155 Sub-clonal antigenic heterogeneity mediates immune evasion in low-grade gliomas

<h3>Background</h3> While low mutational burden and an immunosuppressive microenvironment contribute to immunotherapeutic resistance, the intratumoral heterogeneity (ITH) of low-grade glioma (LGGs) plays a significant role in targeting tumor-specific antigens (TSAs) that would lead to a durable clinical response. While ITH has been heavily investigated in various cancer types, the effect of ITH on the antigenic landscape and immune landscape has largely been unexplored in LGGs. <h3>Methods</h3> We performed whole genome and RNA sequencing on 10 maximally-distanced and spatially-mapped biopsies captured from seven Grade II astrocytoma patients. <i>In silico</i> prediction of TSAs was used to generate a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neoepitopes. This library allowed us to caputure neoantigen-reactive CD8+ T-cells derived from patients’ peripheral blood. Single-cell TCR sequencing allowed us to identify the neoantigen-reactive TCR clonotypes, which were transduced subsequently into Jurkat76 cells and CD8+ T-cells for functional and cytotoxic validation. <h3>Results</h3> Unsupervised hierarchical clustering of RNA-sequencing data revealed two distinct immune clusters across all spatially-mapped astrocytoma samples corresponding to high (immune-hot) and low (immune-cold) levels of immune infiltration. Immune-hot sites tended to be proximal to one another than to immune-cold regions, suggesting that immune infiltration patterns are spatially localized. Interestingly, mutations initially identified in immune-hot biopsies were significantly less prevalent at recurrence when compared to those from immune-cold sites. Differential gene expression analysis of the KEGG Antigen Processing and Presentation gene set revealed significantly higher expression levels of HLA alleles in immune-hot sites when compared to their immune-cold counterparts. Notably, significantly more TSA candidates with a favorable affinity towards their patient-specific HLAs (HLAthena HLA:peptide presentation score &gt; 0.90) were identified in intratumoral sites with greater immune infiltration. Altogether, these results suggest that the immune system exerts a selective pressure that potentially eliminates tumor cells with highly immunogenic mutations. From one patient’s (P375) PBMCs, we identified and sequenced 5 CD8+ T-cell clones recognizing three TSAs derived from mutations in <i>PRMT5</i>, <i>MRPL46</i>, and <i>TP53</i>. TSA-specific T-cell receptor (TCR)-expressing Jurkat76 cells were cultured with HLA-transduced antigen-presenting cells pulsed with each peptide. Jurkat76 cells specific against mutant <i>PRMT5</i> demonstrated a strong TSA-specific IL-2, NFAT, AP-1, and NFκB immune response. <h3>Conclusions</h3> Our study suggests that heterogeneous HLA presentation and antigenic ‘quality’ enable the immune system to sculpt the tumor mutational landscape. Furthermore, we identified a TCR clone that successfully recognizes and induces an immune response against mutant-<i>PRMT5</i>, demonstrating a feasible methodology to develop personalized T-cell-based immunotherapies for LGG patients.