Recognition of Butyrophilin-family Members by gamma-delta T Cells

Abstract

Human gamma-delta (γδ) T cells can respond rapidly with cytotoxic responses to pathogenic infections and malignant cells. Although they are found at low numbers in peripheral blood, these might expand and constitute up to half of the total circulating T cells after infection. Most systemic γδ T cells expressed a recombined heterodimeric T-cell receptor (TCR) with genes of the variable (V) γ9 and δ2 loci, termed Vγ9Vδ2 T cells. Opposed to conventional αβ T cells that recognise processed peptides on major histocompatibility complex (MHC) class I and II and elicit delayed adaptive responses, γδ T cells are innate-like T cells that recognise non-peptidic antigens akin to mucosal-associated invariant T (MAIT) or MHC-like lipid-presenting molecule (CD1)-restricted T cells which recognise vitamin-B derivates or lipids, respectively. The most interesting feature of Vγ9Vδ2 T cells is that they recognise phosphorylated antigens (aka phosphoantigens) which are essential for life including bacteria and particularly abundant in cancer cells. Little is known how γδ T cells recognise phosphoantigens, even though, a few putative ligands have been described. <br /> Butyrophilins (BTNs) are a family of transmembrane molecules capable of regulating the immune activity of innate-like γδ T cells. They dimerise to constitute complexes, some of which may interact with germline-encoding regions of the γδ T cell receptors in murine or human species. For instance, mouse butyrophilin-like (BTNL) molecules shape the Vγ7+ γδ T-cell compartment in the intestine, while human BTNL counterparts selectively activate Vγ4+ γδ T cells of the colon. Here, tetrameric γδTCR clonotype probes are used for a genetic screen to identify a previously unknown molecular ligand essential for recognition of phosphorylated antigens by γδ T cells. This screen identifies BTN2A1 and subsequent experiments elucidate this protein contacts with the Vγ9 domain irrespective of the Vδ recombined segment and is essential to confer reactivity to phosphoantigens together with BTN3A1. Thus, BTN2A1 and BTN3A1 constitute a functional complex of which we found both intra- cellular domains are critically important in maintaining an active conformation. Whereas internal BTN3A1 PRY/SPRY (B30.2) motif senses the antigen, the BTN2A1 intracellular domain appears fundamental to retain association of the complex. Mutagenic alanine screens reveal a dual-ligand binding site for the Vγ9Vδ2 TCR, where conserved residues of the Vγ9 domain bind to BTN2A1 and several residues located at the complementarity-determining regions (CDRs) might react to a putative molecule of the BTN3A family. Thus, this work proposes a phosphoantigen-reactive γδ T cells recognise a dual-ligand binding complex where BTN2A1 contacts the Vγ9 domain and BTN3A1 is a phosphoantigen sensor molecule that plausibly induces the molecular switch necessary to induce immune responses. <br /> Lastly, the influence of tumour infiltrated phosphoantigen-reactive γδ T cells in renal carcinogenic tumour patient-derived organoids (PDO) samples is examined and their relevance in healing disease assessed in comparison to previous clinical studies. These results are of vital importance to better understand the potential of γδ T cells in prospective medical applications.