989 A luciferase-based method to assess antigen-specific T cell responses and antigen presentation to evaluate immunomodulatory checkpoints and therapeutics

• Published in: Journal for immunotherapy of cancer 2022-11, Vol.10 (Suppl 2), p.A1031-A1031
• Main Authors: Jimena Alvarez Freile, Qi, Yuzhu, Jacob, Lisa, Lobo, Maria Franceskin, Harm Jan Lourens, Huls, Gerwin, Bremer, Edwin
• Format: Article
• Language: English
• Subjects: Antigen presentation; Cytokines; Human papillomavirus; Immunotherapy; Lymphocytes; Peptides; T cell receptors
• ISSN: 2051-1426
• DOI: 10.1136/jitc-2022-SITC2022.0989

BackgroundTargeted reactivation of the immune system, e.g. using immune checkpoint inhibitors (ICI), has shown great potential in the treatment of cancer. However, in vitro tools to rapidly investigate the impact of checkpoints in the context of specific T cell receptor (TCR) activation as well therapeutic effects of ICI treatment are lacking. Here, we have developed a simple method using the human papilloma virus 16 (HPV-16) E7-peptide (HLA-A2*02:01) and corresponding antigen-specific NFAT-luciferase model to assess antigen presentation and antigen-specific T cell responses and evaluate the impact of immunosuppressive factors and therapeutics that target such factors.MethodsHPV-16_E711-20 peptide was used as model antigen.1 Jurkat NFAT-luciferase cell line or isolated primary T cells were modified with a previously reported TCR recognizing the E711-20 peptide.2 HLA-A2+/- cancer cells were pulsed and incubated with Jurkat/T cells, with and without the E711-20-TCR. To study E7 presentation endogenously, CaSki cells were used. For the luciferase-based assay, T cell activation was correlated with the luminescence read out. For primary T cells, CD25/CD69 expression (Flow Cytometry), cytokine production (ELISA) and clustering (microscopy) were evaluated. As proof of concept for monitoring immunomodulatory events, cells were also treated with TGF-beta or modified to overexpress inhibitory immune checkpoints (e.g., HLA-G, VISTA).ResultsUpon E711-20 pulsing of HLA-A2+ cells, an E:T ratio dependent increase in luminescence compared to non-pulsed cells (2-25 fold-increase) was observed upon coculture with Jurkat E711-20-TCR but not with parental Jurkat cells. Analogous experiments with CaSki yielded 30% increase in luminescence, demonstrating that the method is valid for measuring endogenous E711-20 processing and MHC presentation. Both HLA-A2+ pulsed cells and CaSki triggered specific T cell activation, illustrated by 5-20 fold-increase in MFI values for CD25 and CD69 expression and T cell clustering. For cytokine production, only the combination peptide: E711-20-TCR significantly triggered TNFa and IFNy production in HLA-A2+ pulsed cells, whereas for CaSki only higher IFNy production was observed. Overexpression of inhibitory molecules, such as HLA-G and VISTA, had a significant negative impact on TCR-induced luminescence compared to EV or non-treated cells. Moreover, treatment with immunosuppressive cytokines such as TGF-beta significantly impacted on luminescenc