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Human CD4+CD25+CD127loFOXP3+Treg Activation by IL-4 and Alloantigen Induces Activated Treg that do not Express CD45RA and Express the IL-5 Receptor Alpha (CD125)

Verma, Nirupama D.1,2; Robinson, Catherine M.1,2; Tran, Giang T.1,2; Hodgkinson, Suzanne J.1,2; Hall, Bruce M.1,2

doi: 10.1097/

1Immune Tolerance Group, INGHAM Institute, Liverpool, Australia; 2South Western Clinical School and Faculty of Medicine, University of New South Wales, Sydney, Australia.

Aim: Transplant tolerance induction requires CD4+CD25+FOXP3+Treg (tTreg). tTreg are not antigen-specific and for therapy large numbers are required. Antigen-specific CD4+CD25+FOXP3+Treg that are present when a host develops transplant tolerance are more potent and alloantigen-specific. In rats, culture of naïve tTreg with rIL-4 and alloantigen induces IL-5Rα expressing antigen-specific Treg. These activated Treg are further expanded by IL-5. Here, we aim to examine if similar activation pathways occur in human CD4+CD25+CD127loFOXP3+ Treg.

Methods: Treg were isolated from healthy human blood using a Human CD4+CD127loCD25+ Regulatory T cell isolation Kit and their purity assayed by multicolour flow cytometry. These enriched tTreg were cultured with human rIL-4 (200Units/ml) and allogeneic stimulator cells to examine proliferation, changes in surface molecules by FACS and expression of cytokine and cytokine receptors by RT-PCR.

Results: tTreg had significant proliferation at 3-7d with rIL-4 and antigen with a peak at 4d. The level of proliferation was less than with rIL-2, however. FACS showed a reduction in CD45RAhi, an increase in CD45RAintermediate population and a marked increase in CD45RA population (43% vs 11%). CD45RO+ and CD45RB+ population also increased. In the CD45RA-/lo population, the expression of CD25 and FOXP3 increased. Nearly all cells continued to express CD62L. RT-PCR showed induction of IL-5Rα (CDw125) but not IFNGR.

Conclusions: Human CD4+CD25+CD127loFOXP3+Treg can be activated by alloantigen and rIL-4 to express the IL-5 receptor, similar to our observation in rats. This suggests IL-5 may promote the activation of antigen-specific Treg. The rIL-4 has two effects; 1. It induces polyclonal activation of tTreg that continue to express CD45RA. 2. The increased CD45RA/CD45RO+ population was consistent with activation of antigen-specific Treg. The activated cells had increased CD25 and FOXP3 expression, markers that distinguish them from naïve tTreg, which are CD45RA+ and have less CD25 and FOXP3 expression. Thus, alternate tTreg activation pathway may produce potent antigen-specific Treg for therapy circumventing requirement for high tTreg numbers needed to induce transplant tolerance.

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