Lymphoma Tumor Microenvironment Research Articles

T-cell help in the tumor microenvironment enhances rituximab-mediated NK-cell ADCC

AbstractRituximab (RTX) and other monoclonal antibodies (mAbs) that bind directly to malignant cells are of great clinical value but are not effective for all patients. A major mechanism of action of RTX is antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Prior in vitro studies in our laboratory demonstrated that T cells contribute to maintaining the viability and cytotoxic potential of NK cells activated by anti-CD20–coated target B cells. Here, we conducted studies using a novel mouse model and clinical correlative analysis to assess whether T-cell help contribute to RTX-mediated NK-cell ADCC in the tumor microenvironment (TME) in vivo. A humanized mouse model was developed using Raji lymphoma cells and normal donor peripheral blood mononuclear cells that allows for control of T-cell numbers in the lymphoma TME. In this model, NK-cell viability and CD16 and CD25 expression dropped after RTX in the absence of T cells but increased in the presence of T cells. RTX therapy was more effective when T cells were present and was ineffective when NK cells were depleted. In patients with indolent lymphoma, fine needle aspirates were obtained before and ∼1 week after treatment with a RTX-containing regimen. There was a strong correlation between CD4+ T cells as well as total T cells in the pretherapy TME and an increase in NK-cell CD16 and CD25 expression after RTX. We conclude that T-cell help in the TME enhances RTX-mediated NK-cell viability and ADCC.

Mechanisms of lymphoma-stromal interactions focusing on tumor-associated macrophages, fibroblastic reticular cells, and follicular dendritic cells.

The interaction between cancer cells and stromal cells contributes to the pathogenesis of various types of tumors in the tumor microenvironment (TME). Macrophages (Mφs), a type of stromal cell, are transformed into tumor-associated Mφs (TAMs) after integrating within solid tumors. TAMs are known to interact with cancer cells and induce tumor progression. Thus, the cancer cells construct an organ-specific TME, which is advantageous for the survival of cancer cells in the TME. The density of stromal cells is known to be involved in the prognosis of patients with lymphomas. A higher density of stromal cells increases the interaction between lymphoma cells and stromal cells, promoting lymphoma progression. This review focuses on stromal cells in lymphoid tissues, such as TAMs, fibroblastic reticular cells (FRCs), and follicular dendritic cells (FDCs). This review also focuses on the signal transduction caused by stromal cells and tumor cells via factors such as cytokines. IL-10 and other cytokines secreted by TAMs activate the JAK/STAT pathway in lymphoma cells of follicular lymphoma, classic Hodgkin lymphoma, and diffuse large B-cell lymphoma. FRCs play roles in tumor promotion in follicular lymphoma and diffuse large B-cell lymphoma. Cytokines/chemokines secreted by FDCs play essential roles in lymphoma cell survival, proliferation, invasion, and migration in follicular lymphoma. In conclusion, TAMs, FRCs, and FDCs play crucial roles in the TME of lymphomas. Furthermore, histological spatial analysis revealing the positional relationship of each cell could highlight lymphoma-stromal interactions.

Efficient 3D imaging and pathological analysis of the human lymphoma tumor microenvironment using light-sheet immunofluorescence microscopy.

Rationale: The composition and spatial structure of the lymphoma tumor microenvironment (TME) provide key pathological insights for tumor survival and growth, invasion and metastasis, and resistance to immunotherapy. However, the 3D lymphoma TME has not been well studied owing to the limitations of current imaging techniques. In this work, we take full advantage of a series of new techniques to enable the first 3D TME study in intact lymphoma tissue. Methods: Diverse cell subtypes in lymphoma tissues were tagged using a multiplex immunofluorescence labeling technique. To optically clarify the entire tissue, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+), clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) and stabilization to harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD) were comprehensively compared with the ultimate dimensional imaging of solvent-cleared organs (uDISCO) approach selected for clearing lymphoma tissues. A Bessel-beam light-sheet fluorescence microscope (B-LSFM) was developed to three-dimensionally image the clarified tissues at high speed and high resolution. A customized MATLAB program was used to quantify the number and colocalization of the cell subtypes based on the acquired multichannel 3D images. By combining these cutting-edge methods, we successfully carried out high-efficiency 3D visualization and high-content cellular analyses of the lymphoma TME. Results: Several antibodies, including CD3, CD8, CD20, CD68, CD163, CD14, CD15, FOXP3 and Ki67, were screened for labeling the TME in lymphoma tumors. The 3D imaging results of the TME from three types of lymphoma, reactive lymphocytic hyperplasia (RLN), diffuse large B-cell lymphoma (DLBCL), and angioimmunoblastic T-cell lymphoma (AITL), were quantitatively analyzed, and their cell number, localization, and spatial correlation were comprehensively revealed. Conclusion: We present an advanced imaging-based method for efficient 3D visualization and high-content cellular analysis of the lymphoma TME, rendering it a valuable tool for tumor pathological diagnosis and other clinical research.

