Mukherjee, Prithviraj

Abstract Description A subset of follicular lymphoma 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. However, little is known on the molecular mechanisms whereby IRF4 controls the crosstalk between B and T cells. To this aim, we leveraged Cγ1cre;irf4fl/fl mice that yield conditional deletion of irf4 (cKO) in germinal center B cells (GCB). After 10 days of immunization, cKO GCB showed significant increase of HLA-DR and CD40 expression, a skewed LZ:DZ ratio towards increased DZ, and suppression of plasma cells compared to WT. Upon irf4 deletion, we also found a significant increase of total CD4 and TFH cells and a decrease in Treg cells. To test if this effect was antigen-dependent, we cocultured sorted GCB from NP-OVA-immunized cKO and WT mice with CSFE-labeled OVA-specific OT-II CD4 T cells at a ratio of 2:1 for 5 days. We observed increased antigen specific proliferation of CD4 T cells with cKO GCB compared to WT. We then performed seahorse assay and mass spectrometry of the same GCB to define if IRF4-dependent B cell metabolic reprogramming influences immune responses. GCB cKO had lower glycolytic ECAR and increased intracellular glucose and non-essential amino acids than WT cells, suggesting a metabolic shift from glycolysis to the TCA cycle. Thus, IRF4 controls immune and metabolic signaling with functional effects on the B-T cell crosstalk. Funding Sources NCI K08CA279652, LRF Clinical Investigator Award (CDA 1020588), the Gerstner Family Career Development Award, the ASH Junior Faculty Scholar Award. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Signal regulatory protein-α (SIRPα) is a key member of the “do-not-eat-me” signaling pathway and is expressed on intratumoral monocytes/macrophages in lymphoma. In this study, we found that SIRPα is also expressed by a subset of malignant B cells in lymphoma. SIRPα+ B cells have a memory phenotype with germinal center (GC) dark-zone features, and RNA velocity analysis showed that SIRPα+ B cells exhibit higher spliced transcripts confirming a more mature phenotype. SIRPα expression is substantially upregulated by treating lymphoma B cells with LPS or CpG and SIRPα+ B cells showed increased proliferation when compared to SIRPα- B cells, which was associated with reduced cellular autophagy. SIRPα+ B cells exhibited increased expression of BAFF signaling pathway molecules as the likely mechanism of increased proliferation and survival. Furthermore, in co-culture, SIRPα+ lymphoma B cells suppressed T-cell function and blocking SIRPa/CD47 signaling reversed T cell inhibition. Clinically, we found that a greater number of SIRPα+ B cells present in the diagnostic tumor biopsies of FL patients was associated with a significantly poorer overall survival. We conclude that SIRPα+ lymphoma B cells are a proliferative component of indolent lymphomas, are associated with T-cell suppression, and increased prevalence of these cells is associated with a poor prognosis. Targeting SIRPα+ B cells in lymphoma may therefore be a promising therapeutic strategy. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description The efficacy of immunotherapy for splenic marginal zone lymphoma (SMZL) remains limited due to the strong immune suppression present in the tumor microenvironment (TME). Tregs play a key role in maintaining TME homeostasis by suppressing effector T cells and malignant B cells. Previously, we identified two Treg subsets CD26+ and CD161+ in SMZL. CD161+ Tregs were more effective at suppressing both CD8+T cells and malignant B cells, whereas CD26+Tregs primarily inhibited CD8+T cells. Patients with higher levels of CD26+ Tregs and fewer CD161+ Tregs tend to have worse outcomes. In this study, we found that cytokine-inducible SH2-containing protein (CISH), a known negative regulator of immune activation, is highly expressed in CD26+Tregs compared to CD161+ Tregs. Co-culturing B cells enhanced the expression of CISH and CD26 in CD4+CD25- Treg cells, knockdown of CISH in Tregs reduced CD26 expression and their immune suppression activity of CD8+T cells, while CRISPR knockout of CISH in Karpas 299 cells led to higher expression CD70, a molecule associated with less immunesuppression. Our findings suggest that increased CISH expression in Tregs could result in increased immune suppression and unfavorable outcome in SMZL, suggesting CISH is a potential therapeutic tagrget in SMZL by dempening immunosuppressive property of the Treg cells hence boosting the antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

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.

