A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools
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) a...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-01-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/15/3/574 |
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| author | Meytal Dror Levinsky Baruch Brenner Michal Yalon Zohar Levi Zvi Livneh Zoya Cohen Tamar Paz-Elizur Rachel Grossman Zvi Ram Ilan Volovitz |
| author_facet | Meytal Dror Levinsky Baruch Brenner Michal Yalon Zohar Levi Zvi Livneh Zoya Cohen Tamar Paz-Elizur Rachel Grossman Zvi Ram Ilan Volovitz |
| author_sort | Meytal Dror Levinsky |
| collection | DOAJ |
| description | 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/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05–0.2%). 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 IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 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. |
| first_indexed | 2024-03-11T09:51:38Z |
| format | Article |
| id | doaj.art-fc4dec3fa4864a59b8a01538f538dc14 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-11T09:51:38Z |
| publishDate | 2023-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-fc4dec3fa4864a59b8a01538f538dc142023-11-16T16:14:10ZengMDPI AGCancers2072-66942023-01-0115357410.3390/cancers15030574A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring ToolsMeytal Dror Levinsky0Baruch Brenner1Michal Yalon2Zohar Levi3Zvi Livneh4Zoya Cohen5Tamar Paz-Elizur6Rachel Grossman7Zvi Ram8Ilan Volovitz9The Cancer Immunotherapy Laboratory, Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, IsraelThe Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, IsraelThe Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, IsraelThe Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, IsraelThe Biomolecular Sciences Department, The Weizmann Institute of Science, Rehovot 7610001, IsraelThe Sackler Faculty of Medicine, The Tel Aviv University, Tel Aviv 6997801, IsraelThe Biomolecular Sciences Department, The Weizmann Institute of Science, Rehovot 7610001, IsraelThe Neurosurgery Department, The Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, IsraelThe Neurosurgery Department, The Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, IsraelThe Cancer Immunotherapy Laboratory, Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, IsraelPersonalized 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/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05–0.2%). 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 IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 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.https://www.mdpi.com/2072-6694/15/3/574cancerpeptide vaccineRNA vaccinepersonalized cancer vaccinecancer testis antigensneoantigens |
| spellingShingle | Meytal Dror Levinsky Baruch Brenner Michal Yalon Zohar Levi Zvi Livneh Zoya Cohen Tamar Paz-Elizur Rachel Grossman Zvi Ram Ilan Volovitz A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools Cancers cancer peptide vaccine RNA vaccine personalized cancer vaccine cancer testis antigens neoantigens |
| title | A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools |
| title_full | A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools |
| title_fullStr | A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools |
| title_full_unstemmed | A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools |
| title_short | A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools |
| title_sort | highly sensitive flow cytometric approach to detect rare antigen specific t cells development and comparison to standard monitoring tools |
| topic | cancer peptide vaccine RNA vaccine personalized cancer vaccine cancer testis antigens neoantigens |
| url | https://www.mdpi.com/2072-6694/15/3/574 |
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