Distinct Minor Neoplastic Follicular T-Cell Subsets Dictate Lymphoma Fates through Functional Specialization

Background: The presence and role of follicular T-cell populations other than T follicular helper (Tfh) cells, such as T follicular regulatory (Tfr) and cytotoxic (Tfc) cells, are gaining increasing attention in certain pathological states. However, the ecosystem of follicular T cells in the tumor microenvironment (TME) has not been fully elucidated. In particular, the significance of minor follicular T-cell subsets in the neoplastic follicular environment remains elusive. Here, we aimed to reveal the landscape of follicular T-cell alterations in various cancers, with a particular emphasis on the follicular lymphoma (FL) TME. Methods: We analyzed single-cell RNA/TCR sequencing data of >500,000 human T cells from FL (obtained from four cohorts) and 25 other cancer types, as well as homeostatic and reactive lymph nodes (LNs), to construct a comprehensive single-T-cell atlas. We investigated differentially expressed genes, RNA velocity, and TCR clonality using this atlas. To determine the functions of neoplastic follicular regulatory (Tnfr) and cytotoxic (Tnfc) T cells, we performed in vitro cytokineproduction and co-culture assays, in combination with cell activation/suppression, cell division, and apoptosis assays, using human FL samples. With the PhenoCycler-Fusion system, we conducted multiplex digital spatial profiling (DSP) of 169 FL samples from two independent cohorts (now being extended to 242 FL samples from three cohorts) for >25 antibodies. We also performed single-cell spatial and protein expression profiling and prognostic analysis. Results: In FL, distinct minor neoplastic follicular T-cell subsets-Tnfr and CD4 (Tnfc4) and CD8 (Tnfc8) Tnfc cells-increased relative to homeostatic LNs. The TCR repertoire analysis revealed that Tnfr cells shared clonotypes with conventional effector regulatory T (Trg) and Tfh cells, whereas Tnfc4 and Tnfc8 cells shared clonotypes with Tfh cells and effector and exhausted (Tcex) cytotoxic CD8 T cells, respectively. In line with these findings, the RNA velocity survey suggested that Tnfr, Tnfc4, and Tnfc8 cells originated from Trg, Tfh, and naïve-like CD8 T cells, respectively. Tnfr and Tnfc cells expressed higher levels of effector genes, including those involved in cytokine release, chemokine response, migration, and PD-1 signaling, than their reactive LN counterparts. The pan-cancer survey revealed that Tfr and CD4 Tfc cells were exclusive to FL, whereas the prevalence and gene expression profiles of CD8 Tfc cells varied across cancers. Tnfr cells were marked by abundant expression of IL10 and IL21, whereas Tnfc cells displayed a unique phenotype, as they concomitantly expressed markers of effector Tfh (e.g., CXCL13, CXCR5, and PDCD1), naïve/stem (e.g., CCR7 and TCF7), central memory (e.g., CD27, CD28, and SELL), and tissue-resident memory (e.g., ITGAE) cells. Hierarchical clustering demonstrated that Tnfc8 cells had transcriptional profiles similar to those of melanoma TCF1 +PD-1 +CD8 + stem-like T cells. DSP of FL detected Tnfr and Tnfc cells frequently localized within and around neoplastic follicles, forming a cellular neighborhood that allowed them to interact closely. Tnfr cells were distributed predominantly near Tfh cells. The functional co-culture assays demonstrated that Tnfr cells suppressed Tfh-cell activation and division, thereby inhibiting Tfh-mediated malignant B-cell activation and survival. Tnfc8 cells showed a higher cell division capability than that of Tcex cells, suggesting that Tnfc8 cells function as a pool of CD8 T cells in neoplastic follicles. The prognostic analysis revealed that Tnfr and Tnfc cell proportions correlated with early disease relapse (i.e., POD24) and predicted a significantly longer time-to-relapse ( P <0.05 for Tnfr and <0.001 for Tnfc cells) in FL. In the multivariate analysis, the prognostic impact of these two cell subsets was independent of the FLIPI. The prognostic analysis findings were confirmed using a validation cohort. Conclusions: Our multi-omics approach identified the expansion of minor neoplastic follicular T-cell subsets that carry unique transcriptional and functional profiles and robust prognostic impacts. These findings deepen our understanding of the biological and immunological roles of non-Tfh follicular T cells in the lymphoma TME and highlights their clinical potential for patient risk stratification and future therapeutic interventions.

Location, location, location: mapping the lymphoma tumor microenvironment using spatial transcriptomics.

Lymphomas are a heterogenous group of lymphoid neoplasms with a wide variety of clinical presentations. Response to treatment and prognosis differs both between and within lymphoma subtypes. Improved molecular and genetic profiling has increased our understanding of the factors which drive these clinical dynamics. Immune and non-immune cells within the lymphoma tumor microenvironment (TME) can both play a key role in antitumor immune responses and conversely also support lymphoma growth and survival. A deeper understanding of the lymphoma TME would identify key lymphoma and immune cell interactions which could be disrupted for therapeutic benefit. Single cell RNA sequencing studies have provided a more comprehensive description of the TME, however these studies are limited in that they lack spatial context. Spatial transcriptomics provides a comprehensive analysis of gene expression within tissue and is an attractive technique in lymphoma to both disentangle the complex interactions between lymphoma and TME cells and improve understanding of how lymphoma cells evade the host immune response. This article summarizes current spatial transcriptomic technologies and their use in lymphoma research to date. The resulting data has already enriched our knowledge of the mechanisms and clinical impact of an immunosuppressive TME in lymphoma and the accrual of further studies will provide a fundamental step in the march towards personalized medicine.

