PURPOSE. Regulatory CD4 ϩ 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 ϩ CD25 ϩ T cells. METHODS. Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGF␤-treated antigen-presenting

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... (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 ϩ CD25 ϩ , and CD4 ϩ CD25 Ϫ 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 ϩ 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, TGF␤-treated APCs, even when the DO11.10 T cells were depleted of CD25 ϩ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4 ϩ CD25 ϩ or CD4 ϩ CD25 Ϫ ACAID T cells. The CD4 ϩ CD25 ϩ , but not the CD4 ϩ CD25 Ϫ , ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25 ϩ cells. CONCLUSIONS. Some of the CD4 ϩ T regs of ACAID arise from CD25 Ϫ precursors, and the induction of ACAID is not dependent on the presence of natural CD4 ϩ CD25 ϩ T regs. (Invest Ophthalmol Vis Sci.