Mutations in CD58 and Other Immune System Related Genes in Hodgkin Lymphoma
Authors: Abdul Razak Fazlyn Reeny , Diepstra Arjan , Visser Lydia , Van den Berg Anke 
Published: October 2019

Abstract
Hodgkin Lymphoma (HL) is a B cell derived malignancy characterized by a minority of tumor cells, known as Hodgkin Reed-Sternberg (HRS) cells. The background is composed of a wide variety of inflammatory cells with T cells representing the largest population. Chemokines and cytokines produced by HRS cells and by the infiltrating cells shape the environment and provide proliferative and survival signals to the HRS cells. Despite this critical dependence on the microenvironment, HRS cells also need to apply mechanisms to escape from both antigen-dependent and innate immune responses. HRS cells have evolved multiple mechanisms to evade cytotoxic T cell (CTL) and natural killer (NK) cell mediated anti-tumor responses. These mechanisms include secretion of immune-suppressive factors (IL10, TGFβ and others), recruitment of regulatory and helper T cells, expression of PDL1 and CD95 and loss of HLA expression. Recent publications show that mutations in immune system related genes might represent a mechanism of HRS cells to evade detection by immune cells. The aim of this study was to validate whole exome sequencing results of seven HL cell lines focusing on immune system associated genes. We previously showed that B2M mutations affect the ATG start codon in L428 (heterozygous) and DEV (homozygous) cells. B2M mRNA levels were reduced in both cell lines as compared to L1236, whereas HLA-A, HLA-B and HLA-C mRNA levels were in the same range. Consistent with these findings we observed no membranous B2M and HLA class I expression by flow cytometry in the two cell lines with mutated B2M genes. In primary diagnostic HL tissue we showed lack of membranous B2M in 51% of the cases. We now studied two additional genes in more detail. CD58 gene mutations were observed in KMH2 and DEV cells. By manual inspection of the alignments using the Integrative Genomics Viewer (IGV), we also noticed a lack of reads of exons 1, 2 and 3 in SUPHD1. Heterozygous mutations and homozygous loss of exons 1-3 were confirmed for all three cell lines. CD58 mRNA levels were low or absent in SUPHD1 and KMH2 cells and normal in DEV. CD58 protein expression as determined by flow, western blot and IHC was low or absent in all 3 mutated HL cell lines in comparison to four cell lines with wild type CD58. Tumor cells of 36 primary HL cases with good treatment outcome showed a strong CD58 expression in all cases. As HL cell lines are derived from end stage HL patients, we next studied CD58 expression in relapsed HL patients. No or weak CD58 staining was observed in HRS cells in 6 out of 45 patients who experienced a relapse. Our results indicate that mutations in CD58 and loss of CD58 expression are common in HL derived cell lines and that loss of CD58 expression in tumor cells is restricted to relapsed HL patients. Heterozygous CSF2RB mutations in KMH2, SUPHD1, DEV and L1236 were validated by RNA-seq and Sanger-seq. As CSF2RB encodes the common β chain (CD131) shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, we also measured the expression of these 3 α chain receptors. We observed the same expression pattern between CD131 and CD116 (GM-CSF α receptor chain) in HL cell lines by flow cytometry suggesting that these mutations mainly affect the GM-CSF receptor. In conclusion, we show that mutations of immune system genes are common in HL. Deleterious mutations in B2M explain the lack of HLA class I expression, indicating that this genetic alteration is responsible for defective antigen presentation. Deleterious mutations or deletions of CD58 exons result in loss of CD58 protein expression. This will lead to loss of binding to CD2 expressed on T cells and will result in a defect in T cell adhesion and activation. Overall these results indicate that mutations are likely to contribute to the immune escape mechanisms applied by the HRS cells.

Disclosures
No relevant conflicts of interest to declare.