Authors: Maryam Shahin; Neil Almeida, BA; Diego Carrera, MD; Erin Simonds, PhD; Payal Watchmaker, PhD; Hideho Okada, MD, PhD (Dayton, OH)

Low-grade gliomas are slow-growing primary brain tumors with potential to recur or progress to higher-grade tumors with a phenotype resistant to chemoradiation. Immunotherapy in the form of peptide vaccines poses a unique advantage, with the goal of eliciting an antigen-specific T cell response which could overcome current challenges in treating low-grade glioma. Currently, there is an ongoing clinical trial at UCSF (NCT02924038) which aims to use a cocktail of 11 glioma- associated peptides (IMA950 Vaccine Peptide) in patients with low-grade glioma. To assess anti-tumor response in low-grade glioma bearing patients undergoing peptide vaccine clinical trials, we aimed to develop an immune monitoring platform for high dimensional cellular analysis using mass cytometry (CyTOF). Peptide-MHC tetramers are tagged with heavy metal labels and used in combination with 30 different antibody markers to allow detailed phenotypic analysis of peptide-reactive T cells. After optimization of our protocol for tetramerization of biotinylated peptide-MHC monomers to avidin-fluorophore, we validated our protocol by comparing the NIH H3.3K27M tetramer and our H3.3K27M tetramer tested against cells transduced to express the H3.3K27M T cell receptor (Jurkat76CD8+siTCR+). Post validation, we stimulated human leukocyte antigen (HLA)-A2+ healthy donor derived PBMCs with two IMA950 peptides (FABP7 and BCAN) in vitro. Using the appropriate tetramer, we successfully identified FABP7 and BCAN reactive T cells and tetramerized our H3.3K27M peptide-MHC monomers to metals for utilization in a CyTOF platform. We developed a tetramer protocol which can identify specific T cell reactive populations that may be found in patients undergoing IMA950 Vaccine Peptide therapy and conjugated tetramers to metals for CyTOF. Next, we plan to tetramerize IMA950 Vaccine Peptide-MHC monomers to metals for utilization in a CyTOF platform to enable immune monitoring in patients. Furthermore, we aim to isolate peptide reactive T cell populations that stain with appropriate tetramer and sequence the T cell receptor.