Splenic marginal zone lymphoma (SMZL) is a B-cell malignancy with a typically indolent course. However, some patients have more aggressive disease and initial treatment can range from observation to chemoimmunotherapy. The reason for this spectrum of disease behavior is incompletely understood but the tumor microenvironment (TME) likely plays a role as recent studies have shown that an optimized antitumor immune response in SMZL patients would allow the immune system to better target malignant B cells and irradicate the disease. Regulatory CD4+T cells (Tregs) are crucial in maintaining TME homeostasis by suppressing effector T cells and malignant B cells and in this study, we therefore explored the key factors influencing the function and development of intratumoral Tregs in SMZL patients. We identified two distinct Treg subsets in the TME of SMZL patients: CD26+Tregs and CD161+Tregs, each with unique phenotypic profiles, functions, and prognostic significance. CD161+Tregs suppress CD8+T cells and malignant B cells more effectively than CD26+Tregs, which only inhibit CD8+T cells. Clinically, patients with inferior outcomes have significantly higher numbers of CD26+Tregs and fewer CD161+Tregs. To delineate differences between these subsets, we performed Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-seq) on biopsies from 7 SMZL patients. We found that cytokine-inducible SH2-containing protein (CISH) is the most highly expressed gene in CD26+Tregs compared to CD161+Tregs, aside from FOXP3. CISH has been reported to be a negative regulator of immune effector cell activation in tumor models. Although CISH deficiency leads to a loss of Foxp3 expression and reduced Treg suppressive function, the mechanism by which CISH modulates Treg function, especially immune suppression in hematologic malignancies, remains largely unknown. To further investigate the mechanism of CISH in Treg function in MZL patients, we conducted CITE-seq analysis on a larger cohort of MZL patients (n=12), divided into groups based on outcomes (EFS24 failed, n=4; EFS24 achieved, n=8). The CITE-seq analysis results showed significantly higher CISH expression in Tregs from patients with inferior outcomes (P=1.07E-06). We then explored factors influencing CISH expression and found that TCR engagement and treatment with cytokines (IL-15, IL-2, and IL-7) elevated CISH expression in Tregs. While we previously observed that lymphoma B cells could enhance Foxp3 expression in CD4+CD25-T cells, this study we found that B cells could also enhance CISH and CD26 expression in CD4+CD25-T cells in vitro, indicating that a high frequency of B cells in the environment may lead to increased CISH expression in Treg cells leading to an unfavorable outcome in patients. We further investigated the role of CISH in the function of Treg cells. Following CISH knockdown in Treg cells, we observed decreased CD26 expression and reduced suppressive function on CD8+ T cell proliferation. Using CRISPR-Cas9, we knocked out CISH in the Karpas 299 cell line, a human T cell lymphoma line that constitutively expresses CD25, Foxp3, and CISH. CISH-KO Karpas 299 cells showed significantly less apoptosis and Foxp3 expression compared to CISH-WT cells. Bulk RNA-seq analysis of CISH-KO and CISH-WT Karpas 299 cells revealed that CISH-KO cells showed enhanced DNA replication and cell cycle pathway activity. Taken together, our results suggest that high expression of CISH in Tregs may predict an unfavorable outcome in SMZL patients. Malignant B-cells in the TME of SMZL patients may promote expression of CD26 and CISH in Tregs, thereby modulating Treg function. Targeting CISH could decrease the immunosuppressive activity of Tregs in the TME, potentially optimizing antitumor immunity in SMZL patients.