Oncolytic virus oHSV2 combined with PD-1/PD-L1 inhibitors exert antitumor activity by mediating CD4 + T and CD8 + T cell infiltration in the lymphoma tumor microenvironment

A novel therapeutic regimen showed that the oncolytic type II herpes simplex virus (oHSV2) was able to prevent colorectal cancer growth, recurrence, and metastasis. However, no study has yet explored whether oHSV2 has an impact on the development of diffuse large B-cell lymphoma (DLBCL). We chose the clinical chemotherapeutic drug doxorubicin (DOX) as a positive control to evaluate the effect of oHSV2 infection on the apoptotic, invasive, and proliferative capacity of DLBCL cells. We next further explored the therapeutic efficacy of oncolytic virus oHSV2 or DOX in DLBCL tumor bearing BALB/c mice, and evaluated the infiltration of CD8 + T cells and CD4 + T cells in tumor tissues. A pathological approach was used to explore the effects of oHSV2 on various organs of tumor bearing mice, including the heart, liver, and kidney. Next, SU-DHL-4 cells were co-cultured with cytotoxic T lymphocytes (CTLs) to mimic the tumor immune microenvironment (TME), to explore the impact of oHSV2 on the immune environment at the cellular level, and then analyzed the relationship between oHSV2 and the PD-1/PD-L1 immune-checkpoint. Subsequently, we further validated the efficacy of combined oHSV2 and PD-L1 treatment on transplanted tumor growth in mice at the in vivo level. DLBCL cells were sensitive to the action of the oncolytic virus oHSV2, and the decline in their proliferative activity showed a time-and dose-dependent manner. oHSV2 and DOX intervention preeminently increased the cell apoptosis, restrained cell proliferation and invasion, with the greatest changes occurring in response to oHSV2 infection. oHSV2 application effectively improved the immune status of the tumor microenvironment, favoring the invasion of CD8 + T and CD4 + T cells, thereby enhancing their antitumor effects. Besides, oHSV2 treatment has a safety profile in the organs of tumor bearing mice and indeed inhibits the PD-1/PD-L1 immune checkpoint in DLBCL. Interestingly, the combination of oHSV2 and PD-L1 antibodies results in more profound killing of DLBCL cells than oHSV2 infection alone, with a significant increase in the proportion of CD4 + T cells and CD8 + T cells. The antitumor effect was the best after combining oHSV2 and PD-L1 antibodies, suggesting that the combination therapy of oHSV2 and PD-L1 would have a better prospect for clinical application.

Discovering the fibroblastic reticular cell in the immune tumor microenvironment in lymphoma.

The study of the cellular and molecular microenvironment in B cell lymphoma, especially diffuse large B cell lymphoma (DLBCL), has led to prognostic and therapeutic algorithms that may improve patient outcomes. Emerging gene signature panels provide a granular understanding of DLBCL based on the immune tumor microenvironment (iTME). In addition, some gene signatures identify lymphomas that are more responsive to immune-based treatment, indicating that the iTME has a biological signature that could affect outcomes when targeted. In this issue of the JCI, Apollonio et al. report on fibroblastic reticular cells (FRCs) as potential targets in aggressive lymphoma. FRCs interacted with lymphoma cells and induced a state of chronic inflammation that suppressed immune function by impeding optimal T cell migration and inhibiting CD8+ T cell lytic function. These findings suggest that manipulating the iTME by directly targeting FRCs may enhance responses to immunotherapy in DLBCL.

Abstract 5147: Mice with T cells deficient in the Cytosolic Branched Chain Aminotransferase reduce the tumor burden of low and high immunogenic strains of Lymphoma

