B7 co-stimulation antagonizes RORγt⁺ regulatory T cells and tolerance to gut microbiota

Introduction/Rationale: Regulatory T (Treg) cells recognizing dietary or microbiota-derived antigens express RORγt and are essential for intestinal immune tolerance. A recent paradigm-shifting finding revealed that these cells require major histocompatibility complex class II (MHCII) on RORγt⁺ antigen-presenting cells (APCs) to provide signal one. Here, we evaluated signal two and found that broad blockade of co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) unexpectedly provoked the expansion of microbiota-specific RORγt⁺ Treg cells.

Methods: Cutting-edge transgenic, knockout, and adoptive-transfer mouse models were employed to define how the induction of RORγt⁺ Treg cells is regulated by MHCII (signal one) from RORγt⁺ APCs and B7 co-stimulation (signal two) from non-RORγt⁺ APCs to maintain immune tolerance to gut microbiota. Flow cytometry, ex vivo T cell differentiation, histology, and colitis models induced by Helicobacter hepaticus plus anti–IL-10R antibody, combined with co-stimulation blockade and CTLA4-Ig treatment, were integrated to dissect the interplay between RORγt⁺ APCs and RORγt⁺ Tregs.

Results: Mechanistically, B7/CD28-dependent Akt signaling antagonizes the core transcriptional program of RORγt⁺ Treg cells through the Forkhead box protein O1 (Foxo1) pathway. The expansion of RORγt⁺ Tregs was recapitulated by CTLA4-Ig but required MHCII (signal one) on RORγt⁺ APCs together with inhibition of B7 co-stimulation (signal two) from non-RORγt⁺ APCs. CTLA4-Ig treatment during pathobiont colonization and IL-10R blockade restored microbiota-specific RORγt⁺ Treg generation and protected against experimental colitis, reducing IL-17⁺ and TNF⁺ T cells.

Conclusion: These results mechanistically define that B7 co-stimulation limits microbiota-specific RORγt⁺ Treg cells, while identifying CTLA4-Ig as an approach to enhance immune tolerance and reduce inflammatory cytokines in the intestine when acting in concert with RORγt⁺ APCs, provoking a novel therapeutic strategy for patients with chronic intestinal inflammation.