Abstract A17: The immune checkpoints TIGIT and PD-1 are markedly upregulated in NLPHL compared to classical Hodgkin Lymphoma

Hodgkin lymphoma (HL) comprises two distinct disease entities based on clinical, morphologic and genotypic characteristics. Relative to classical Hodgkin Lymphoma (cHL), nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is rare, and its tumor microenvironment (TME) is very poorly characterized. With the exception of rituximab, there are no targeted treatments nor advances in the treatment of NLPHL for decades. Unlike cHL, the utility of immune-checkpoint blockade (ICB) has not been evaluated in NLPHL. Diagnostic samples were collected from 49 NLPHL patients from 4 Australian centres and compared with stage-matched cHL patients (and with normal lymph nodes). An integrative transcriptomic, proteomic, T-cell clonal and functional analysis was performed to enable a comparison of the composition of the TME and its contribution to immune-evasion in NLPHL with cHL. 730 cancer-immune related genes were digitally quantified. Relative to cHL, gene set enrichment analysis identified T-cell receptor (TCR) and immune-checkpoint signaling pathway dysregulation in NLPHL. Most striking was prominent differential expression of the immune-transcriptome, particularly enrichment for programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) in NLPHL versus cHL. These were also over expressed compared to normal lymph nodes. Consistent with this, there was also upregulation of numerous T-cell markers (CD247, CD3D, GZMK, CD28, EOMES) in NLPHL. In contrast, immunosuppressive macrophage (CD163, CD36, CD68, COLEC12, MARCO) and regulatory T-cell genes (FOXP3) were higher in cHL. Importantly, PD-L1 and CD155 (respective ligands for PD-1 and TIGIT) were expressed at the surface of NLPHL and cHL malignant B-cells. Multispectral immunofluorescent microscopy showed intratumoral TIGIT+CD4+ and PD-1+CD4+ T-cells were markedly increased in NLPHL versus cHL and localised within NLPHL follicles. Expanded populations of intratumoral CD4+ T-cell clones were predominantly PD-1+ and frequently also TIGIT+. Multi-parameter flow cytometry and dimensionality reduction was used to establish the distribution of immune checkpoints within circulating T-cell subsets. PD-1+TIGIT+CD4+ T-cells were raised in circulating Treg, TH1 and TH2 subsets in NLPHL versus cHL, and PD-1+TIGIT+ TH2 T-cells displayed raised levels of the exhaustion marker EOMES, collectively indicating systemic T-cell dysfunction. To functionally demonstrate the utility of ICB to stimulate T-cells, an assay using cHL and/