Treg-expressed CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1 on antigen-presenting cells

概要

〔目的(Purpose)〕
Foxp3-expressing CD4十CD25+ regulatory T cells (Tregs) constitutively and highly express the immune checkpoint receptor cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose Treg-specific deficiency causes severe systemic autoimmunity. As a key mechanism of Treg-mediated suppression, Treg-expressed CTLA-4 downregulates the expression of CD80/CD86 costimulatory molecules on antigen-presenting cells (APCs). Accuinulating evidence also indicates that CD80 not only interacts, in trans, with CD28 and CTLA-4 expressed on T cells but also, in cis, with programmed death ligand-1(PD-L1)protein expressed by APCs. These cis-CD80/PD-Ll heterodimers on APCs limit the availability of free PD-L1, thereby interfering with the trans-binding between PD-L1 and its receptor PD-1 expressed on T cells. It can be asked then whether Treg-expressed CT LA-4 is able to control the availability of PD-L1 through modulating CD80 expression on APCs. Therefore, it needs to be determined how CTLA-4 precisely modifies the expression profile of APCs for the suppression of conventional T cells (Tconvs).

〔方法ならびに成績(Methods/Results)〕
Methods: In order to determine the role of the cytoplasmic tail portion of CTLA-4 in its cell-extrinsic Rmction, we used tailless (TL)CTLA-4 transgenic (TLC4Tg) mice, whose T cells expressed a mutant CTLA-4 protein without its cytoplasmic tail portion. For functional comparison with TL Tregs, CTLA-4 knockout (KO) and wild-type (WT) CTLA-4-expressing Tregs were isolated from female CTLA-4fiox/n°x, Foxp3,RES*Crc heterozygous, Rosa-RFP-reporter mice, which harbored both WT and KO Tregs without developing spontaneous autoimmunity. For tracking the CD80/CD86 molecules expressed on the dendritic cell (DC) surface following their interaction with Tregs, we generated three types of gene-transduced murine DC lines by using CD80-GFP or CD86-GFP fusion protein or GFP alone. Moreover, to examine whether CTLA-4-driven uptake of APC membrane proteins by Tregs might also occur in vivo, purified WT or KO fregs irom CD45.2 CRF mice, together with Tconvs from CD45.1 congenic mice, were transferred into syngeneic CD45.1 RAG2 KO mice for assessing the capture of CD45.1 protein by the transferred CD45.2+WT or KO Tregs. Meanwhile, in order to investigate the effect of CTLA-4-dependent trogocytosis on cis-CD80/PD-Ll heterodimers expressed by DCs, we cocultured these Tregs with freshly purified murine splenic DCs. For analysis, we labeled DCs first with l-l11A anti-PD-Ll monoclonal antibody (mAb). which was previously reported to detect total (CD80-bound and fi.ee) PD-L1 molecules, and then with 10F.9G2 PD-L1 mAb, which competes with CD80 for binding and stains free PD-L1, Results: Here we showed that Treg-expressed CTLA-4 facilitated Treg-APC conjugation and immune synapse formation. The immune synapses thus formed provided a stable platform whereby Tregs were able to deplete CD80/CD86 molecules on APCs by extracting them via CTLA-4-dependent trogocytosis. The depletion occurred even with Tregs solely expressing a mutant CTLA-4· form lacking the cytoplasmic portion required for its endocytosis. Thus, the tail portion of CTLA-4 is dispensable for CD80/CD86 downregulation on APCs and Treg suppressive function. The CTLA-4-dependent trogocytosis of CD80/CD86 also accelerated in vitro and in vivo passive transfer of other membrane proteins and lipid molecules from APCs to Tregs without their significant reduction on the APC surface. Furthermore, CD80 downregulation or blockade by Treg-expressed membrane CTLA-4 or soluble CTLA-4-immunoglobulin (CTLA-4-Ig), respectively, disrupted cis-CD80/ PD-L1 heterodimers and increased free PD-L1 on dendritic cells (DCs), expanding a phenotypically distinct population of CD801。free PD-Llhl DCs.

〔総括(Conclusion)〕
Tregs are able to inhibit the T-cell stimulatory activity of APCs by reducing their CD80/CD86 expression via CTLA-4-dependent trogocytosis. This CD80/CD86 reduction on APCs is able to exert dual suppressive effects on T-cell immune responses by limiting CD80/CD86 costimulation to naive T cells and by increasing free PD-L1 available for the inhibition of programmed death-1(PD-l)-expressing effector T cells. Blockade of CTLA-4 and PD-1/PD-L1 in combination may therefore synergistically hinder Treg-mediated immune suppression, thereby effectively enhancing immune responses including tumor immunity.