Abstract Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma associated with an increase in serum immunoglobulin M (IgM) monoclonal protein. The underlying causes of the disease are still not fully understood, but cells in the bone marrow (BM) microenvironment (TME) are anticipated to play a significant role in disease progression. We previously reported that myeloid-derived suppressor cells (MDSCs), a phenotypically heterogeneous group of cells known for their ability to suppress immune responses, are expanded in the bone marrow of patients with WM and that the expansion of MDSCs is particularly notable for increased numbers of polymorphonuclear MDSCs (PMN-MDSCs). In the present study, we evaluated the ability of WM cells to attract MDSCs to the tumor microenvironment (TME). We also evaluated whether PMN-MDSCs suppress the cytotoxic function of T-cells and thereby promote the survival of WM cells. Our study confirms the expansion of MDSCs, notably PMN-MDSCs in BM of WM patients as compared to normals. We also found that PMN-MDSCs expressing CD66b was a predominant population in WM patients. To determine the phenotype and transcriptome of both PMN-MDSCs and monocytic MDSCs (M-MDSCS) in WM, we compared BM aspirates from WM patients to normal bone marrow (NBM) and performed cellular indexing of transcriptomes and epitopes by sequencing (CITEseq) analysis (WM=3, NBM=2) followed by qPCR validation. We found that MDSCs in WM express significantly higher levels of HIF1-α overall (p<0.02) compared to NBM. Furthermore, PMN-MDSCs are more abundant in WM bone marrow than M-MDSCs and express significantly higher levels of CXCR4 (p<0.001), myeloperoxidase (MPO; p<0.0003), and azuricidin (AZU1; p<0.0004), suggesting an activated phenotype. To determine whether WM cells attract MDSCs to the BM, we performed a transwell assay and showed that WM cells (BCWM.1 cell line) attracted MDSCs, particularly PMN-MDSCs, to a greater extent than M-MDSCs (n=3; p<0.001). We next evaluated whether MDSCs prevent cytotoxic T-cells from lysing WM cells. In-vitro co-culture studies with BCWM.1 cells and T-cytotoxic cells (B:CD8-T) showed that T-cytotoxic cells were able to lyse the WM cell line by 46.4% , (median range 46.4 - 47.8%, p=0.001). However, when BCWM.1 cells and T-cytotoxic cells were co-cultured in the presence of MDSCs (i.e B:CD8-T:M), the specific lysis of BCWM.1 cells was reduced by 12.6% respectively (range 2.2- 12.6%, p<0.001). Furthermore, we observed that B:CD8-T:M co-culture significantly reduced CD8-T cell activation as compared to activated T cells alone (17.9%) (median range 17.8- 18%, p<0.001). In contrast, no affect was observed with B: CD8-T coculture by 91% (range 90.6- 91.4% p<.001) confirming that MDSCs suppress CD8-T cell activation. Our study therefore provides evidence that MDSCs in WM have an activated phenotype and that PMN-MDSCs in WM are attracted by malignant cells in the bone marrow. Furthermore, we find that the increased presence of PMN-MDSCs in the WM bone marrow environment suppresses T-cell lysis of malignant cells and may thereby contribute to disease progression. These findings provide a strong rationale for targeting MDSCs as a potential therapeutic strategy in WM.

Abstract The role of the bone marrow (BM) microenvironment in regulating the antitumor immune response in Waldenstrom macroglobulinemia (WM) remains limited. Here we transcriptionally and phenotypically profiled non-malignant (CD19− CD138−) BM cells from WM patients with a focus on myeloid derived suppressive cells (MDSCs) to provide a deeper understanding of their role in WM. We found that HLA-DRlowCD11b+CD33+ MDSCs were significantly increased in WM patients as compared to normal controls, with an expansion of predominantly polymorphonuclear (PMN)-MDSCs. Single-cell immunogenomic profiling of WM MDSCs identified an immune-suppressive gene signature with upregulated inflammatory pathways associated with interferon and TNF signaling. Gene signatures associated with an inflammatory and immune suppressive environment were predominately expressed in PMN-MDSCs. In vitro WM PMN-MDSCs demonstrated robust T-cell suppression, and their viability and expansion was notably enhanced by G-CSF and TNFα. Furthermore, BM malignant B-cells attracted PMN-MDSCs to a greater degree than monocytic MDSCs. Collectively, these data suggest that malignant WM B cells actively recruit PMN-MDSCs which promote an immunosuppressive BM microenvironment through a direct T cell inhibition, while release of G-CSF/TNFα in the microenvironment further promotes PMN-MDSC expansion and in turn immune suppression. Targeting PMN-MDSCs may therefore represent a potential therapeutic strategy in patients with WM.

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.