Table S2 contains a summary of the B cell receptor sequencing data of lymphoma samples. It includes 1) an overview of the investigated samples, 2) a list of characteristics of the dominant clone of each lymphoma sample, 3) a list comparing the frequencies of V family usage in WT samples to the lymphoma frequency, 4) the frequencies of FR and CDR mutations in samples analyzed by full-length BCR sequencing.

Table S6 contains clinical parameters and sequencing results of human DLBCL samples.

Table S4 contains the individual mutations of PPMBC and MBC lymphomas.

This file contains the Supplementary Figures S1 to S12.

Supplementary Table S1 - Differentially expressed genes and PAGE analysis in Myd88 L252P GCB

Gating strategies, validation of results using alternative models or definitions, QC controls, etc.

Table S5 contains statistical values of gene set enrichment analyses of the KEGG, BIOCARTA and HALLMARK gene set collections.

Table S3 contains B cell receptor repertoire sequencing data and lists the individual sublcones of the dominant clones of MBC, SMBC and PPMBC lymphomas.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has substantially improved the outcome of patients suffering from relapsed and/or refractory (r/r) aggressive B cell lymphoma. However, around 60% of patients do not show long-term remissions after CAR-T cell therapy. Recent studies have indicated a relevant role of the lymphoma microenvironment (LME) in response and resistance to CAR-T cell therapy. However, targeting the LME in aggressive B cell lymphoma to boost CAR-T cell efficacy has not yet been sufficiently explored. We therefore aimed to unravel the immunosuppressive capacity of the LME and its myelo-monocytic cell compartment with the ultimate goal to identify potential therapeutic targets and enhance CAR-T cell response. Methods: To elucidate hallmarks associated with an immunosuppressive LME and CAR-T cell resistance in patients with r/r B cell lymphoma, we applied multi-dimensional analyses to pre- and post-CAR-T cell-treated human lymphoma specimens (n = 41), including bulk RNA sequencing, single-cell RNA sequencing of 47,078 live cells and Imaging Mass Cytometry (IMC). To validate our findings and explore the potential of new therapeutic targets, we utilized ex vivo co-culture experiments, a fully murine CD19 CAR-T cell therapy platform in an immunocompetent, autochthonous DLBCL mouse model and performed bulk RNA sequencing and IMC of diseased spleens. Results: In our cohort of CAR-T cell treated patients (n = 104) durable response, defined as complete remission six months after CAR-T cell therapy, resulted in prolonged progression-free and overall survival. In CAR-T cell non-durable responding lymphoma patients, we identified a prognostically relevant lymphoma-associated myelo-monocytic (LAMM) signature including genes such as CD14, CD68, MARCO, ITGAM, IL1B, IL10 and S100A9. Furthermore, non-durable response was characterized by increased hypoxia and reduced (CD8+) T cell infiltration. In particular, in-depth profiling using single-cell RNA sequencing and IMC revealed a distinct CSF1R+CD14+CD68+ LAMM cell population associated with non-durable response and poor clinical outcome in CAR-T cell-treated patients with r/r B cell lymphoma. Importantly, high LAMM and low CD8+ T cell infiltration prior to CAR-T cell therapy showed a reduced progression-free survival when compared to low LAMM and high CD8+ T cell infiltration in r/r B cell lymphoma samples. Next, in ex vivo co-culture experiments we demonstrated that CSF1R+ LAMM cells strongly inhibit the proliferation and the cytotoxic capacity of CAR-T cells. To elaborate on LAMM-T cell interaction at a molecular level, we performed inference analysis of cell-cell communication in our single-cell RNA sequencing dataset using CellphoneDB which revealed that LAMM cells exert their immunosuppressive function by direct interaction with T cells via prostaglandin E2 (PGE2) and EP2/EP4 receptor signaling. Most strikingly, applying a fully autochthonous DLBCL CAR-T cell mouse model, we demonstrated that the combination of CD19 CAR-T cell therapy with CSF1R blockade switches an immunosuppressive LME into a T cell-enriched LME, which was accompanied by a follicular architecture and blood vessel normalization of diseased spleens indicated by IMC analysis. Finally, we showed that the combination of CSF1R inhibition and CD19 CAR-T cell therapy displayed synergistic treatment effects and prolonged survival with long-lasting, complete remissions. Conclusion: Our multiomic data and preclinical models provide strong evidence that CSF1R+ LAMM cells contribute to CAR-T cell failure in r/r aggressive B cell lymphoma and that CSF1R inhibition synergistically improves CD19 CAR-T cell response, promotes an immunosupportive microenvironment and restores anti-lymphoma immunity. Given that CSF1R inhibitors have already been clinically evaluated and FDA-approved in other malignancies, this therapeutic combination has the potential for rapid clinical translation. Based on our findings, we propose to test the combination of CAR-T cell therapy and CSF1R inhibitors in patients with r/r aggressive B cell lymphoma within prospective clinical trials.

