ACAID Tolerogenic APC Induce Two Types Of CD4+ Treg Cells By Two Different Mechanisms

Abstract

Purpose: : ACAID induction, in vivo or in vitro, generates three types of Tregs: efferent CD8⁺, afferent CD4⁺CD25⁺ and CD4⁺CD25^-. Previously, we showed CD8⁺ Treg cells required F4/80 protein expression on the APC and iNKT cells for their development. The purpose of this study was to determine if the generation of ACAID induced afferent CD4⁺ Treg cells subpopulations were also dependent on the described ACAID mechanisms.

Methods: : ACAID tolerogenic APC (Tol APC) were generated from C57BL/6, PD-L1 KO, or F4/80 KO by overnight culture of peritoneal exudate cells (PEC) with TGFβ2 and Ovalbumin (OVA). Spleen cells from primed OVA tg (OTII) mice, Jα281 KO or F4/80 KO mice were depleted of natural T regs by incubation with CD25 antibody (PC61.5). The remaining natural T reg-depleted cells were co-cultured (5D) with OVA pulsed-Tol APC. Following culture the T cells were stained for select surface markers (CD4, CD25, LAP-1, FoxP3, PD-L1) and analysed by flow cytometry. The efferent suppressor function of the CD25⁺ or CD25^- CD4⁺T cells was tested in a Local Adoptive Transfer (LAT) assay and afferent suppressor function was tested in a footpad assay and proliferation and assessed by bromodeoxyuridine (BrdU) incorporation into the draining lymph nodes.

Results: : We observed that post ACAID culture, there were two distinct populations of CD4⁺ T reg cells. ACAID induced CD4⁺CD25^- Tregs were LAP-1⁺ FoxP3^- and PD-L1^lo and exhibited only afferent suppressor function. CD4⁺CD25⁺ Tregs were LAP-1^- FoxP3⁺ and PD-L1^hi and exhibited both afferent and efferent suppressor function. Moreover, ACAID induced CD4⁺CD25⁺ Tregs required PD-L1 and not F4/80KO and iNKT cells.

Conclusions: : Thus the ACAID Tol APC induces two populations of T reg cells by two different mechanisms. The CD4⁺CD25^- Treg cells are dependent on F4/80 protein, iNKT cells, but not PD-L1 for their generation. The CD4⁺CD25⁺ Treg cells are generated by the activation of the TCR in the presence of TGFβ and PD-L1⁺ APC and supports the notion that PD-L1 is involved in the generation of inducible Treg cells within immune privileged sites where TGFβ is high.This research was supported in part by NIH grant: EY011983, EY016476

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