Abstract Current immunotherapies rely on modulating host immunity to induce an anti-tumor immune response. The efficacy of such a response depends on the immunogenicity of the tumor, which varies greatly between different cancers or the same cancer in different individuals. While most lymphomas are immunogenic, non-malignant T cells that infiltrate the lymphoma microenvironment are subjected to immunosuppressive signals and areas of nutrient depletion, which leads to T cell exhaustion and inhibition of T cell function. Metabolic enzymes, such as the cytosolic branched chain aminotransferase, BCATc, exert immunosuppressive effects limiting T cells ability to function. Previous research described BCATc as a negative regulator of T cell activation, thus identifying BCATc as a potential target for immunotherapeutic modulation in the tumor microenvironment (TME). In the current study we exploit a low (EL4) and high (EL4-OVA) immunogenic murine lymphoma strains to explore how a loss of BCATc in T cells impacts their ability to produce anti-lymphoma immunity in mice. We used 12 week old male mice with BCATc deleted from T cells (T-BCATcKO) and control T-BCATcfl/fl mice that express the transgenic floxed version of BCATc. Mice were injected with 2.5x105 cells belonging to the low (EL4) or high (EL4-OVA) immunogenic strains and tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. Tumors were then lysed, and the protein profile of each tumor was determined by monitoring changes in expression of protein markers of T cell exhaustion (TOX, CD39), apoptosis (BAX, PUMA), or signaling proteins, such as the ribosomal S6 protein, the protein kinase B (AKT), and the energy sensor the AMP-activated protein kinase (AMPK). T-BCATcKO mice showed significant reduction in tumor growth compared to control mice regardless of the lymphoma strain being tested. The mouse cohort with the biggest number of tumor free mice was the T-BCATcKO cohort injected with EL4 cells (13% of the mice were tumor free on day 9). However, EL4-OVA cells grew into smaller tumors across all cohorts. Mice had comparable food intake but T-BCATcKO mice, injected with EL4 cells, had a significantly lower body weight. In T-BCATcKO mice, the reduced EL4 tumor mass correlated with upregulated AMPK and AKT along with higher expression of PUMA. The reduced EL4-OVA tumor masses correlated with downregulated S6 and decreased TOX expression, but high expression of BAX compared to the corresponding tumors isolated from T-BCATcfl/fl mice. These findings suggest that T cells deficient in BCATc can reduce lymphoma growth regardless of the immunogenicity of the lymphoma stain likely by establishing catabolic state and inducing apoptosis. Thus, BCATc may play an immunosuppressive role in the lymphoma TME and the absence of BCATc from T cells may help T cells combat lymphoma. Citation Format: Lucas D. Figueroa, Tanner Wetzel, Leighton Wheeler, Christie Adam, Michael Boyer, Elitsa Ananieva. Mice with T cells deficient in the Cytosolic Branched Chain Aminotransferase reduce the tumor burden of low and high immunogenic strains of Lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5147.

Anticancer polypyrrole-polyethylenimine drug-free nanozyme for precise B-cell lymphoma therapy

As an alternative strategy for cancer treatment, the combination of cancer nanomedicine and immunotherapy is promising with regard to efficacy and safety; however, precise modulation of the activation of antitumor immunity remains challenging. Therefore, the aim of the present study was to describe an intelligent nanocomposite polymer immunomodulator, drug-free polypyrrole-polyethyleneimine nanozyme (PPY-PEI NZ), which responds to the B-cell lymphoma tumor microenvironment, for precision cancer immunotherapy. Earlier engulfment of PPY-PEI NZs in an endocytosis-dependent manner resulted in rapid binding in four different types of B-cell lymphoma cells. The PPY-PEI NZ effectively suppressed B cell colony-like growth in vitro accompanied by cytotoxicity via apoptosis induction. During PPY-PEI NZ-induced cell death, mitochondrial swelling, loss of mitochondrial transmembrane potential (MTP), downregulation of antiapoptotic proteins, and caspase-dependent apoptosis were observed. Deregulated AKT and ERK signaling contributed to glycogen synthase kinase-3-regulated cell apoptosis following deregulation of Mcl-1 and MTP loss. Additionally, PPY-PEI NZs induced lysosomal membrane permeabilization while inhibiting endosomal acidification, partly protecting cells from lysosomal apoptosis. PPY-PEI NZs selectively bound and eliminated exogenous malignant B cells in a mixed culture system with healthy leukocytes ex vivo. While PPY-PEI NZs showed no cytotoxicity in wild-type mice, they provided long-term and efficient inhibition of the growth of B-cell lymphoma-driven nodules in a subcutaneous xenograft model. This study explores a potential PPY-PEI NZ-based anticancer agent against B-cell lymphoma.

Graphdiyne oxide nanosheets exert anti-lymphoma effect by killing cancer stem cells and remodeling tumor microenvironment

Inflammatory and immunosuppressive tumor microenvironment (TME) is responsible for accelerating lymphoma initiation and progression. Graphdiyne oxide (GDYO) nanosheets have been shown to restrain tumor inflammation, remodel tumor microenvironment and suppress tumor growth. However, whether GDYO exerts anti-lymphoma effect has been elusive. Here, we first delineated the inflammatory and immunosuppressive signature of lymphoma TME by bioinformatic analysis. We then demonstrated the toxic effects of GDYO on multiple types of lymphoma cells in vitro. Further xenograft experiments showed that GDYO significantly eliminated tumor initiation and growth in vivo. Moreover, single-cell transcriptome sequencing of tumor tissue revealed GDYO treatment remarkably decreased the frequencies of cancer stem cells, immunosuppressive Treg cells and pro-inflammatory myeloid cells. Mechanistically, GDYO treatment markedly inhibited the Mif-Ackr3 signaling in tumor cells and CAFs, reduced expression of inflammatory cytokines in CAFs, mitigated the number of Treg cells and CSCs, which ultimately suppressed the growth of lymphoma. Finally, we showed that GDYO showed excellent biosafety and biocompatibility. Together, our study demonstrates that GDYO exerts the duplex anti-lymphoma effects by simultaneously killing the cancer stem cells and remodeling the lymphoma TME, which provide a reference for the potential application of GDYO nanomaterials as a novel nanomedicine to treat lymphoma in the future.

