Pasqualucci, Laura

Abstract Germinal center (GC)-derived lymphomas arise within a specialized immune ecosystem, yet whether malignant cells direct its remodeling remains unclear. Integrating genomic, transcriptomic, spatial, and functional analyses across follicular lymphoma and diffuse large B-cell lymphoma, we define a developmental framework linking malignant B-cell differentiation state to tumor microenvironment (TME) organization. Rather than segregating by histology, lymphomas align along a shared differentiation continuum in which proliferative dark zone (DZ) states associate with immune-depleted TMEs, whereas post-GC/memory B-cell (MBC) states drive inflamed but immunosuppressed TMEs. Spatial profiling reveals collapse of GC architecture and emergence of suppressive myeloid niches along this trajectory. Single-cell lineage reconstruction uncovers a DZ-to-MBC developmental axis underlying lymphoma evolution. Genetic reprogramming in vivo demonstrates that malignant differentiation state is sufficient to instruct TME remodeling. Collectively, these findings establish malignant differentiation state as a causal determinant of immune architecture across GC-derived lymphomas and identify lineage-directed immune reprogramming as a potential therapeutic strategy.

Abstract Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Although patients with FL have high response rates to therapy, most develop increasingly resistant disease. In addition, transformation into an aggressive lymphoma is associated with unfavorable outcomes. Many novel agents are under investigation, and early clinical data are encouraging. Aligning treatment with the underlying tumor biology and sequencing of therapies remain key clinical challenges. At the Lymphoma Research Foundation’s biannual 2024 Follicular Lymphoma Scientific Workshop, experts convened to discuss the role of chemotherapy in the context of new therapies, the impact of early progression on treatment sequencing, novel end points in clinical trials, disease biology and the tumor microenvironment, and new treatments on the horizon. This report focuses on updates in FL biology, first-line treatment, the role of progression of disease in 24 months, clinical trial design, and redefining cure in FL.

Abstract Twenty percent of patients with follicular lymphoma relapse early with poor outcomes; however, the molecular mechanisms underlying this aggressive behavior are unknown. Using a multiomic approach, we show that patients with follicular lymphoma with elevated IRF4 expression (IRF4hi) have increased transformation risk, dysregulated immune signaling, and a suppressive tumor microenvironment. Loss- and gain-of-function experiments in IRF4hi lymphoma cells, along with chromatin profiling, demonstrate that IRF4 impairs their interaction with T cells by repressing antigen presentation and co-receptor gene modules while promoting the expression of cytokines that antagonize T-follicular helper cell and regulatory T-cell functions. Additionally, IRF4 rewires tumor metabolism and restricts glucose availability to immune cells. Silencing of IRF4 inhibits tumor cell growth and restores immune surveillance mechanisms, thus representing a promising target for therapy. Our data suggest that IRF4hi lymphoma cells co-opt a developmental mechanism used to exit the germinal center response in promoting a more aggressive cancer via engagement of multiple immune-evasive mechanisms. Significance: We show that IRF4 controls immune and metabolic signaling with functional effects on the cross-talk between B and T cells, resulting in a suppressive immune microenvironment when IRF4 is overexpressed in lymphoma. This underscores the importance of IRF4 as a pivotal therapeutic target to restore antitumor immunosurveillance for IRF4-associated lymphomas.

