A. S. Montoya, M. Frank, P. Jiang, M. Zhang, H. Nie, E. Bontekoe, J. K. Slone, L. L. Posta, K. L. Singampalli, D. D. Dixit, W. Xiao, T. Cascone, J. Zhang, E. Dondossola, L. E. Kavraki, M. Gillison, P. L. Wenzel, P. B. Lillehoj, J. V. Heymach, and A. Reuben, “216 Unbiased microfluidic isolation of antigen-specific T cells in solid tumors,” Journal for ImmunoTherapy of Cancer, vol. 12, no. Suppl 2, pp. A248–A248, 2024.
Background Tumor antigen-specific CD8+ T cells offer a compelling method for therapeutic targeting of solid tumors. However, efforts to identify tumor antigen-specific T cells have been hampered by limitations such as the need to pre-select antigens, inherently biasing such analyses. Here, we propose a method called ATTACH (Assessment of T cells Tethered to Antigen Class I/II Histocompatibility) to enrich for tumor antigen-specific T cells using tumor cells as de facto ‘tetramer pools’, thereby allowing for rapid, versatile, and unbiased isolation of antigen-specific T cells.Methods Lewis lung carcinoma cells expressing the ovalbumin antigen (LLC-OVA) were seeded overnight into microfluidic slides. The following day, OT-I alone or in combination with C57BL/6 CD8 splenocytes were added and a low, constant rate of fluidic shear stress was applied to remove nonspecific/weakly bound T cells. Fluorescent microscopy was used to quantify the retention of T cells followed by recovery for downstream functional analyses. Experiments were repeated using human antigens and TCR-engineered cells.Results Enumeration of fluorescent cells pre- and post-ATTACH confirmed a 5-fold antigen-dependent enrichment of OT-I in presence versus absence of OVA (23.25% vs 4.36%, p=0.0004). CD8 co-receptor blockade confirmed retention was mediated through TCR/pMHC binding (8.79% vs 2.86% p=0.075). With a 1:1 ratio of OT-I to C57BL/6 CD8+ T cells, a 2-fold enrichment of antigen-specific T cells (16.40% vs. 7.7%, p=0.0151) was achieved. Furthermore, enriched antigen-specific populations recovered using ATTACH exhibited a 12-fold increase in IFN-g secretion (p=0.022) and 3-fold higher cytotoxic potential as measured by cleaved caspase-3/7. Moreover, experiments using human TCR-engineered T cells showed a 5-fold enrichment for antigen-specific T cells (25.89% vs 5.48%, p=0.058).Conclusions Here, we demonstrate the feasibility of enriching for antigen-specific T cells recognizing solid tumor antigens using a microfluidic platform. Additional platform optimization and validation experiments are underway.
Publisher: http://dx.doi.org/10.1136/jitc-2024-SITC2024.0216