Targeting The Tumor Microenvironment in Lymphomas: Emerging Biological Insights and Therapeutic Strategies.

This review aims to discuss recent advances in elucidating the tumor microenvironment (TME) in B lymphomas and resultant novel therapeutic development. While tumor morphology, immunophenotype, and molecular profile are established factors that predict outcome and guide therapy, the prognostic impact of infiltrating, non-tumor cells is now emerging. This is simultaneously facilitating the development of new therapies that target non-tumor cells. The tumor microenvironment (TME) is a complex ecosystem composed of infiltrating cells and byproducts, extracellular matrix, and other non-cellular tissues. In lymphomas, our current understanding of the role of the TME is principally informed by studies in B-cell lineage diseases. As we improve our understanding of lymphoma biology, the importance of the impact of the non-tumor cell microenvironment is becoming more apparent. This lays the foundation for the investigation and development of novel therapies and combination strategies that target non-tumor cells and tumor cell/non-tumor cell interactions.

Tumor Immune Microenvironment in Lymphoma: Focus on Epigenetics.

Lymphoma is a neoplasm arising from B or T lymphocytes or natural killer cells characterized by clonal lymphoproliferation. This tumor comprises a diverse and heterogeneous group of malignancies with distinct clinical, histopathological, and molecular characteristics. Despite advances in lymphoma treatment, clinical outcomes of patients with relapsed or refractory disease remain poor. Thus, a deeper understanding of molecular pathogenesis and tumor progression of lymphoma is required. Epigenetic alterations contribute to cancer initiation, progression, and drug resistance. In fact, over the past decade, dysregulation of epigenetic mechanisms has been identified in lymphomas, and the knowledge of the epigenetic aberrations has led to the emergence of the promising epigenetic therapy field in lymphoma tumors. However, epigenetic aberrations in lymphoma not only have been found in tumor cells, but also in cells from the tumor microenvironment, such as immune cells. Whereas the epigenetic dysregulation in lymphoma cells is being intensively investigated, there are limited studies regarding the epigenetic mechanisms that affect the functions of immune cells from the tumor microenvironment in lymphoma. Therefore, this review tries to provide a general overview of epigenetic alterations that affect both lymphoma cells and infiltrating immune cells within the tumor, as well as the epigenetic cross-talk between them.

Tumor Microenvironment of Lymphomas and Plasma Cell Neoplasms: Broad Overview and Impact on Evaluation for Immune Based Therapies.

Lymphomas and plasma cell neoplasms are a heterogenous group of malignancies derived from lymphocytes. They are a significant cause of patient morbidity and mortality. Advances in morphologic, immunophenotypic and molecular techniques have led to better understanding of the pathogenesis and diagnosis of these neoplasms. Advances in treatment, particularly immune-based therapies, increasingly allow for targeted therapies of these diseases. Mechanistic studies using animal models and clinical trials have revealed the importance of the tumor microenvironment on disease pathogenesis, progression, and response to therapy in these malignancies. Simultaneous progress in diagnostic techniques has made it feasible to generate high-resolution, high-throughput data from the tumor microenvironment with spatial context. As the armamentarium of targeted therapies and diagnostic techniques grows, there is potential to harness these advances to better stratify patients for targeted therapies, including immune-based therapies, in hematologic malignancies.

CD8+ T Lymphocytes Immune Depletion and LAG-3 Overexpression in Hodgkin Lymphoma Tumor Microenvironment Exposed to Anti-PD-1 Immunotherapy.

Background: Resistance to anti-PD-1 remains a considerable clinical challenge for the treatment of patients with classical Hodgkin lymphoma (cHL), and mechanisms of anti-PD-1 resistance remain unknown. This pilot study aims to investigate the tumor microenvironment in patients with cHL relapsing after anti-PD-1. Methods: This study investigated tumor samples of eight patients with cHL, including four patients exposed to anti-PD-1 with a paired longitudinal histological analysis before and after anti-PD-1, and four patients not exposed to anti-PD-1 who served as control for the cellular biological investigations. Fresh cells tumor microenvironment analysis included phenotypic characterization of their T cell surfaces immune checkpoint markers PD-1, PD-L1, ICOS, TIM-3, LAG-3, 41-BB and BTLA. Tumor tissues immunohistochemistry staining included CD30, CD4, CD8, CD68, CD163, PD-L1, PD-1, LAG-3 and TIM-3. Findings: Paired longitudinal tumor tissues analysis in the tumor microenvironment found a CD8+ lymphocytes tumor depletion and an increase of LAG-3 staining after anti-PD-1 exposure. The fresh cells analysis of the tumor microenvironment in patients exposed to anti-PD-1 found CD8+ lymphocyte depletion, with an elevated CD4+/CD8+ lymphocytes ratio (median ratio 9.77 in exposed anti-PD-1 versus 2.39 in not-exposed anti-PD-1 patients; p = 0.0943). On the cell surfaces of CD4+ lymphocytes, the median positive expression of LAG-3 was significantly higher in the samples exposed to anti-PD-1 compared to the controls (15.05 [IQR:17.91-10.65] versus 3.84 [IQR 1.87-6.57]; p = 0.0376). Interpretation: This pilot study proposes hypotheses for understanding the resistance to immunotherapies in patients with Hodgkin lymphoma. Hodgkin lymphoma exposed to anti-PD-1 correlated in tumor microenvironment with an immune depletion of CD8+ T lymphocytes and overexpression of LAG-3 on CD4+ helper T lymphocytes.