Chimeric antigen receptor T cell (CAR T) therapy is a potent treatment for relapsed/refractory (r/r) B cell lymphomas but provides lasting remissions in only -40% of patients and is associated with serious adverse events. We identify an upregulation of CD80 and/or CD86 in tumor tissue of (r/r) diffuse large B cell lymphoma (DLBCL) patients treated with tisagenlecleucel. This finding leads to the development of the CAR/CCR (chimeric checkpoint receptor) design, which consists of a CD19-specific first -generation CAR co -expressed with a recombinant CTLA-4-linked receptor with a 4-1BB co -stimulatory domain. CAR/CCR T cells demonstrate superior efficacy in xenograft mouse models compared with CAR T cells, superior long-term activity, and superior selectivity in in vitro assays with non-malignant CD19+ cells. In addition, immunocompetent mice show an intact CD80-CD19+ B cell population after CAR/CCR T cell treatment. The results reveal the CAR/CCR design as a promising strategy for further translational study.

Although several promising approaches for the treatment of relapsed/refractory diffuse large B-cell lymphoma (rrDLBCL) have been approved recently, it remains unclear which patients will ultimately achieve long-term responses. Circulating tumor (ct)DNA sequencing has emerged as a valuable tool to assess minimal residual disease (MRD). Correlations between MRD and outcomes have been shown in previously untreated DLBCL, but data on the repeated assessment of MRD in the dynamic course of rrDLBCL is limited. Here, we present an approach leveraging cost- and time-sensitivity of digital droplet (dd)PCR to repeatedly assess MRD in rrDLBCL and present proof-of-principle for its ability to predict outcomes.

Diffuse large B cell lymphoma (DLBCL) is a highly heterogeneous disease and is the most common lymphoid malignancy in adults (Swerdlow et al., 2016, Alizadeh et al., 2000). It is traditionally divided into two subtypes based on the cell-of-origin: Activated B cell-like (ABC) DLBCL and germinal center B cell-like (GCB) DLBCL (Alizadeh et al., 2000). DLBCL can be further subdivided into clusters A53, ST2, N1, BN2, EZB, and MCD based on the mutational profiles of the tumors (Schmitz et al., 2018, Wright et al., 2020). While cure rates of ~65% are achievable in DLBCL patients with frontline combination chemo-immune therapy (e.g. R-CHOP), treating relapsed or refractory disease remains a challenge, particularly in ABC-DLBCL and MCD patients (Pfreundschuh et al., 2011, Tilly et al., 2015, Schmitz et al., 2018). Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional repressor that is frequently mutated in MCD patients, with many of these mutations predicted to be loss-of-function (Schmitz et al., 2018). Besides human DLBCL tumors, in previously published MCD mouse models, which harbor a B cell-specific loss of Prdm1, expression of mutant Myd88p.L252P, overexpression of BCL2, and in some cases expression of a Cd79b ITAM mutation, Irf2bp2 is also one of the most frequently co-mutated genes (Knittel et al., 2016, Flümann et al., 2023, Flümann et al., 2024). However, the role of IRF2BP2 in lymphoma biology and as a potential tumor suppressor gene in DLBCL has not been investigated. To understand the role of IRF2BP2 in the germinal center reaction, we analyzed the B cell compartment of autochthonous mice which harbor a B cell specific knockout of Irf2bp2 at a premalignant age. We observed a dramatic decrease in germinal center B cells in Irf2bp2 knockout mice compared to controls, as well as a higher relative memory B cell output and reduced class switch recombination. This preliminary dataset suggests that Irf2bp2 may play a role in the dynamics of the germinal center reaction and B cell differentiation. Moreover, we show that CRISPR/Cas9-mediated loss of IRF2BP2 in human ABC-DLBCL cell lines leads to increased proliferation and NF-KB signaling, compared to IRF2BP2-proficient cells. Additionally, we find that IRF2BP2 knockout ABC-DLBCL cells express higher interleukin-1 beta (IL1β) and are sensitive to anti-IL1β inhibition both in vitro and in vivo, while IRF2BP2-proficient cells remain insensitive. Furthermore, murine lymphoma cell lines derived from MCD mouse models, which harbor a spontaneous Irf2bp2 mutation are also sensitive to anti-IL1β inhibition in vitro and retain sensitivity over several weeks. Our findings suggest that IL1β is the primary mediator of increased NF-KB signaling upon IRF2BP2 perturbation and anti-IL1β therapy could be a potential treatment strategy for IRF2BP2-mutant patients.

