{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T11:34:34Z","timestamp":1784633674134,"version":"3.55.0"},"reference-count":60,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,1,17]],"date-time":"2023-01-17T00:00:00Z","timestamp":1673913600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Israel Science Foundation","award":["1652\/20"],"award-info":[{"award-number":["1652\/20"]}]},{"name":"Chaya Charitable Trust","award":["1652\/20"],"award-info":[{"award-number":["1652\/20"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and\/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3\/CD4\/CD8\/IFN\u03b3\/Viability+CD14+CD19 (dump)] demonstrate background IFN\u03b3 secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05\u20130.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFN\u03b3-positive CD4 T cell frequencies decreased to 0.019% \u00b1 0.028% and CD8 T cells to 0.009% \u00b1 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.<\/jats:p>","DOI":"10.3390\/cancers15030574","type":"journal-article","created":{"date-parts":[[2023,1,18]],"date-time":"2023-01-18T02:31:11Z","timestamp":1674009071000},"page":"574","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools"],"prefix":"10.3390","volume":"15","author":[{"given":"Meytal","family":"Dror Levinsky","sequence":"first","affiliation":[{"name":"The Cancer Immunotherapy Laboratory, Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel"},{"name":"The Neurosurgery Department, The Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel"},{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baruch","family":"Brenner","sequence":"additional","affiliation":[{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"The Institute of Oncology, Davidoff Cancer Center, The Rabin Medical Center, Beilinson Hospital, Petach Tikva 4941492, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michal","family":"Yalon","sequence":"additional","affiliation":[{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"The Pediatric Hematology-Oncology Department, Safra Children\u2019s Hospital, Sheba Medical Center, Ramat Gan 52621, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zohar","family":"Levi","sequence":"additional","affiliation":[{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"The Gastroenterology Department; The Rabin Medical Center, Petach Tikva 4941492, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zvi","family":"Livneh","sequence":"additional","affiliation":[{"name":"The Biomolecular Sciences Department, The Weizmann Institute of Science, Rehovot 7610001, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoya","family":"Cohen","sequence":"additional","affiliation":[{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"},{"name":"The Felsenstein Medical Research Center, The Rabin Medical Center, Petach Tikva 4941492, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamar","family":"Paz-Elizur","sequence":"additional","affiliation":[{"name":"The Biomolecular Sciences Department, The Weizmann Institute of Science, Rehovot 7610001, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7741-375X","authenticated-orcid":false,"given":"Rachel","family":"Grossman","sequence":"additional","affiliation":[{"name":"The Neurosurgery Department, The Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel"},{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zvi","family":"Ram","sequence":"additional","affiliation":[{"name":"The Neurosurgery Department, The Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel"},{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5776-4755","authenticated-orcid":false,"given":"Ilan","family":"Volovitz","sequence":"additional","affiliation":[{"name":"The Cancer Immunotherapy Laboratory, Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel"},{"name":"The Neurosurgery Department, The Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel"},{"name":"The Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1038\/nature22991","article-title":"An Immunogenic Personal Neoantigen Vaccine for Patients with Melanoma","volume":"547","author":"Ott","year":"2017","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1038\/nature23003","article-title":"Personalized RNA Mutanome Vaccines Mobilize Poly-Specific Therapeutic Immunity against Cancer","volume":"547","author":"Sahin","year":"2017","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1255","DOI":"10.1074\/mcp.RA119.001524","article-title":"Identification of Tumor Antigens among the HLA Peptidomes of Glioblastoma Tumors and Plasma","volume":"18","author":"Shraibman","year":"2019","journal-title":"Mol. 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