Introduction: Little is known about the characteristics of the CD4+ T cells residing in close proximity to the tumor cells in classical Hodgkin lymphoma (cHL). We recently established that these CD4+ T cells interact with tumor cells by binding of CD2 to CD58 and T cell receptor to HLA class II in vitro and in situ. Formation of this immunological synapse results in strong T cell activation under normal conditions, but the CD4+ T cells in cHL are only weakly activated and typically lack expression of the activation marker CD26. The aim of this study was to further characterize these T cells.
Methods: CD4+CD26- and CD4+CD26+ T cell subsets were sorted from 19 cHL lymph node derived cell suspensions (tumor cells HLA class II positive) and analyzed by RNA sequencing and T cell receptor variable gene segment usage. In addition, co-expression of genes of interest was investigated at the single cell level using previously generated single-cell RNA sequencing (scRNA-seq) data and at the protein level by immunohistochemistry.
Results: Gene set variation analysis showed an enrichment of memory Treg, Th17 and T follicular helper cell gene signatures in CD4+CD26- T cells, while naïve and Th1/17 gene cell signatures were enriched in CD4+CD26+ T cells. Although CD4+CD26- T cells displayed an antigen experienced phenotype, the T cell receptor variable gene segment usage was polyclonal and did not differ from CD4+CD26+ T cells, indicating lack of clonal expansion. 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) with a moderate to high expression level were chosen for subsequent co-expression analysis using scRNA-seq data. This revealed that the majority of CD4+CD26- T cells expressed TOX2 either alone or in combination with any of the other selected genes. Protein expression of TOX and TOX2, transcription factors crucial for the acquisition of an exhausted T cell phenotype, was accentuated in T cells that physically interact with tumor cells. More than 50% of these rosetting T cells were positive for TOX in 63% and TOX2 in 79% of cHL cases.
Conclusion: CD4+ T cells residing in close proximity to cHL tumor cells are polyclonal, antigen experienced and exhausted. We propose that TOX and TOX2, transcription factors known to induce expression of a variety of immune checkpoints, are attractive therapeutic targets for cHL.
Johanna Veldman, Jessica Rodrigues Plaça, Lauren Chong, Martijn Terpstra, Mirjam Mastik, Léon C. van Kempen, Tomohiro Aoki, Christian Steidl, Anke van den Berg, Lydia Visser, Arjan Diepstra