Diffuse large B -cell lymphoma (DLBCL) is the most common aggressive lymphoma and constitutes a highly heterogenous disease. Recent comprehensive genomic pro filing revealed the identity of numerous molecularly de fined DLBCL subtypes, including a cluster which is characterized by recurrent aberrations in MYD88 , CD79B, and BCL2 , as well as various lesions promoting a block in plasma cell differentiation, including PRDM1 , TBL1XR1, and SPIB . Here, we generated a series of autochthonous mouse models to mimic this DLBCL cluster and speci fically focused on the impact of Cd79b mutations in this setting. We show that canonical Cd79b immunoreceptor tyrosine -based activation motif (ITAM) mutations do not accelerate Myd88 - and BCL2 -driven lymphomagenesis. Cd79b -mutant murine DLBCL were enriched for IgM surface expression, reminiscent of their human counterparts. Moreover, Cd79b -mutant lymphomas displayed a robust formation of cytoplasmic signaling complexes involving MYD88, CD79B, MALT1, and BTK. These complexes were disrupted upon pharmacological BTK inhibition. The BTK inhibitor -mediated disruption of these signaling complexes translated into a selective ibrutinib sensitivity of lymphomas harboring combined Cd79b and Myd88 mutations. Altogether, this in-depth cross -species comparison provides a framework for the development of molecularly targeted therapeutic intervention strategies in DLBCL.

Diffuse large B cell lymphoma (DLBCL) is a genetically highly heterogeneous disease. Yet, to date, the vast majority of patients receive standardized frontline chemo-immune-therapy consisting of an anthracycline backbone. Using these regimens, approximately 65% of patients can be cured, whereas the remaining 35% of patients will face relapsed or refractory disease, which, even in the era of CAR-T cells, is difficult to treat. To systematically tackle this high medical need, it is important to design, generate and deploy suitable in vivo model systems that capture disease biology, heterogeneity and drug response. Recently published, large comprehensive genomic characterization studies, which defined molecular sub-groups of DLBCL, provide an ideal framework for the generation of autochthonous mouse models, as well as an ideal benchmark for cell line-derived or patient-derived mouse models of DLBCL. Here we discuss the current state of the art in the field of mouse modelling of human DLBCL, with a particular focus on disease biology and genetically defined molecular vulnerabilities, as well as potential targeting strategies.

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Introduction: Recent efforts have established distinct subtypes of DLBCL by clustering cases based on their mutational profiles. Mutations associated with the MCD/C5 cluster include MYD88 p.L265P, CD79B p.Y196X and BCL2 amplifications. Several recurring MCD-associated mutations affect transcription factors involved in regulating B cell differentiation (BCL6, SPIB, PRDM1, TBL1XR1). We previously established an autochthonous mouse model mimicking B cell-specific expression of MYD88L265P, BCL2 amplification and loss of PRDM1. These animals develop a disease that resemble many features of MCD DLBCL and thereby provide a valuable tool for DLBCL research. Here, we aimed to advance this existing model by introducing a Cd79b ITAM mutation. Methods: We generated an allele that allows the conditional expression of Cd79b p.Y195H (the murine orthologue of CD79B p.Y196H) from the endogenous locus and bred it to our established DLBCL mouse models. The developing lymphomas were characterized by exome, transcriptome and B cell receptor sequencing. We performed flow cytometric analyses, phosphoproteomics and proximity ligation assays to assess the activation status of the BCR pathway in our models. Lastly, we conducted MRI-guided treatment experiments with the BTK inhibitor ibrutinib. Results: By including a B-cell-specific Cd79b p.Y195H mutation, we refined our mouse model to gain novel insights into MCD/C5 DLBCL characteristics. Mice developed highly proliferative, (oligo-)clonal lymphomas. While mouse models with an engineered loss of Prdm1 formed lymphomas that were B220+CD138- and enriched for pre-memory and light zone gene signatures, the B220-CD138+ tumors developing in Prdm1-deficient lines showed plasmablastic features on a transcriptional level. The Cd79b status had no effect on the putative precursor population of the malignant B cells, however the highest frequency of spontaneous mutations in genes associated with MCD DLBCL was observed Prdm1-deficient lymphomas carrying both Myd88 and Cd79b activating mutations. Futhermore, the presence of the Cd79b p.Y195H allele increased BCR pathway activation levels in both the Prdm1-proficient and -deficient mouse lines. This activated state of the BCR pathway in Cd79b-mutated murine lymphoma translated into an increased sensitivity of those tumors to BTK inhibition by ibrutinib. Conclusions: Taken together, we refined existing Myd88 p.L252p and BCL2-driven MCD/C5 DLBCL mouse models by co-expressing the Cd79b p.Y195H mutation. Cd79b-mutant murine lymphomas exhibited increased BCR activation levels, resulting in an increased sensitivity towards BTK inhibition, when compared to Cd79b wt control tumors. These findings indicate that patients with CD79B ITAM mutations might be particularly sensitive to BTK inhibitor treatment. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Basic and Translational Science Conflicts of interests pertinent to the abstract. H. C. Reinhardt Employment or leadership position: CDL Therapeutics Consultant or advisory role: Roche, Novartis, BMS, AbbVie, Vertex, Merck Research funding: AstraZeneca, Gilead

