Tang, Xinyi

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 T follicular helper (TFH) cells are a subset of CD4+ T cells that reside within the follicles of secondary lymphoid organs and play an essential role in the germinal center (GC) reaction. To examine the role TFH cells in follicular lymphoma (FL), we first characterized TFH cells using single cell analysis with the high-throughput technologies of mass cytometry (CyTOF) and the Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). Clustering analysis revealed that TFH cells form subsets with unique phenotypes, and the expression of many common markers including CXCR5, ICOS, IL-21, TOX2, CD57, GZMK, XCL1, XCL2, CXCL13 and CD40L varies on TFH subsets. We found that TFH cells are phenotypically more heterogenous in FL than benign tonsil tissues by both proteomic (n=82) and transcriptomic (n=8) profiling. Each FL patient showed different dominant TFH subsets, in contrast to tonsil donors which were highly homogenous. Some TFH subsets were only present in FL and absent in tonsil tissues, suggesting the existence of FL-specific TFH cells. Cytokines (IL-2, IL12, IL-17 and IL-21) and activated B cells upregulated TFH markers including PD-1, CXCR5 and IL-21 to induce the development of TFH subsets such as IL-21+ or IL-21+IFN-g+ TFH cells, which was inhibited by rapamycin, an immunosuppressor. We observed that the TFH cell frequency significantly correlated with the number of lymphoma B cells. Futhermore, while TFH cells as a whole showed no correlation with prognosis, TFH subsets such as CD57+ TFH cells were significantly associated with an inferior patient outcome. Taken together, our results indicate that TFH cells are highly heterogenous with different tumor-specific TFH subsets present in FL and associated with patient outcomes in FL.

Abstract The tumor microenvironment (TME) plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed a cohort of 82 FL patients with biopsy specimens collected before the first treatment regimen was administered. Our results showed that there was a difference in the T-cell phenotype in patients with various TMEs. Patients with TMEs rich in monocyte/macrophages/NK cells had more CD8+ cells and tended to have fewer TN, but more TEMRA and TEXH cells. Patients enriched with T cells exhibited a T-cell phenotype skewed toward to TCM and patients with B cell dominance tended to have significant higher number of TFH cells. T-cell phenotypes with more terminally differentiated, senescent and exhausted cells were highly represented in patients with less T cells when compared to patients with more T cells. We observed that CD57+ TFH cells was significantly more abundant in patients who had disease progression than patients who had complete response to therapy, suggesting a role for CD57+ TFH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that increased numbers of CD57+ TFH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57+ TFH cells exhibited a substantially different transcriptome from CD57− TFH cells. Genes that were differentially upregulated in CD57+ TFH cells when compared to CD57− TFH cells included genes involved in cell survival, compromised inflammatory response, and metabolic activation. Taken together, our results indicate different tumor microenvironments among patient groups that is associated with variable T-cell phenotypes.

Abstract The role and immunological constitution of the tumor microenvironment (TME) in splenic marginal zone lymphoma (sMZL) is largely unknown and studies on the phenotype and subset composition of intratumoral T cells are lacking. In the present study, we evaluated 36 sMZL spleen specimens by single cell analysis to gain a better understanding of the TME in sMZL. Using mass cytometry (CyTOF), we observed that the TME in sMZL is distinct from that of control non-malignant reactive spleen (rSP). We found that the number of TFH cells varied greatly in sMZL, ICOS+ TFH cells were more abundant in sMZL than rSP, and TFH cells positively correlated with increased numbers of memory B cells. Treg cell analysis revealed that TIGIT+ Treg cells are enriched in sMZL and correlate with suppression of TH17 and TH22 cells. Intratumoral CD8+ T cells were comprised of subsets of short-lived, exhausted and late-stage differentiated cells, thereby functionally impaired. We observed that T-cell exhaustion was present in sMZL and TIM-3 expression on PD-1low cells identified cells with severe immune dysfunction. Gene expression profiling by CITE-seq analysis validated this finding. Taken together, our data sugget that the TME as a whole, and T-cell population specifically, are heterogenous in sMZL and immune exhaustion is one of the major factors impairing T-cell function.

Abstract Purpose: Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied. Experimental Design: Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays. Results: We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition. Conclusions: IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease.