{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,24]],"date-time":"2026-01-24T02:08:31Z","timestamp":1769220511176,"version":"3.49.0"},"posted":{"date-parts":[[2024,11,4]]},"group-title":"Neuroscience","reference-count":53,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,11,4]],"date-time":"2024-11-04T00:00:00Z","timestamp":1730678400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,11,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease of the central nervous system caused by JC virus (JCV). Survival is dependent on early diagnosis and ability to re-establish anti-viral T cell immunity. Adoptive transfer of polyomavirus-specific T cells has shown promise; however, there are no readily available HLA-matched anti-viral T cells to facilitate rapid treatment.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Objective<\/jats:title>\n                  <jats:p>\n                    Identify epitopes of the JCV major capsid protein VP1 that elicit an immune response in the context of human leukocyte antigen allele A*02:01 (HLA-A2) and isolate cognate T cell receptors (TCRs) from healthy donors. Evaluate individual VP1-specific TCRs for their capacity to be expressed in T cells and clear JCV\n                    <jats:italic>in vitro<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    PBMCs from HLA-A2+ healthy donors were stimulated with peptide libraries tiled across the JCV VP1 protein. Multiple rounds of stimulation were performed to identify the antigens that induced the largest expansion and CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cell response (measured as INF\n                    <jats:italic>\u03b3<\/jats:italic>\n                    , TNF\n                    <jats:italic>\u03b1<\/jats:italic>\n                    , CD137, and CD69 expression). High-affinity, antigen-specific CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells were isolated based on intensity of tetramer binding for downstream single-cell TCR sequencing. Candidate TCRs were selected based on tetramer binding affinity and activation assays. Promising TCRs were introduced into the T cell genome via viral transduction for\n                    <jats:italic>in vitro<\/jats:italic>\n                    validation including peptide-pulsed K562 cells and astrocyte cells, and JCV-infected astrocytes.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    Four conserved JCV VP1 epitopes (amino acids 100-108, 251-259, 253-262, and 274-283) presented by HLA-A2 were identified. VP1(100-108) consistently elicited the highest level of IFN-\n                    <jats:italic>\u03b3<\/jats:italic>\n                    production from multiple donors and this peptide is in a highly conserved region of VP1. We next identified fourteen high avidity TCRs specific for VP1(100-108). When virally transduced into primary human T cells, seven of these TCRs demonstrated specific binding to VP1(100-108):HLA-A2 tetramers, and four showed increased IFN-\n                    <jats:italic>\u03b3<\/jats:italic>\n                    response when incubated with peptide. Primary CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells expressing two of these TCRs cleared both HLA-A2 positive K562 cells and HLA-A2 positive SVG astrocyte cell line presenting exogenously added VP1 peptide at a range of E:T ratios. In addition, both TCR-transduced T cell populations effectively lysed JCV-infected astrocytes.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>\n                    We identified JCV VP1 epitopes that are immunogenic in the context of HLA-A2 MHC-I, including epitopes that have not been previously described. The VP1(100-108) epitope was used to isolate HLA-A2-restricted TCRs. When cloned into primary human CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells, these TCRs recognized VP1 (100-108)-presenting targets, and the transduced T cells conferred cytotoxic activity and eliminated K562 and astrocyte cells displaying the VP1(100-108) peptide and not sham peptide, as well as JCV-infected astrocytes. Taken together, these data suggest that JCV VP1-specific TCRs could be appealing therapeutics for HLA-A2+ individuals with PML in whom intrinsic T cell immunity cannot be rescued.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2024.11.04.621904","type":"posted-content","created":{"date-parts":[[2024,11,4]],"date-time":"2024-11-04T23:20:23Z","timestamp":1730762423000},"source":"Crossref","is-referenced-by-count":1,"title":["Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1972-7301","authenticated-orcid":false,"given":"Sasha","family":"Gupta","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tijana","family":"Martinov","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ashley","family":"Thelen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Megumi","family":"Sunahara","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shwetha","family":"Mureli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angie","family":"Vazquez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Josiah","family":"Gerdts","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ravi","family":"Dandekar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Irene","family":"Cortese","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Camille","family":"Fouassier","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elaine","family":"Schanzer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fyodor D.","family":"Urnov","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander","family":"Marson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brian R.","family":"Shy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip D.","family":"Greenberg","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8705-5084","authenticated-orcid":false,"given":"Michael R.","family":"Wilson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024110800001397000_2024.11.04.621904v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(71)91777-6"},{"key":"2024110800001397000_2024.11.04.621904v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41582-020-00427-y"},{"key":"2024110800001397000_2024.11.04.621904v1.3","doi-asserted-by":"publisher","DOI":"10.1007\/s11481-010-9203-1"},{"key":"2024110800001397000_2024.11.04.621904v1.4","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.05031-11"},{"key":"2024110800001397000_2024.11.04.621904v1.5","doi-asserted-by":"crossref","unstructured":"McEntire, C.R.S. , et al., Characteristics of Progressive Multifocal Leukoencephalopathy Associated With Sarcoidosis Without Therapeutic Immune Suppression. 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