Understanding the Immune-Stroma Microenvironment in B Cell Malignancies for Effective Immunotherapy.

Cancers, including lymphomas, develop in complex tissue environments where malignant cells actively promote the creation of a pro-tumoral niche that suppresses effective anti-tumor effector T cell responses. Research is revealing that the tumor microenvironment (TME) differs between different types of lymphoma, covering inflamed environments, as exemplified by Hodgkin lymphoma, to non-inflamed TMEs as seen in chronic lymphocytic leukemia (CLL) or diffuse-large B-cell lymphoma (DLBCL). In this review we consider how T cells and interferon-driven inflammatory signaling contribute to the regulation of anti-tumor immune responses, as well as sensitivity to anti-PD-1 immune checkpoint blockade immunotherapy. We discuss tumor intrinsic and extrinsic mechanisms critical to anti-tumor immune responses, as well as sensitivity to immunotherapies, before adding an additional layer of complexity within the TME: the immunoregulatory role of non-hematopoietic stromal cells that co-evolve with tumors. Studying the intricate interactions between the immune-stroma lymphoma TME should help to design next-generation immunotherapies and combination treatment strategies to overcome complex TME-driven immune suppression.

The tumor microenvironment of lymphomas: Insights into the potential role and modes of actions of checkpoint inhibitors.

The tumor microenvironment (TME) - a term comprising non-neoplastic cells and extracellular matrix as well as various cytokines, chemokines, growth factors, and other substances in the vicinity of tumor cells - is an integrative part of most tumors including lymphomas. Interactions between lymphoma cells and the TME are vital for survival and proliferation of the former. In addition, lymphoma cells often reprogram the TME to protect them from defense mechanisms of the host's immune system. In this review, we will introduce the role of the tumor microenvironment (TME) for lymphoma cells looking at direct cell-cell interactions as well as cytokine-related communications. The immunomodulative/immunosuppressive role of the TME is more and more coming into the focus of potential new targeted therapies, and thus a special attention will be given to the interactions of immune checkpoints such as programed cell death protein 1 and L1 (PD-1/PD-L1), T-cell immunoglobulin and mucin-domain containing protein-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), and cytotoxic T-lymphocyte-associated protein-4 (CTLA4) with the TME, as well as their expression by both lymphoma cells and cells of the TME. Aspects of the TME will be discussed for indolent and aggressive B-cell lymphomas, Hodgkin lymphomas, and T-cell lymphomas. In addition, the potential influence of other immunomodulators such as lenalidomide will be briefly touched. The complex role of the TME is in the focus of new therapeutic options. In order to exploit its full therapeutic potential, however, a thorough understanding of TME biology and interaction between lymphoma cells and the TME, as well as the host's immune system and the TME is necessary.

Highly Multiplexed Single Cell Spatial Analysis of the Tumor Microenvironment in Lymphoma Predicts Clinical Outcomes

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Although a majority of patients are cured with standard chemo-immunotherapy, up to 40% of DLBCL patients have refractory disease or develop relapse following R-CHOP regimen, warranting development of novel, more effective therapeutic strategies for this cohort[1]. The composition of immune cells in the tumor microenvironment (TME) and tumor PD-L1 expression have been reported to predict DLBCL outcomes, however PD1 inhibitors demonstrate response rates of less than 10%.[2-5] We hypothesize that a better characterization of spatial architecture of the tumour microenvironment (TME) in lymphoma will help explain why DLBCL has poor responses to PD1 inhibitors and guide future targeted immunotherapies for these patients. Methods Here we characterized the TME in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution [6]. Using a panel of 32 antibodies, IMC was performed 41 tissue microarray cores from 33 DLBCL cases. IMC images were analyzed for relevant immunophenotypes, the spatial architecture of those phenotypes and compared to clinical outcomes to identify immune contexture based biomarkers. Results Phenograph was used to cluster tumor and immune cells based on phenotype. Immune cells represented 33% of the cells broken down to CD4 (36%), CD8 (30%), macrophages (26%) and TREG(8%) (Figure A).Immune cell infiltration in individual tumor samples ranged from 7% to 75% with marked heterogeneity between samples. Analysis of immune marker expression on tumor cells identified co-expression of PD-L1/CCR4/TIM3 to be highly prognostic for overall survival (p=0.003, Figure B-C) To characterize the patterns of spatial interaction in the TME, we developed an unsupervised multivariate model to construct spatial meta-clusters based on average distances from CD8 to the centroids of 5 nearest endothelial cells, TREG, CD4 T cells, macrophages, and tumor cells (Figure D). Spatial analysis revealed 11 meta-clusters for CD8 T cell interactions (Figure E). Each CD8 spatial interaction pattern is distinctive with case to case heterogeneity (Figures F). Risk assessment analyses of spatial clusters 1, 2 and 4 ("hazardous") had almost 3 times higher odds of being identified in refractory cases compared to clusters 3, 5 and 6 ("protective") (Figure G). In the "protective" spatial neighborhoods, we observed the presence of activated CD8, Th1-like CD4, and less suppressive TREGphenotypes, with opposite in "hazardous" areas (Figures H). TIM-3 expression was high both on T cells and tumor cells in the "hazardous" neighborhoods. Finally, we show that sub-setting our analysis of CD8 phenotypes based on their spatial location to other cells improved our ability to predict overall survival in the cohort. Conclusion These results are the first to demonstrate that spatial profiling of immune architecture in DLBCL is associated with clinical outcomes, and that spatial analysis of immune cells should be explored as a potential biomarker for patients treated with immunotherapies.

