Anti-CD45RB treatment induces t cell regulation by increasing CD4+CD25+CD45RB low cells

Abstract

<b>Introduction:</b> We have shown that 3 doses of anti-CD45RB mAb induce longterm graft survival and same donor strain tolerance in ∼50% of C57BL/6 recipients of Balb/c islets. Anti-CD45RB treatment promotes tolerance by inducing a shift in CD45 isoform expression from CD45RB^High to CD45RB^Low and up-regulating CTLA-4 expression. Here, we examine whether anti-CD45RB treatment induces expression of regulatory cells. <b>Methods:</b> Lymphocytes were isolated from Balb/c mice (n = 3) and phenotyped by immunostaining and flow cytometry analysis following anti-CD45RB mAb treatment (3 doses: 100 μg/iv/dose). <b>Results:</b> Flow cytometry analysis showed a ∼55% increase of CD25 expression on CD4+ lymphocytes after treatment with 3 doses of anti-CD45RB. In addition, the CD25+CD45RB^LowCD4+ subset showed a threefold increase after anti-CD45RB treatment. Control untreated allogenic lymphocytes from C57BL/6 mice (n = 2) were then used as stimulator cells in mixed lymphocyte reactions (MLR) to demonstrate whether anti-CD45RB treated lymphocytes (unseparated population) could regulate proliferation. Results show that anti-CD45RB treated T cells do not proliferate when compared to untreated lymphocytes. In addition, when anti-CD45RB treated lymphocytes were cocultured with increased amounts of untreated T cells (1:1, 1:2, and 1:4 ratios), proliferation was increased in a dose-dependent manner (∼50, ∼75, and ∼90%, respectively) when compared to untreated cells. <b>Conclusions:</b> A short course of treatment with anti-CD45RB mAb induces a significant increase (300%) of CD25+CD45RB^LowCD4+ cells. This T cell subset has been shown to have regulatory properties in both mice and humans. In addition, anti-CD45RB treated lymphocytes inhibit thymidine incorporation and proliferation of allogeneic lymphocytes. Finally, when cocultured with untreated T cells, anti-CD45RB treated lymphocytes regulate the proliferation of allogeneic responder lymphocytes in a dose-dependent manner.