{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:21:44Z","timestamp":1785410504341,"version":"3.56.0"},"posted":{"date-parts":[[2025,12,9]]},"group-title":"Immunology","reference-count":25,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T00:00:00Z","timestamp":1765238400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["P50CA196530"],"award-info":[{"award-number":["P50CA196530"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["K12CA215110"],"award-info":[{"award-number":["K12CA215110"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["K08CA270191"],"award-info":[{"award-number":["K08CA270191"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000005","name":"United States Department of Defense","doi-asserted-by":"crossref","award":["KC230193\/HT9425&#8208;24&#8208;1&#8208;0759"],"award-info":[{"award-number":["KC230193\/HT9425&#8208;24&#8208;1&#8208;0759"]}],"id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/027327e78","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) is approved for the treatment of advanced melanoma but is limited by the need for patients to undergo surgical tumor resection. Malignant pleural effusions (MPE) may represent a more accessible source of tumor-reactive T cells. Here, we characterize the cellular composition as well as the transcriptional and functional properties of T cells from MPE compared with pulmonary metastasis and blood from a patient with melanoma.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    The immune cellular composition was immunophenotyped by high-dimensional flow cytometry from synchronously collected MPE, a lung metastasis, and blood from a patient with metastatic melanoma. Sorted CD3\n                    <jats:sup>+<\/jats:sup>\n                    T cells were profiled by single-cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq). TCR reactivity to autologous tumor was evaluated through\n                    <jats:italic>in vitro<\/jats:italic>\n                    activation assays with TCR-transduced Jurkat and autologous cancer cells. The killing capacity of\n                    <jats:italic>ex vivo<\/jats:italic>\n                    expanded T cells of autologous cancer cells was assessed through\n                    <jats:italic>in vitro<\/jats:italic>\n                    cytotoxicity assays.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    MPE had higher proportions of CD45\n                    <jats:sup>+<\/jats:sup>\n                    immune cells and CD3\n                    <jats:sup>+<\/jats:sup>\n                    T cells (70.5% vs 50%) compared with tumor and was enriched for effector CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells, CCR7\n                    <jats:sup>\u2212<\/jats:sup>\n                    CD45RA\n                    <jats:sup>\u2212<\/jats:sup>\n                    effector memory CD4\n                    <jats:sup>+<\/jats:sup>\n                    T cells, and quiescent CD25\n                    <jats:sup>high<\/jats:sup>\n                    CD127\n                    <jats:sup>low<\/jats:sup>\n                    regulatory CD4\n                    <jats:sup>+<\/jats:sup>\n                    T cells. MPE T cells exhibited lower levels of co-inhibitory receptors (PD-1, LAG-3, TIGIT, TIM-3) expression relative to tumor. ScRNA-seq showed enrichment of NK-like effector CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cells in MPE. Pseudotime analysis indicated that MPE T cells were less exhausted than tumor T cells. The clonal repertoire of MPE and tumor highly overlapped, including 62.2% of predicted neoantigen-specific (NeoTCR) clonotypes. Notably, clonally-related NeoTCR T cells in MPE exhibited higher cytotoxic and stemness, and lower exhaustion signatures compared with sister clones in the tumor. Two of four selected NeoTCR clonotypes transduced in Jurkat cells demonstrated MHC class I-restricted reactivity in co-culture with autologous cancer cells. MPE T cells also readily expanded in the presence of high-dose IL-2 and demonstrated MHC class I-dependent killing of autologous cancer cells.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>MPE harbors polyclonal, tumor-reactive T cells with lower features of terminal exhaustion and higher cytotoxic potential relative to tumor T cells. MPE may therefore serve as a more accessible source for TIL therapy.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.05.692662","type":"posted-content","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T01:40:26Z","timestamp":1765330826000},"source":"Crossref","is-referenced-by-count":0,"title":["Phenotypic and functional characterization of tumor-reactive T cells in malignant pleural effusions"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5638-1151","authenticated-orcid":false,"given":"Noam S.","family":"Gaiger","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jack","family":"Coburn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9576-9589","authenticated-orcid":false,"given":"Mark N.","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heng-Le","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gavitt A.","family":"Woodard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4932-9873","authenticated-orcid":false,"given":"Harriet M.","family":"Kluger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1948-3711","authenticated-orcid":false,"given":"David A.","family":"Schoenfeld","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8764-8384","authenticated-orcid":false,"given":"Benjamin Y.","family":"Lu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025121202550827000_2025.12.05.692662v1.1","doi-asserted-by":"publisher","DOI":"10.1186\/1479-5876-10-169"},{"key":"2025121202550827000_2025.12.05.692662v1.2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2210233"},{"key":"2025121202550827000_2025.12.05.692662v1.3","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-15-1879"},{"key":"2025121202550827000_2025.12.05.692662v1.4","doi-asserted-by":"publisher","DOI":"10.1097\/CJI.0b013e31819c8bda"},{"issue":"2","key":"2025121202550827000_2025.12.05.692662v1.5","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1038\/d41573-025-00001-5","article-title":"2024 FDA approvals","volume":"24","year":"2025","journal-title":"Nat Rev Drug Discov"},{"key":"2025121202550827000_2025.12.05.692662v1.6","doi-asserted-by":"crossref","first-page":"e431608","DOI":"10.1200\/EDBK_431608","article-title":"Adoptive Cell Therapy for Solid Tumors: Current Status in Melanoma and Next-Generation Therapies","volume":"44","year":"2024","journal-title":"Am Soc Clin Oncol Educ Book"},{"issue":"1","key":"2025121202550827000_2025.12.05.692662v1.7","doi-asserted-by":"crossref","first-page":"2371556","DOI":"10.1080\/2162402X.2024.2371556","article-title":"Candidate tumor-specific CD8(+) T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis","volume":"13","year":"2024","journal-title":"Oncoimmunology"},{"key":"2025121202550827000_2025.12.05.692662v1.8","doi-asserted-by":"crossref","unstructured":"Mahmood K , Wang HM , Ji ZC , Giovacchini CX , Wahidi MM , Dorry M , et al. 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