CD4 Lymphocytes and T Regulatory Cells Show Distinct Phenotypes in Follicular Lymphoma and Classical Hodgkin Lymphoma Which May Account for Differences in Responses to Checkpoint Therapy

Physiologically T-regulatory cells (Treg) suppress immune responses against self-antigens preventing autoimmunity. However, in cancer they are believed to suppress anti-tumor immune response, and the presence of Treg in the tumor microenvironment has been associated with adverse outcome in most cancers. Treg are integral part of tumor microenvironment in lymphoma, and can use different mechanisms to inhibit both adaptive and immune response (including CD4, CD8, DC, macrophages, B-cells and NK cells) modulating therefore the interaction between lymphoma and the host microenvironment. Increased numbers of Treg have been associated with adverse clinical outcome in follicular lymphoma (FL) but a more favorable outcome in classical Hodgkin lymphoma (CHL). In this study we examined Treg phenotype in detail using multiparameter flow cytometry in lymph node specimens with normal histology (NLN) and involved by FL and CHL. We report that both CD4 positive T-cells and Treg subset show distinct phenotypes in different disease entities suggesting different biological functions. Single cell suspensions were prepared from NLN (n=10) and lymph node biopsies involved by FL (n=10) and CHL (n=10), and high dimensional multiparameter flow cytometric immunophenotyping was performed using antibodies against immune checkpoints (TIGIT, TIM3, PD-1, CD96, LAG3, CTLA4, CD73). To characterize the CD4 T-cell compartments we used a dimensionality reduction algorithm for non-linear data representations (tSNE). We studied the CD4+FoxP3-CD25- and Treg characterized by CD4+FoxP3+CD25+ phenotype separately and in both the compartments we identified and characterized subpopulations specific for each of the disease cohorts. tSNE representations CD4 positive T-cells and Treg showed different distributions in NLN, FL and HL. In NLN, CD4 cells broader heterogeneity without distinct clusters whereas in FL and CHL CD4 positive T-cells and Treg (Figure 2 A-B) showed highly polarized phenotypes which were distinct from each other and nearly absent within normal Treg compartment. We observed a strong expression of the immune checkpoint regulators TIM3, LAG3, CTL4, Tbet and PD1 in CD4 T-cells derived from HL tissues. In contrast FL CD4 T-cells were mainly characterized by an up regulation of PD1, CTLA4 and TIGIT. The TIGIT+ cells in FL samples are mainly CXCR5+PD1bright and they up regulate CTLA-4 in the FoxP3+ compartment. In all the neoplastic tissues the FoxP3+CD25+ T-Reg express mainly an activated/memory phenotype (CD45RO+CTLA4+), but while in HL microenvironment they show a TH1 phenotype (CXCR3+Tbet+), in FL they mainly express PD1+CXCR5+ (Figure 1B). Additionally, the dominant population of regulatory T-cells derived from HL samples down modulate PD1 expression, show high level of expression of TH1-associated transcription factor Tbet and have high proliferation index, while in FL, PD1 is brightly expressed, Tbet is not expressed and proliferation index is low in the dominant population (Figure 1A). The distinct phenotypic differences of Treg in FL lymphoma and CHL may account for the better prognosis seen in CHL with increased Treg which has TH1 like phenotype, therefore predicted to have anti-tumor activity. The differences seen in the expression immune checkpoint regulators both on CD4 positive T-cells and Treg subset may explain different rate responses seen in FL compared to CHL with checkpoint therapy. Disclosures Roshal: Physicians' Education Resource: Other: Provision of services; Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services. Dogan:Corvus Pharmaceuticals: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Roche: Consultancy, Research Funding; Celgene: Consultancy; Seattle Genetics: Consultancy.

Identification of a novel monocytic phenotype in Classic Hodgkin Lymphoma tumor microenvironment.

