Comparative Evaluation of T-cell Receptors in Experimental Glioma-Draining Lymph Nodes

Background. Glioblastomas, the most common primary malignant brain tumors, are considered immunologically cold malignancies due to growth in an immune sanctuary site. While peptide vaccines have shown to generate intra-tumoral antigen-specific T cells, the identification of these tumor-specific T cells is challenging and requires detailed analyses of tumor tissue. Several studies have shown that CNS antigens may be transported via lymphatic drainage to cervical lymph nodes, where antigen-specific T-cell responses can be generated. Therefore, we investigated whether glioma-draining lymph nodes (TDLN) may constitute a reservoir of tumor-reactive T cells. Methods. We addressed our hypothesis by flow cytometric analyses of chicken ovalbumin (OVA)-specific CD8(+) T cells as well as T-cell receptor beta (TCR ss) next-generation-sequencing (TCR ss-NGS) of T cells from tumor tissue, TDLN, spleen, and inguinal lymph nodes harvested from experimental mouse GL261 glioma models. Results. Longitudinal dextramer-based assessment of specific CD8(+) T cells from TDLN did not show tumor model antigen reactivity. Unbiased immunogenomic analysis revealed a low overlap of TCR ss sequences from glioma-infiltrating CD8(+) T cells between mice. Enrichment scores, calculated by the ratio of productive frequencies of the different TCR ss-CDR3 amino- acid (aa) rearrangements of CD8(+) T cells derived from tumor, TDLN, inguinal lymph nodes, and spleen demonstrated a higher proportion of tumor-associated TCR in the spleen compared to TDLN. Conclusions. In experimental glioblastoma, our data did not provide evidence that glioma-draining cervical lymph nodes are a robust reservoir for spontaneous glioma-specific T cells highlighting the requirement for detailed analyses of glioma-infiltrating T cells for the discovery of tumor-specificTCR.