Diffuse large B-cell lymphomas have spatially defined, tumor immune microenvironments revealed by high-parameter imaging.

Diffuse large B-cell lymphoma (DLBCL) not otherwise specified is the most common aggressive non-Hodgkin lymphoma and a biologically heterogeneous disease. Despite the development of effective immunotherapies, the organization of the DLBCL tumor-immune microenvironment (TIME) remains poorly understood.We interrogated the intact TIME of 51 de novo DLBCLs with triplicate sampling to characterize 337 995 tumor and immune cells using a 27-plex antibody panel that captured cell lineage, architectural, and functional markers. We spatially assigned individual cells, identified local cell neighborhoods, and established their topographical organization in situ. We found that the organization of local tumor and immune cells can be modeled by 6 composite cell neighborhood types (CNTs). Differential CNT representation divided cases into 3 aggregate TIME categories: immune-deficient, dendritic cell-enriched (DC-enriched), and macrophage-enriched (Mac-enriched). Cases with immune-deficient TIMEs have tumor cell-rich CNTs, in which the few infiltrating immune cells are enriched near CD31+ vessels, in keeping with limited immune activity. Cases with DC-enriched TIMEs selectively include tumor cell-poor/immune cell-rich CNTs with high numbers of CD11c+ DCs and antigen-experienced T cells also enriched near CD31+ vessels, in keeping with increased immune activity. Cases with Mac-enriched TIMEs selectively include tumor cell-poor/immune cell-rich CNTs with high numbers of CD163+ macrophages and CD8 T cells throughout the microenvironment, accompanied by increased IDO-1 and LAG-3 and decreased HLA-DR expression and genetic signatures in keeping with immune evasion. Our findings reveal that the heterogenous cellular components of DLBCL are not randomly distributed but organized into CNTs that define aggregate TIMEs with distinct cellular, spatial, and functional features.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Augmenting emergency granulopoiesis with CpG conditioned mesenchymal stromal cells in murine neutropenic sepsis.

Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin-Sca1+C-kit+CD150-CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

GRK2 enforces androgen receptor dependence in the prostate and prostate tumors.

Metastatic tumors that have become resistant to androgen deprivation therapy represent the major challenge in treating prostate cancer. Although these recurrent tumors typically remain dependent on the androgen receptor (AR), non-AR-driven tumors that also emerge are particularly deadly and becoming more prevalent. Here, we present a new genetically engineered mouse model for non-AR-driven prostate cancer that centers on a negative regulator of G protein-coupled receptors that is downregulated in aggressive human prostate tumors. Thus, prostate-specific expression of a dominant-negative G protein-coupled receptor kinase 2 (GRK2-DN) transgene diminishes AR and AR target gene expression in the prostate, and confers resistance to castration-induced involution. Further, the GRK2-DN transgene dramatically accelerates oncogene-initiated prostate tumorigenesis by increasing primary tumor size, potentiating visceral organ metastasis, suppressing AR, and inducing neuroendocrine marker mRNAs. In summary, GRK2 enforces AR-dependence in the prostate, and the loss of GRK2 function in prostate tumors accelerates disease progression toward the deadliest stage.

Checkpoint blockade in Hodgkin and non-Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is characterized by nearly universal genetic alterations in 9p24.1, resulting in constitutive expression of PD-1 ligands. This likely underlies the unique sensitivity of cHL to PD-1 blockade, with response rates of ∼70% in relapsed/refractory disease. There are now numerous clinical trials testing PD-1 inhibitors in earlier stages of treatment and in combination with many other therapies. In general, non-Hodgkin lymphomas (NHLs) do not display a high frequency of 9p24.1 alterations and do not share cHL's vulnerability to PD-1 blockade. However, a few entities have genetic or immunologic features that may predict sensitivity to immune checkpoint blockade. These include primary mediastinal B cell lymphoma, primary central nervous system lymphoma, and primary testicular lymphoma, which harbor frequent alterations in 9p24.1, as well as Epstein Barr virus (EBV)-infected lymphomas, where EBV infection leads to increased PD-L1 expression. Although these subtypes may be specifically vulnerable to PD-1 blockade, the majority of NHLs appear to be minimally sensitive to PD-1 blockade monotherapy. Current investigations in NHL are therefore focusing on targeting other checkpoints or studying PD-1-based combination therapy. Looking forward, additional insight into the most common mechanisms of resistance to immune checkpoint inhibitors will be important to guide rational clinical trial design. In this review, we describe the biological basis for checkpoint blockade in cHL and NHL and summarize the clinical data generated to date. Guided by our rapidly evolving understanding of the pathobiology of various lymphoma subtypes, we are hopeful that the role of checkpoint inhibitors in lymphoma treatment will continue to grow.

Therapeutic targeting of the inflammome.

Inflammatory responses can vary depending on a myriad of factors including: (1) the initiating stimulus or trigger, (2) the cell types involved in the response, and (3) the specific effector cytokine-chemokine milieus produced. The compilation of these and other factors in a given mechanistic context is sometimes referred to as the "inflammome". Humans and other higher-order mammals have evolved (over time) several discrete inflammomes to counter the effects of pathogens. However, when these inflammomes are induced inappropriately, they drive the development of chronic inflammatory diseases. The vast majority of biological anti-inflammatory treatments currently being developed are focused on the post hoc inhibition of downstream effectors by anti-cytokine monoclonal antibodies and receptor antagonists. This prevailing "end-point treatment" has even directed a new disease classification paradigm, namely a cytokine-based disease classification, as opposed to a traditional diagnosis based on a particular tissue or organ system dysfunction. Although this approach has a number of advantages, it omits the processes that led to the generation of the inflammatory effectors in the first place. In this review, we will expand the cytokine-based disease taxonomy into an inflammome-based taxonomy that includes interventions that subvert a priori cytokine development and can complement post hoc inhibition.

Serine lipids of Porphyromonas gingivalis are human and mouse Toll-like receptor 2 ligands.

The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2(-/-)) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2(-/-) mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

RKIP contributes to IFN-γ synthesis by CD8+ T cells after serial TCR triggering in systemic inflammatory response syndrome.

Systemic inflammatory response syndrome (SIRS) is associated with the development of severe medical complications, including progression to multiple organ dysfunction syndrome and even death. To date, only marginal improvements in terms of therapeutic options have been established for patients affected by SIRS. Raf kinase inhibitor protein (RKIP) is a regulator of MAPK and NF-κB signaling cascades, which are both critical for production of the proinflammatory cytokines responsible for SIRS initiation. By testing a T cell-dependent mouse model of SIRS that utilizes staphylococcal enterotoxin A specific for Vß3(+) T cells, we show that RKIP is necessary for the exaggerated production of IFN-γ from SIRS splenocytes. This effect was not due to differences in T cell expansion, IL-10 production, or APC priming, but rather a cell-intrinsic defect lying downstream of the TCR in staphylococcal enterotoxin A-specific CD8(+) T cells. Importantly, mice lacking RKIP were still able to proliferate, survive, and contribute to cytokine production in response to pathogen associated molecular pattern-TLR-mediated stimuli, despite the TCR-dependent defects seen in our SIRS model. Finally, by blocking RKIP in wild-type SIRS splenocytes, the IFN-γ response by CD8(+) Vß3(+) T cells was significantly diminished. These data suggest that RKIP may be a potential therapeutic target in SIRS by curbing effector cytokine production from CD8(+) T cells during serial TCR triggering.