Reprogramming of cellular metabolism is a hallmark of cancer leading to improved cellular fitness and providing a selective advantage to malignant cells. Recently, we found that follicular lymphoma (FL) patients with poor prognosis display increased transcriptional and protein expression of IRF4 (IRF4 high), altered immune signaling and an immunosuppressive tumor microenvironment (TME). Furthermore, high expression of IRF4 rewired B cell metabolism towards glycolysis ( Mondello P et al, Blood 2022;140 Suppl 1:168-169). However, it's unknown whether this metabolic reprogramming may be responsible in part for the impairment of immune cells in the TME, which also rely on glucose for their proliferation and function. Using RNA-seq on 88 FL patients, we found a negative correlation between IRF4-regulated genes associated with glycolysis and tumor infiltration by major immune cell subsets in IRF4 high compared to IRF4 low tumors ( Fig 1A), suggesting that IRF4-driven tumor glycolysis may reinforce an immune suppressed phenotype. Especially, we observed that ALDOA and GPI genes were inversely correlated with expression of CD4 + T cells (r=0.34, p=0.003; and r=0.38, p=0.015, respectively) and T reg cells (r=0.3, p=0.05; and r=0.35, p=0.027, respectively) in IRF4 high but not in IRF4 low tumors. As radioactive glucose is the cornerstone of PET assessment, we investigated the correlation between baseline SUV values and IRF4 expression. Remarkably, we found significantly higher levels of FDG uptake in FL IRF4 high compared to IRF4 low cases (p= 0.003), supporting the glycolytic features of Iymphoma expressing IRF4. To define the metabolic activity and function of lymphoma cells with different IRF4 status, we performed seahorse assay in TMD8 and SUDHL2 cells (lymphoma cells with IRF4 high expression) transfected with siIRF4 or siCtr for 72 hours. In line with the IRF4-dependence on glycolysis, TMD8 transfected with siCtr had a low baseline glycolytic ECAR, while the same value doubled after silencing IRF4. Notably, after adding oligomycin, which stimulates glycolysis, TMD8 control cells showed a lower glycolytic capability compared with those deprived of IRF4, probably due to the already maximal addiction to glycolysis ( Fig 1B left). We then investigated the glycolytic demand of germinal center (GC) B cells differently expressing IRF4. Using irf4 fl/fl mice ( Klein et al, Nat Immunol 2006) crossed with Cγ 1cre animals ( Cγ 1cre; irf4 fl/fl) to induce conditional deletion of irf4 in GC B cells, we tested ex-vivo sorted GC B cells from Cγ 1cre; irf4 fl/fl, Cγ 1cre; irf4 fl/+ and WT mice immunized for 10 days. This time point is critical for studying IRF4 as it corresponds to the GC reaction peak when GC B cells express elevated IRF4 levels promoting terminal differentiation to plasma cells. As expected, GC B cells minimally relied on glycolysis and showed a low ability to respond to oligomycin in WT mice ( Weiser et al, Nat Immunol 2020). However, GC B cells with partial or complete loss of irf4 demonstrated a superior ability to response to increased glycolytic demand ( Fig 1B right), further supporting the conclusion that IRF4 influences B cell dependency on glycolysis. As the malignant cell metabolism can alter nutrient availability in the TME, we explored if lymphoma cells differently expressing IRF4 may change glucose levels in the media and in turn impact T cells activity. We found low glucose and high lactate levels in the media of cell lines with IRF4 high expression, while the opposite was observed for those that were IRF4 low. We then cocultured primary human CD4 + T cells with TMD8 cells transfected with either siIRF4 or siCtr for 3 days at three different concentrations of D-glucose: high (22.2 mM, Gluc hi), normal (5.5 mM, Gluc nor) or absent (0 mM, Gluc nil). In line with our prior findings, coculture of B cells in which IRF4 was knocked out showed a significant decrease in abundance of T reg cells and an increase in T FH cells compared to control in presence of Gluc hi or Gluc nor. In contrast, there was no difference in the overall amount of either T cell subtype irrespective of IRF4 status with Gluc nil, supporting the prerequisite of microenvironmental glucose as primary fuel for T cell survival and function. In conclusion, IRF4 rewires the metabolism in B cells with profound functional implications on the TME. Our data suggest that glucose metabolism represents a functional vulnerability that might be exploited therapeutically.

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 irf4 fl/fl mice with conditional deletion of irf4 ( Klein et al, Nat Immunol 2006) and Cγ 1cre mice ( Cγ 1cre; irf4 fl/fl) to induce recombination in GC B cells. After 10 days of immunization (GC reaction peak), Cγ 1cre; irf4 fl/fl and Cγ 1cre; irf4 fl/+ GC B cells showed significant increase of HLA-DR and CD40 expression compared to WT. As expected, Cγ 1cre; irf4 fl/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 T FH cells and decrease of T reg cells ( Fig 1B). Notably, mice with loss of irf4 showed a significant decrease in exhausted T FH 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 T reg and T FH 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 Cγ 1cre; irf4 fl/fl, Cγ 1cre; irf4 fl/+ 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 Cγ 1cre; irf4 fl/fl and Cγ 1cre; irf4 fl/+ GC B cellscompared to WT,as indicated by decrease in CSFE expression . We confirmed an increase of T FH cells and a decrease of T reg 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.

Abstract With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with recognized variability in clinical outcome, genetic features, and cells of origin. To date, transcriptional profiling has been used to highlight similarities between DLBCL tumor cells and normal B-cell subtypes and associate genes and pathways with unfavorable outcome. Given the genetic heterogeneity in DLBCL, there are likely to be subsets of tumors with different pathogenetic mechanisms and possible treatment targets. To identify DLBCL subtypes that were sufficiently robust to be captured by multiple methods, we analyzed the profiles of 176 newly diagnosed DLBCLs using three different clustering algorithms (hierarchical clustering (HC), self-organizing maps (SOM), and probabilistic clustering (PC)), the top 5% of genes with the highest reproducibility across duplicate samples and largest variation across patient tumors, and a resampling-based method (consensus clustering) that automatically selects the most stable numbers of clusters with each algorithm. Three discrete subsets of DLBCLs -- “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” and “Host Response” (HR) -- were identified, characterized using gene set enrichment analysis and confirmed in an independent series of newly diagnosed DLBCLs with available array data. There was an association between cluster membership and examined genetic abnormalities in DLBCL. BCL2 translocations were more common in the OxPhos cluster whereas BCL6 translocations were more frequent in the BCR/proliferation cluster. Translocations of either type were uncommon in the HR cluster. Patients with HR DLBCLs were also significantly younger than those with OxPhos or BCR/proliferation tumors; HR patients also had a significantly higher incidence of splenic and BM involvement. The unique characteristics of HR tumors - fewer known genetic abnormalities and prominent host immune and inflammatory cell transcripts -prompted us to assess host immune cells in study tumors using morphologic and immunohistochemical approaches. HR DLBCLs contained significantly higher numbers of morphologically distinct CD2+/CD3+ tumor-infiltrating lymphocytes and interdigitating S100+/GILT+/CD1a-/CD123- dendritic cells. The HR cluster shared features of histologically defined T-cell/histiocyte-richBCL, including fewer known genetic abnormalities, younger age at presentation and frequent splenic and bone marrow involvement. The current study identifies tumor microenvironment and host inflammatory response as defining features in DLBCL, provides insights into the nature of the host immune response in a major DLBCL subtype and suggests rational treatment targets in newly identified tumor groups.