CD4+ T cells in the tumor micro-environment of classical Hodgkin lymphoma are TOX and TOX2 expressing, antigen experienced, polyclonal and exhausted

The CD4+ T cells in the tumor micro-environment (TME) of classical Hodgkin lymphoma (cHL) tumor cells are characterized by lack of the activation marker CD26. In this study we characterized sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. Gene set variation analysis revealed that CD4+CD26- T cells have characteristics of antigen experienced T cells, with enrichment of memory Treg, Th17 and Tfh and lack of naïve T cell gene signatures compared to CD4+CD26+ T cells. Interestingly, CD4+CD26- T cells displayed a polyclonal T cell receptor variable gene segment usage that did not differ from CD4+CD26+ T cells. Differential gene expression analysis revealed a significant enrichment of 100 genes in CD4+CD26- T cells. Seven genes (TOX, TOX2, CXCL13, CTTN, PDCD1, CD200 and NFIA) were chosen for subsequent co-expression analysis using previously generated single-cell RNA sequencing data. The majority of CD4+CD26- T cells expressed TOX2 alone or together with one or more of the other selected genes. Immunohistochemistry revealed staining of more than 50% of rosetting T cells for TOX in 63% and TOX2 in 79% cHL cases, suggesting that direct interaction of these T cells with tumor cells induces their expression. In conclusion, CD4+CD26- T cells in the TME of cHL are antigen experienced and polyclonal with an exhausted phenotype. We propose that TOX and TOX2, two transcription factors crucial for the acquisition of this exhausted phenotype are attractive therapeutic targets in cHL.