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4142 IRF4 Expression in Germinal Center and Lymphoma B Cells Regulates Antigen-Dependent Immune Response

Program: Oral and Poster Abstracts
Session: 603. Lymphoid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research, Lymphomas, non-Hodgkin lymphoma, B Cell lymphoma, Diseases, Lymphoid Malignancies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Patrizia Mondello, MD, PhD, MSc1, Prithviraj Mukherjee1*, Geoffrey Nelson2*, Surendra Dasari, PhD3*, Christine Hachfeld1*, Vaishali Bhardwaj, PhD1, Xinyi Tang1*, Zhi-Zhang Yang1*, Zhiquan Wang, PhD1, Megan Weivoda, PhD1, Mark Shlomchik4*, Laura Pasqualucci, MD5*, Karen Adelman2* and Stephen M Ansell, MD, PhD1

1Division of Hematology, Mayo Clinic, Rochester, MN
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Cambridge, MA
3Department of Quantitative Health Sciences, Mayo Clinic, Rochester, MN
4Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA
5Institute for Cancer Genetics, Columbia University, New York, NY

A substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. We showed that this unfavorable group displays increased expression of IRF4, dysregulated immune signaling and an immunosuppressive microenvironment. At the molecular level, IRF4 controls expression of antigen presentation and immune molecules (e.g. HLA-DR, CD40, PDL1). Furthermore, silencing of IRF4 triggers anti-lymphoma immunity (Mondello P et al, Blood 2022;140 Suppl 1:168-169). However, little is known on the molecular mechanisms whereby IRF4 controls the crosstalk between B and T cells.

To address this question, first we performed RNA-seq, ATAC-seq and CUT&TAG of active (H3K27Ac, H3K4me3) and repressive (H3K27me3, H3K4me1) histone marks in TMD8 and HBL1 (lymphoma cells with high IRF4 expression) transfected with two pairs of siIRF4 or siCtr for 72 hours. Gene expression revealed a broad change in transcription, with 432 upregulated genes and 136 downregulated genes in siIRF4 compared to siCtr. Notably, the genes with increased expression following IRF4 silencing coded for antigen presentation, immune molecules and cytokines (e.g. HLA-DR, CD86, IL6, IL10). Accordingly, we observed significant enrichment of antigen presentation, interferon and immune response signatures (Fig 1A). Integrating RNA-seq with chromatin accessibility showed a significant shift towards transcriptional upregulation at genes nearest to upregulated ATAC peaks, with significant enrichment at enhancers (n=771). Conversely, transcriptional suppression was enriched at enhancers (n=2,458) of downregulated peaks. k-means analyses of all union peaks, generated by merging ATAC with CUT&TAG peaks, revealed a significant gain of H3K4me3, with concordant loss of H3K4me1 at enhancers with upregulated gene expression. In contrast, H3K4me3 was severely decreased at suppressed peaks. TF motif analysis revealed an enrichment for PU.1 and E2A at IRF4-bound regions, suggesting their cooperation in regulating immune signaling.

Since antigen presentation and other immune molecules are critical components of the immune synapse, we explored the role of IRF4 on the interaction between germinal center (GC) B and T cells by crossing irf4fl/fl mice with conditional deletion of irf4 (Klein et al, Nat Immunol 2006) and 1cre mice (1cre;irf4fl/fl) to induce recombination in GC B cells. After 10 days of immunization (GC reaction peak), 1cre;irf4fl/fl and 1cre;irf4fl/+ GC B cells showed significant increase of HLA-DR and CD40 expression compared to WT. As expected, 1cre;irf4fl/fl mice displayed a significantly skewed light-zone:dark-zone ratio in favor of increased dark-zone, which are the cells that most reflect the action of BCL6, with concordant suppression of plasma cells. Additionally, we observed a significant increase of CD4+ T cells after partial or complete deletion of irf4, with upregulation of TFH cells and decrease of Treg cells (Fig 1B). Notably, mice with loss of irf4 showed a significant decrease in exhausted TFH cells. To test if this effect was dependent on MHC:TCR interaction, we cocultured CD4+ T cells with TMD8 cells transfected with two siIRF4 or siCtr in presence of blocking antibodies for MHC class I, MHC class II or both. Blocking one or the other MHC class I or II partially rescued the IRF4 effect on Treg and TFH cells respectively as well as reduced anti-lymphoma cytotoxicity. Further experiments blocking IRF4-dependent IL6 and IL10 identified these two cytokines as responsible for the additional effects on T cells. To confirm this as an antigen-dependent immune response we coculture sorted GC B cells from NP-OVA-immunized 1cre;irf4fl/fl, 1cre;irf4fl/+ and WT mice with CSFE-labeled OVA-specific OT-II CD4+ T cells at a ratio of 2:1 in an organoid system for 5 days. We observed induced antigen specific proliferation of CD4+ T cells in the presence of 1cre;irf4fl/fl and 1cre;irf4fl/+ GC B cells compared to WT, as indicated by decrease in CSFE expression. We confirmed an increase of TFH cells and a decrease of Treg cells, supporting the conclusion that IRF4 directly controls the antigen-dependent immune response.

In summary, IRF4 expression in GC and lymphoma B cells regulates immune signaling with PU.1 and E2A, thereby modulating antigen-dependent immune responses with potential implications for lymphomagenesis. Future studies investigating targeting of IRF4 in lymphoma are warranted.

Disclosures: Ansell: ADC Therapeutics, Affimed, Bristol-Myers Squibb Company, Pfizer Inc, Regeneron Pharmaceuticals Inc, Seagen Inc, Takeda Pharmaceuticals USA Inc.: Other: Contracted Research.

*signifies non-member of ASH