Recognition of synthetic polyanionic ligands underlies spontaneous reactivity of Vγ1 γδTCRs

Although gamma delta TCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several V gamma 1-containing TCRs, including the V gamma 1V delta 6 TCR of gamma delta NKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self-ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by V gamma 1V delta 6 and related TCRs, but not by TCRs from several other gamma delta or innate-like alpha beta T cells, and demonstrated that both gamma and delta chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo V gamma 1 gamma delta T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of V gamma 1V delta 6 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.