T cell receptors (TCRs) have been classically divided into two categories: αβ and γδ TCRs that are expressed on the surface of the eponymous lineages of T cells. Despite numerous functional differences, the architecture of both TCR lineages is very similar. They are formed by two polypeptide chains with two domains each: one constant (C) domain, proximal to the cell surface, and one variable (V) domain, directly involved in antigen (Ag) recognition. Usually the Ag-binding site corresponds to the surface formed by three Complementarity-Determining Regions (CDR) from each chain. Recently, it has been shown that the genomes of marsupials and monotremes contain another cluster of genes encoding a novel type of a TCR-like chain, the μ chain. Surprisingly, genomic DNA seemed to suggest that, upon somatic recombination, the resulting polypeptide could contain two variable and one constant domains.
Using single cell RNA sequencing we identified a subset of T cells in the spleen of the opossum (Monodelphis domestica) that express the μ chain. Simultaneously, we detected TCRγ chain transcripts in all the TCRμ+ cells, suggesting that γ and μ chains co-expressed in a novel lineage of cells, γμ T cells. Using the sequences of paired γ and μ transcripts we generated recombinant γμTCRs and determined their crystal structures. We have confirmed that both chains form a heterodimeric T cell receptor with an unusual asymmetrical architecture where the Vγ-Cγ domains contact Vμj-Cμ domains and the additional V domain (Vμ) sits unpaired atop of the assembly. The Vμ domain shares molecular similarities with a single-chain antibody VH domain and carries a long flexible CDR3μ loop that represents the only segment that significantly varies between different γμTCRs identified, pointing towards its function as the sole Ag recognition determinant.