ACAID CD8+ T Regulatory Cells Express Foxp3 and CD94

Abstract

Purpose: : Eye–derived peripheral tolerance following the anterior chamber injection of antigen (anterior chamber–associated immune deviation, ACAID) generates CD8⁺ T regulatory cells (Tregs) that suppress the expression of immunity in the periphery. In– vitro generated ACAID CD8⁺ Tregs have similar suppressive functions in vivo and express CD103. The purpose of this study was to further characterize the in–vitro generated ACAID CD8⁺ Tregs.

Methods: : OT–1 spleen T cells were co–cultured (3 days) with TGF–ß2–treated OVA–pulsed APC to generate ACAID CD8⁺ Tregs or with control APC to generate non–Tregs. OT–1 Tregs and non–Tregs were immunostained for molecules (CD8, CD103, CD25, CTLA4, CD28, CD94, Foxp3 and GITR) known to have critical regulatory functions in other Tregs.

Results: : The expression of CD103 was 66.2±2.8% and 29.1±5.4% in Tregs and non–Tregs, respectively. There was no significant difference in the expression of CTLA4 and CD28. But interestingly, the expression of CD94 and Foxp3 were enhanced in OT–1Tregs. On the other hand the expression of CD25 and GITR were down regulated. In Tregs and non–Tregs respectively: CTLA4 0.28% vs. 0.81%; CD28 1.00% vs. 1.37%; CD94 8.04% vs. 1.30%; Foxp3 88.1% vs. 0.33%; CD25 2.24% vs. 21.2% and GITR 67.8% vs. 91.9%.

Conclusions: : The ACAID CD8⁺ Tregs can be identified by CD103, Foxp3 and CD94 expression. Thus, ACAID OT–1CD8⁺ Tregs express different cell surface and intracellular molecules from non–Tregs. The ACAID CD8⁺ Tregs also differ in its expression of known regulatory molecule from published studies on CD4⁺ CD25⁺ Tregs. The prediction is that the ACAID efferent Tregs use novel regulatory pathways to suppress CD4⁺ effector cells and these pathways differ from those used by afferent Tregs.

Keywords: ACAID • flow cytometry • immunomodulation/immunoregulation