Abstract A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients. Significance: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8–11). This article is highlighted in the In This Issue feature, p. 1

Pharmacological Screen IC50 values

Introduction: Adoptive immunotherapies such as chimeric antigen receptor (CAR) T cell therapy have strongly improved the outcome of patients with diffuse large cell lymphoma (DLBCL) that are relapsed or refractory after standard chemo-immunotherapy. However, approx. 50% of patients with DLBCL are not durably responding to CAR T cell therapy. CAR T cell expansion is associated with durable responses. However, the molecular mechanisms that mediate suppression of CAR T cells leading to resistance still remain elusive. Methods: We performed bulk RNA sequencing of DLBCL patients before CAR T cell therapy. We performed gene set enrichment analysis (GSEA) using signatures derived from published genes associated with an immunosuppressive lymphoma microenvironment. To investigate CAR T cell therapy in an immunocompetent autochthonous mouse model we established murine CD19-redirected CAR-T cells that were generated using splenic T cells isolated from mice harbouring a C57BL/6N background. We treated DLBCL derived from PPMBC (PRDM1-KO, Myd88 + BCL2 overexpression, CD19: Cre) mice with this murine CD19 CAR-T cells in combination with an anti-CSF1R targeted antibody compared to controls. Results: We found increased immunosuppressive metabolic characteristics in CD19 CAR T refractory DLBCL patients indicated by increased glycolysis, hypoxia and elevated reactive oxygen species (ROS) in GSEA analysis. This metabolic signature was associated with an enriched monocytic-myeloid cell signature and a lack of expansion of effector T cells in CD19 CAR T refractory DLBCL. CSF1-CSFR1 (CD115) signaling is one major pathway that mediates the differentiation of myeloid derived cells into immunosuppressive MSCs. CD115-positive MSCs are important immune regulators in the tumor microenvironment that mediate inhibition of T cells and induce the proliferation of Tregs. We therefore hypothesize that combined treatment with a CSFR1 inhibitor will enhance CAR T cell expansion and thus improve CAR T cell response. We show that CSF1R blockade shifts the immunosuppressive lymphoma microenvironment into an proinflammatory environment in CD19 CAR T cell treated mice with DLBCL and abrogation of the expansion of lymphoma associated myelo-monocytic suppressor cells (LAMMs). This shift into an immunosupportive lymphoma microenvironment was accompanied with an increase of T cell expansion within the tumor and expansion of CD19-CAR T cells. We next evaluated whether the combination of CD19-CAR T cell therapy with CSF1R inhibition improves therapeutic outcome PPMBC DLBCL mice. Strikingly, we show that CSF1R Inhibition displays synergistic efficacy in combination with CD19 CAR T cell therapy. Conclusions: Our data strongly indicates that CSF1R inhibition improves CD19-CAR T expansion, promotes an immunosupportive microenvironment and could enhance CD19-CAR T cell therapy efficacy in patients with DLBCL. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Microenvironment No conflicts of interests pertinent to the abstract.