Abstract LB-190: Single-cell polyfunctionality of circulating CD4+ T cells correlates with T cell receptor repertoire diversity of PBMCs, indicating potential synergistic activity and peripheral biomarker predictive of efficacy of intratumor injection of the toll-like receptor 4 agonist G100
Authors: Mackay Sean , Chen Jon , Patino Jeremy , Seo Y. David , Pollack Seth , Zhou Jing 
Published: August 2019

Abstract
Predictive biomarker plays a crucial role in identifying cancer patients who might benefit from a therapy and facilitating a personalized medication. Polyfunctional T cells (co-secretion of 2+ proteins per cell) measured by single-cell IsoCode chip proteomics was demonstrated to predict patient response to CAR-T therapy. Herein, we utilized the IsoCode chip technology to study the efficacy of G100 on eliciting systemic polyfunctional T cells and the association of T cell polyfunctionality with the diversity of T cell receptor (TCR) repertoire in patients with soft tissue sarcomas (STS). PBMC were collected pre- and post-treatment from patients with metastatic STS treated with intratumoral (IT) G100. CD4+ and CD8+ T cells were enriched by microbeads, stimulated with anti-CD3/CD28 at 37°C, 5% CO2 for 24 hours and loaded into an IsoCode chip containing ~12,000 microchambers pre-patterned with a complete copy of a 32-plex antibody array. After 16-hour incubation, protein secretions were analyzed from ~1000 single cells; polyfunctional profile was evaluated across 5 groups: Effector: Granzyme B, TNF-α, IFN-γ, MIP-1α, Perforin, TNF-β; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGF-β1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β. Polyfunctional strength index (PSI) was calculated as the percentage of polyfunctional cells multiplied by the intensities of secreted proteins. TCR deep sequencing of PBMCs was performed to evaluate the TCR repertoire diversity. Both CD4+ and CD8+ T cells had heterogeneous polyfunctional profiles pre- and post-treatment. CD4+ T cells showed a marked increase in polyfunctionality while CD8+ T cells exhibited a polyfunctional decrease after treatment. A greater variety of proteins associated with antitumor immunity was detected in CD4+ T cells post treatment. TNF-α, IL-8, Granzyme B, IL-5, MIP-1β and sCD137 primarily drove the enhanced PSI of CD4+ T cells. More importantly, the pre-treatment CD4 PSI positively correlated with the TCR diversity of PBMCs (P = 0.0185). Interestingly, the functional subgroups of effector, stimulatory and regulatory secretions, but not chemoattractive and inflammatory secretions, contributed to the significant association between pretherapy CD4 PSI and PBMC diversity. Single-cell multiplexed proteomics sensitively identifies circulating polyfunctional T cells induced by IT injection of G100 in patients with STS. The pre-treatment PSI of blood CD4+ T cells correlates with TCR diversity, providing a potential peripheral metric to predict patient response to G100 for the development of more effective therapeutics.
Citation Format: Sean Mackay, Jon Chen, Jeremy Patino, Y. David Seo, Seth Pollack, Jing Zhou. Single-cell polyfunctionality of circulating CD4+ T cells correlates with T cell receptor repertoire diversity of PBMCs, indicating potential synergistic activity and peripheral biomarker predictive of efficacy of intratumor injection of the toll-like receptor 4 agonist G100 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-190.