Induction of eye-derived tolerance does not depend on naturally occurring CD4+CD25+ T regulatory cells.

PURPOSE Regulatory CD4+ T cells (T regs) arise in the spleens of mice with anterior chamber-associated immune deviation (ACAID), an eye-derived tolerance evoked by injection of antigen into the ocular anterior chamber (AC). The current study was conducted to investigate the possibility that these T regs express CD25 and are derived from natural CD4+CD25+ T cells. METHODS Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGFbeta-treated antigen-presenting cells (APCs), and the expression of CD25 assayed by flow cytometry. OVA-specific ACAID T regs were obtained from the spleens of DO11.10 mice with ACAID to OVA. Immunomagnetic enrichment was used to sort out CD4+CD25+, and CD4+CD25- ACAID T cells before they were injected into OVA-immunized mice or examined for mRNA expression of the regulatory T-cell transcription factor Foxp3. In addition, before AC injection of OVA, systemic depletion of CD25+ T cells was performed with injections of anti-IL-2 receptor antibody into the mice. RESULTS OVA-specific T cells from DO11.10 mice expressed CD25 when exposed to OVA-pulsed, TGFbeta-treated APCs, even when the DO11.10 T cells were depleted of CD25+ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4+CD25+ or CD4+CD25- ACAID T cells. The CD4+CD25+, but not the CD4+CD25-, ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25+ cells. CONCLUSIONS Some of the CD4+ T regs of ACAID arise from CD25- precursors, and the induction of ACAID is not dependent on the presence of natural CD4+CD25+ T regs.