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Numerous studies reported that T cell hybridomas expressing several V\u03b31-containing TCRs, including the V\u03b31V\u03b46 TCR of \u03b3\u03b4NKT 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\u03b31V\u03b46 and related TCRs, but not by TCRs from several other \u03b3\u03b4 or innate-like \u03b1\u03b2 T cells, and demonstrated that both \u03b3 and \u03b4 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\u03b31 \u03b3\u03b4 T cells. Taken together, these results explain long-standing observations on the spontaneous reactivity of V\u03b31V\u03b46 TCR and demonstrate an unexpected antigen presentation-independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.<\/jats:p>","DOI":"10.1002\/jlb.2ma1219-392r","type":"journal-article","created":{"date-parts":[[2020,1,14]],"date-time":"2020-01-14T06:34:07Z","timestamp":1578983647000},"page":"1033-1044","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Recognition of synthetic polyanionic ligands underlies \u201cspontaneous\u201d reactivity of V\u03b31 \u03b3\u03b4TCRs"],"prefix":"10.1093","volume":"107","author":[{"given":"Josefine","family":"Dunst","sequence":"first","affiliation":[{"name":"Department of Medicine, Division of Immunology and Allergy, Karolinska Institutet, Karolinska University Hospital , Stockholm, Sweden"},{"name":"Center for Molecular Medicine, Karolinska Institutet , Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vassilis","family":"Glaros","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Immunology and Allergy, Karolinska Institutet, Karolinska University Hospital , Stockholm, Sweden"},{"name":"Center for Molecular Medicine, Karolinska Institutet , Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lukas","family":"Englmaier","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Immunology and Allergy, Karolinska Institutet, Karolinska University Hospital , Stockholm, Sweden"},{"name":"Center for Molecular Medicine, Karolinska Institutet , Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick A.","family":"Sandoz","sequence":"additional","affiliation":[{"name":"Department of Applied Physics, KTH Royal Institute of Technology , Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bj\u00f6rn","family":"\u00d6nfelt","sequence":"additional","affiliation":[{"name":"Department of Applied Physics, KTH Royal Institute of Technology , Stockholm, Sweden"},{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institute , Solna, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"Kisielow","sequence":"additional","affiliation":[{"name":"Institute of Molecular Health Sciences, ETH , Zurich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6672-1914","authenticated-orcid":false,"given":"Taras","family":"Kreslavsky","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Immunology and Allergy, Karolinska Institutet, Karolinska University Hospital , Stockholm, Sweden"},{"name":"Center for Molecular Medicine, Karolinska Institutet , Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2020,1,13]]},"reference":[{"key":"2023012312521385500_jlb10532-bib-0001","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1146\/annurev-immunol-042617-053028","article-title":"Evolution of alternative adaptive immune systems in vertebrates","volume":"36","author":"Boehm","year":"2018","journal-title":"Annu Rev Immunol"},{"key":"2023012312521385500_jlb10532-bib-0002","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1038\/s41590-018-0304-y","article-title":"\u03b3\u03b4 TCR ligands: the quest to solve a 500-million-year-old mystery","volume":"20","author":"Willcox","year":"2019","journal-title":"Nat Immunol"},{"key":"2023012312521385500_jlb10532-bib-0003","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.smim.2010.04.001","article-title":"gammadeltaTCR ligands and lineage 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