into naïve mice (Winn assay) (Winn, 1960). Tregs could also be injected intravenously once or several times, according to the research needs. The effect of the adoptively transferred Tregs on tumor growth is then measured by caliper or by in vivo imaging techniques.
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