Background Antigen-specific T-cell responses play a crucial role in tumor immunology.^{1 2} Assessing T-cell cytolytic potential in vitro is a first step in developing clinically relevant drugs that target T-cell biology. Target antigen presentation can drive T-cell cytolytic functions; however, the development of antigen-specific T cell in vitro assays is often complicated by assay run time, throughput, and variability of data.³ Here, we aim to develop and optimize a high-throughput protocol for maximizing antigen-specific T-cell cytolytic activity to provide a platform to measure the effects of drug treatment on immune responses.

Methods Melanoma-associated antigen recognized by T cells (MART-1) or a control scrambled peptide was added to HCT116 colon cancer or A375 malignant melanoma cells at increasing concentrations and time points before they were plated onto 96-well flat-bottom plates. MART-1–specific CD8+ T cells⁴ were unstimulated or prestimulated overnight with 20 ng/mL interleukin 2 (IL-2), IL-15, or both IL-2 and IL-15, and added to the 96-well plates. Cocultures were incubated for 24, 48, and 72 hours prior to endpoint analysis. To assess cytolytic potential, cytokines in the supernatant (granulocyte-macrophage colony-stimulating factor, granzyme A, granzyme B, interferon γ [IFN-γ], IL-2, perforin, and tumor necrosis factor α [TNF-α]) were quantified using the U-PLEX CAR-T Cell Combo 2 assay.⁵ Target cell viability was assessed by flow cytometry using annexin V and 7-aminoactinomycin D. The assay was miniaturized into 384-well plates using established parameters after reproducible 96-well plate results were obtained.

Results Optimization assays showed that overnight MART-1 pulsing of A375 cells at 1000 ng/mL provided the best window of MART-1–specific CD8+ T-cell cytolytic activity compared with control-pulsed target cells. Granzyme B, IFN-γ, and TNF-α levels increased at 24 hours and decreased with longer incubations. Compared with unstimulated MART-1–specific CD8+ T cells, prestimulation with IL-2, IL-15, or both IL-2 and IL-15 increased the assay window; prestimulation with IL-2 showed the highest activity. Flow cytometry analysis of target cell viability successfully recapitulated cytokine results, showing an increase in apoptosis and death at optimal conditions. Analysis of key cytokines showed the same trends in 384-well plates as findings observed in the 96-well plates.

Conclusions We successfully developed and optimized an antigen-specific T-cell killing assay suitable for high-throughput (96- and 384-well) analysis. This assay can be utilized for screening effects of immuno-oncology and inflammation and autoimmunity targets on CD8+ T cell–mediated cytolytic activity.

Acknowledgements Thanks to Bihui Melidosian for input into early assay development and James Kearns for providing cell lines.

References

1. Huuhtanen J, Chen L, Jokinen E, et al. Evolution and modulation of antigen-specific T cell responses in melanoma patients. Nat Commun 2022;13:5988.

2. Pittet M, Valmori D, Dunbar P, et al. High frequencies of naive Melan-A/MART-1–specific CD8+ T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J Exp Med 1999;190:705–716.

3. Phetsouphanh C, Zaunders J, Kelleher AD. Detecting antigen-specific T cell responses: from bulk populations to single cells. Int J Mol Sci 2015;16:18878–18893.

4. Charles River Antigen-specific T Cells [https://www.criver.com/products-services/cell-sourcing/human-peripheral-blood/antigen-specific-t-cells?region=3601].

5. U-PLEX CAR-T Cell Combo 2 [https://www.mesoscale.com/en/products/u-plex-car-t-cell-combo-2-human-sector-1-pl-k15600k/].