Evidence that Regulator T cells of Eye–Derived Tolerance Can Arise from CD25–Negative Precursors

Abstract

Abstract: : Purpose: Regulatory T cells (T reg) arise in the spleens of mice with anterior chamber associated immune deviation (ACAID), a form of eye–derived tolerance that is evoked by injection of antigen into the anterior chamber (AC) of the eye. One type of ACAID T reg is CD4+, suppresses induction of delayed hypersensitivity (DH) when transferred to naïve mice, and resembles a similar CD4+ T reg that is generated in vitro by exposing naive T cells to antigen–pulsed antigen presenting cells (APC) treated with TGF–b. We investigated whether the T regs generated in–vitro as well as CD4+ ACAID T regs induced in vivo, are derived from, or functionally related to naturally occurring CD4+CD25+ T cells. Methods: The expression of CD25 on DO11.10 T cells when stimulated in vitro to OVA–pulsed, TGFb–treated APCs was analyzed by FACS. For induction of T reg in–vitro, DO11.10 T cells depleted of CD25+ cells prior to in vitro stimulation were stimulated with OVA–pulsed, TGFb–treated APCs. CD4+CD25+ T cells or CD4+CD25–T cells from spleens of mice pre–treated with AC injections of OVA were assayed for the capacity to suppress DH induction when transferred into naïve mice. ACAID was assayed in mice depleted systemically of CD25+ cells following injection into the AC of OVA. Results: DO11.10 T cells were found to express CD25 when stimulated in vitro to OVA–pulsed, TGFb–treated APCs. However, DO11.10 T cells depleted of CD25+ cells prior to in vitro stimulation still differentiated into T regs when exposed to OVA–pulsed, TGFb–treated APCs. In addition, CD4+ T cells from spleens of mice pre–treated with AC injections of OVA suppressed DH induction when transferred into naïve mice, whether the transferred CD4+ cell suspension was enriched for– or depleted of CD25+ cells. Finally, an injection of OVA into the AC of mice depleted systemically of CD25+ cells prevented the mice from acquiring OVA–specific DH when immunized subsequently, just as does a similar injection in non–depleted mice. Conclusions: The CD4+ T regs of ACAID can arise from CD25– precursors, and that induction of ACAID does not require the presence of natural CD4+CD25+ T regs.

Keywords: ACAID • immune tolerance/privilege • immunomodulation/immunoregulation