P-194 Use of CEACAM5 Synthetic Peptides to Induce Activation of CD8+ Suppressor Regulatory T Cells

Abstract

CD8+ regulatory T (TrE) cells can be activated by intestinal epithelial cells (IECs) through the complex formed by the non-classical I molecule, CD1d and gp180 both expressed on IECs. mAb B9, an anti-gp180 Ab, can block this induction. We have previously demonstrated sequence homology between gp180 and CEACAM5. Furthermore, CEACAM5 has properties attributed to gp180, such as CD1d and CD8α binding and activation of CD8-associated LcK. Furthermore, the N70, 81A CEACAM5 mutant, which is devoid of the sugar bridge, appeared to lose the capacity to induce the phosphorylation of Lck when compared to CEACAM5. We suggest that unique set of interactions between CEACAM5, CD1d and CD8α can render CD1d more class I-like, facilitating antigen presentation by CD1d to T cells and the activation of CD8+ Tregs. Moreover, we showed that CEACAM5-activated CD8+ T cells acquire suppressive function. The aim of this study is to determine which amino acids of CEACAM5 are functional in the activation of CD8+ T cells in order to design synthetic peptides with immunoregulatory function. We generated an overlapping peptide library of CEACAM5 N domain to identify the amino acids that contribute to its functionality (19 peptides with offset of 5). We tested the ability of the entire pool of peptides, 3 combinations of peptides and each single peptide to phosphorylate CD8 associated p56Lck kinase both in human isolated CD8+ T cells and in a murine T cell line transfected with human CD8α, 3G8, using In-cell Western Blot assay. OKT8, an anti-CD8 antibody, was used as positive control. The entire collection of peptides induced phosphorylation of Lck compared to OKT8 and to the full length CEACAM5 in CD8+ T cells but not in CD4+ T cells. When pooled peptides were tested, pool 3>>2 (pool 2: G30-T55; pool 3: P66-Y107) resulted in the activation of CD8+ T cells. The peptides 12 (T56-N70) and P14 (I66-Q80) within pool 3 induces phosphorylation of LcK to the greatest extent. Our data suggest the crucial role of the N domain sugar bridge site in CEACAM5 binding to CD8α and in the activation of CD8+ Tregs. Furthermore, we identified the regions of the N domain where the functional amino acid residues are located. These finding are critical in designing peptides that can be used to induce CD8+ TrE cells as in Crohn’s disease in which a defect of these cells have been described.