Identification of a Novel Function of B2M Involving T-Cell Exhaustion Via P-Selectin Ligand Upregulation in Diffuse Large B-Cell Lymphoma

• Published in: Blood 2024-11, Vol.144, p.331-331
• Main Authors: Takata, Katsuyoshi, IIoka, Hidekazu, Tomida, Shuta, Liu, Yuting, Okuda, Shujiro, Usuda, Hiroyuki, Kawasaki, Takashi, Kitadate, Akihiro, Takahashi, Naoto, Duns, Gerben, Sato, Yasuharu, Steidl, Christian, Ennishi, Daisuke
• Format: Article
• Language: English
• ISSN: 0006-4971
• DOI: 10.1182/blood-2024-202199

Background: Despite improvement of outcomes with current immuno-chemotherapies, approximately 40% of diffuse large B-cell lymphoma (DLBCL) patients suffer from recurrence or relapse, making it a refractory lymphoma. We previously reported that down-regulation of major histocompatibility complex (MHC) molecules contributes to a cold immune microenvironment and treatment resistance (Ennishi D & Takata K et al. Cancer Discov. 2019). However, the detailed mechanisms by which MHC reduction induces a cold immune microenvironment remain unclear. Here, we studied the functional consequences of loss of MHC class I and its major component beta-2-microglobulin (B2M) with a focus on the host immune microenvironment. Methods: We used the CRISPR-Cas9 gene editing system to knock out (KO) the B2M gene in two DLBCL-derived cell lines expressing MHC class I (Pfeiffer and SU-DHL-8). mRNA sequencing was performed and gene signatures that were commonly up- and down-regulated in both cell lines were identified. Concurrently, we conducted multicolor immunohistochemical (IHC) analysis using DLBCL patient samples (N=100) to examine the correlation between candidate molecules, MHC class I, and CD8+ T-cells. We also performed ligand stimulation assays and co-culture experiments in the generated isogeneic cell line systems, and generated an in vivo syngeneic mouse (A20 B2M KO) model to investigate antibody treatment. Results: Filtering the mRNA sequencing data (B2M-wt vs. heterozygous and homozygous KO) with P < 0.05 and a fold change > 1.5 revealed 383 up-regulated genes in Pfeiffer and 111 in SU-DHL-8, with four genes commonly up-regulated in both cell lines. Among these, the SELPLG (P selectin ligand) gene was the top upregulated gene. Additional B2M KO in other DLBCL cell lines (SU-DHL-4 and SU-DHL-10) confirmed the up-regulation of SELPLG. To explore mechanistic links between B2M and SELPLG, we performed gene set enrichment analysis (co-upregulated genes and pathways in B2M KO group), co-immunoprecipitation (co-IP), and ligand stimulation assays, and found that B2M binds to TNFRSF12A (TWEAKR) and up-regulates SELPLG via the NF-kB pathway. IHC of DLBCL patient samples showed an exclusive relationship between MHC class I and SELPLG expression in tumor cells (P < 0.001) without any links to DLBCL subtypes (GCB or non-GCB). Multi-color IHC revealed that SELPLG-positive samples had significantly more CD8+ PD1+ T-cells near tumor cells compared to SELPLG-negative samples, suggesting