A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

Overview

Journal Cancers (Basel)

Publisher MDPI

Specialty Oncology

Date 2023 Feb 11

PMID 36765532

Authors

Meytal Dror Levinsky

Baruch Brenner

Michal Yalon

Zohar Levi

Zvi Livneh

Zoya Cohen

Tamar Paz-Elizur

Rachel Grossman

Zvi Ram

Ilan Volovitz

Affiliations

Soon will be listed here.

Abstract

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.

References

Lamoreaux L, Roederer M, Koup R . Intracellular cytokine optimization and standard operating procedure. Nat Protoc. 2007; 1(3):1507-16. DOI: 10.1038/nprot.2006.268. View

Resop R, Uittenbogaart C . Human T-Cell Development and Thymic Egress: An Infectious Disease Perspective. For Immunopathol Dis Therap. 2017; 6(1-2):33-49. PMC: 5489135. DOI: 10.1615/ForumImmunDisTher.2015014226. View

Sahin U, Derhovanessian E, Miller M, Kloke B, Simon P, Lower M . Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature. 2017; 547(7662):222-226. DOI: 10.1038/nature23003. View

Boon T, Coulie P, Van den Eynde B, van der Bruggen P . Human T cell responses against melanoma. Annu Rev Immunol. 2006; 24:175-208. DOI: 10.1146/annurev.immunol.24.021605.090733. View

Graves A, Padilla M, Hokey D . OMIP-022: Comprehensive assessment of antigen-specific human T-cell functionality and memory. Cytometry A. 2014; 85(7):576-9. PMC: 4231567. DOI: 10.1002/cyto.a.22478. View

Tripathi S, Lahesmaa R . Transcriptional and epigenetic regulation of T-helper lineage specification. Immunol Rev. 2014; 261(1):62-83. PMC: 4255756. DOI: 10.1111/imr.12204. View

Chakrabarty S, Snyder J, Shen J, Azmi H, Hu P, Chen Q . Human CD14hi monocytes and myeloid dendritic cells provide a cell contact-dependent costimulatory signal for early CD40 ligand expression. Blood. 2010; 117(5):1585-94. PMC: 3318775. DOI: 10.1182/blood-2008-01-130252. View

Wu T, Womersley H, Wang J, Scolnick J, Cheow L . Time-resolved assessment of single-cell protein secretion by sequencing. Nat Methods. 2023; 20(5):723-734. DOI: 10.1038/s41592-023-01841-y. View

Schoenbrunn A, Frentsch M, Kohler S, Keye J, Dooms H, Moewes B . A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg. J Immunol. 2012; 189(12):5985-94. DOI: 10.4049/jimmunol.1201090. View

10.

Neidleman J, Luo X, McGregor M, Xie G, Murray V, Greene W . mRNA vaccine-induced T cells respond identically to SARS-CoV-2 variants of concern but differ in longevity and homing properties depending on prior infection status. Elife. 2021; 10. PMC: 8545397. DOI: 10.7554/eLife.72619. View

11.

Shraibman B, Barnea E, Melamed Kadosh D, Haimovich Y, Slobodin G, Rosner I . Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma. Mol Cell Proteomics. 2019; 18(6):1255-1268. PMC: 6553928. DOI: 10.1074/mcp.RA119.001524. View

12.

Karlsson A, Martin J, Younger S, Bredt B, Epling L, Ronquillo R . Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells. J Immunol Methods. 2003; 283(1-2):141-53. DOI: 10.1016/j.jim.2003.09.001. View

13.

Janetzki S, Britten C, Kalos M, Levitsky H, Maecker H, Melief C . "MIATA"-minimal information about T cell assays. Immunity. 2009; 31(4):527-8. PMC: 3762500. DOI: 10.1016/j.immuni.2009.09.007. View

14.

Lee H, Nam K, Seo S, Kim Y, Kang H, Kwon B . 4-1BB cross-linking enhances the survival and cell cycle progression of CD4 T lymphocytes. Cell Immunol. 2003; 223(2):143-50. DOI: 10.1016/s0008-8749(03)00169-2. View

15.

Wimmers F, Aarntzen E, Duiveman-deBoer T, Figdor C, Jacobs J, Tel J . Long-lasting multifunctional CD8 T cell responses in end-stage melanoma patients can be induced by dendritic cell vaccination. Oncoimmunology. 2016; 5(1):e1067745. PMC: 4760336. DOI: 10.1080/2162402X.2015.1067745. View

16.

Perfetto S, Chattopadhyay P, Lamoreaux L, Nguyen R, Ambrozak D, Koup R . Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods. 2006; 313(1-2):199-208. DOI: 10.1016/j.jim.2006.04.007. View

17.

Keskin D, Anandappa A, Sun J, Tirosh I, Mathewson N, Li S . Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial. Nature. 2018; 565(7738):234-239. PMC: 6546179. DOI: 10.1038/s41586-018-0792-9. View

18.

Meng X, Sun X, Liu Z, He Y . A novel era of cancer/testis antigen in cancer immunotherapy. Int Immunopharmacol. 2021; 98:107889. DOI: 10.1016/j.intimp.2021.107889. View

19.

Spijkerman R, Hesselink L, Hellebrekers P, Vrisekoop N, Hietbrink F, Leenen L . Automated flow cytometry enables high performance point-of-care analysis of leukocyte phenotypes. J Immunol Methods. 2019; 474:112646. DOI: 10.1016/j.jim.2019.112646. View

20.

Pereira B, Chin S, Rueda O, Vollan H, Provenzano E, Bardwell H . The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes. Nat Commun. 2016; 7:11479. PMC: 4866047. DOI: 10.1038/ncomms11479. View