Classic Hodgkin lymphoma (CHL) characteristically shows few malignant cells in a microenvironment comprised of mixed inflammatory cells. Although CHL is associated with a high cure rate, recent studies have associated poor prognosis with absolute monocyte count in peripheral blood and increased monocyte/macrophages in involved lymph nodes. Thus, the role of monocytic infiltration and macrophage differentiation in the tumor microenvironment of CHL may be more relevant than absolute macrophage numbers to defining prognosis in CHL patients and potentially have therapeutic implications. Most studies identify tumor-associated macrophages (TAMs) using markers (e.g., CD68) expressed by macrophages and other mononuclear phagocytes, such as monocytes. In contrast, Class A Scavenger Receptor (SR-A/CD204) is expressed by tissue macrophages but not monocytic precursors. In this study, we examined SR-A expression in CHL (n = 43), and compared its expression with that of other macrophage markers. We confirmed a high prevalence of mononuclear cells that stained with CD68, CD163, and CD14 in CHL lymph nodes. However, SR-A protein expression determined by immunohistochemistry was limited to macrophages localized in sclerotic bands characteristic of nodular sclerosis CHL. In contrast, SR-A protein was readily detectable in lymph nodes with metastatic tumor, extra-nodal CHL, T cell/histiocyte-rich large B cell lymphoma, and resident macrophages in non-malignant tissues, including spleen, lymph node, liver and lung. The results of SR-A protein expression paralleled the expression of SR-A mRNA determined by quantitative RT-PCR. These data provide evidence that tumor-infiltrating monocyte/macrophages in CHL have a unique phenotype that likely depends on the microenvironment of nodal CHL.

Abstract CT153: Pretreatment immunoscore and an inflamed tumor microenvironment (TME) are associated with efficacy in patients (Pts) with refractory large B cell lymphoma treated with axicabtagene ciloleucel (Axi-Cel) in ZUMA-1

Abstract Background: Axi-cel is an FDA-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after ≥ 2 prior therapies. In Phase 2 of ZUMA-1, the objective response rate was 83% (58% complete responses [CRs]) (Locke et al. Lancet Oncol. 2018). Previous work showed that pre-existing expression of immune-related genes (Immunosign® 21 with NanoString®) may be associated with response to axi-cel (Rossi et al. AACR 2018. #LB-016). This expanded post-hoc analysis includes novel analysis associating T cell density with efficacy. Methods: Pts received a target dose of 2.0 × 106 CAR T cells/kg in ZUMA-1 (NCT02348216). Pretreatment (before conditioning) tumor tissues were analyzed by immunohistochemistry for density of T cell subsets (CD3+, CD8+). A prespecified bioinformatics algorithm was used to generate a numerical index (Immunoscore®; Galon et al. J Pathol. 2014) and analyses cutoffs. Higher Immunoscore® represents increased intra-tumor T cell infiltration. TME was evaluated for Immunosign® 21 and for additional myeloid- and dendritic cell-related genes. The association between Immunoscore®, CD3+, or CD8+ T cell density and CR was evaluated, as was the association of Immunoscore® with Immunosign® 21. Results: Pretreatment tumor biopsy samples from 25 pts were analyzed (data cutoff Aug 11, 2017). Of pts with high Immunoscore®, 77% (7/9) had CR and 11% (1/9) were nonresponders. Of those with low Immunoscore®, 38% (6/16) were nonresponders and 50% (8/16) had CR. The pts in this category who achieved CR had a lower median pretreatment tumor burden by sum of product diameters. Overall, higher pretreatment Immunoscore® was associated with achievement of CR (P = .048). Pretreatment intra-tumor densities of CD3+ and CD8+ T cells separately quantified, were also positively associated with achievement of CR (P = .02 and .036 by t test). In addition, higher pretreatment Immunosign® 21 was associated with clinical efficacy. Values for Immunosign® 21 and Immunoscore® demonstrated 82% concordance (95% CI, 65-93; r2 = 0.451). Finally, TME gene expression analysis indicated a pan-inflammatory profile, including myeloid- and dendritic cell-related gene expression, in pts who achieved CR with higher Immunosign® 21 and Immunoscore®. Conclusions: These results link pre-existing T cell involved lymphoma TME with clinical outcome in patients post axi-cel. They also suggest that factors intrinsic to tumor biology may influence CAR T cell efficacy through the immune microenvironment. In addition, this treatment modality may overcome an unfavorable TME in a subset of patients with low disease burden. Finally, these results support CAR T cell treatment optimizations designed to overcome an immune detrimental TME. John Rossi and Jérôme Galon contributed equally. Citation Format: John M. Rossi, Jérôme Galon, Edmund Chang, Regis Perbost, Nathalie Scholler, Sarah Turcan, Corinne Danan, Frederick L. Locke, Sattva S. Neelapu, David B. Miklos, Caron A. Jacobson, Lazaros J. Lekakis, Yi Lin, Armin Ghobadi, Francesca Milletti, William Y. Go, Adrian Bot. Pretreatment immunoscore and an inflamed tumor microenvironment (TME) are associated with efficacy in patients (Pts) with refractory large B cell lymphoma treated with axicabtagene ciloleucel (Axi-Cel) in ZUMA-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT153.