Purpose Programmed death ligand 1 (PD-L1) is an immunomodulatory molecule expressed by antigen-presenting cells and select tumors that engage receptors on T cells to inhibit T-cell immunity. Immunotherapies targeting the PD-1/PD-L1 pathway have shown durable anti-tumor effects in a subset of patients with solid tumors. PD-L1 can be expressed by Reed-Sternberg cells comprising classical Hodgkin lymphoma (CHLs) and by malignant B cells comprising EBV-positive post-transplant lymphoproliferative disorders (PTLDs). We sought to determine whether the expression of PD-L1 represents a general strategy of immune evasion among aggressive B-cell lymphomas and virus- and immunodeficiency-associated tumors. Experimental Design Using novel antibodies and formalin-fixed, paraffin-embedded (FFPE) tissue biopsies, we examined 237 primary tumors for expression of PD-L1 protein. Results Robust PD-L1 protein expression was found in the majority of nodular sclerosis CHL, mixed cellularity CHL, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma. Within these tumors, PD-L1 was highly expressed by malignant cells and tumor-infiltrating macrophages. In contrast, neither the malignant nor the non-malignant cells comprising nodular lymphocyte-predominant Hodgkin lymphoma, DLBCL-not otherwise specified, Burkitt lymphoma, and HHV8-associated Kaposi sarcoma expressed detectable PD-L1. Conclusion Certain aggressive B-cell lymphomas and virus- and immunodeficiency-associated malignancies associated with an ineffective T-cell immune response express PD-L1 on tumor cells and infiltrating macrophages. These results identify a group of neoplasms that should be considered for PD-1/PD-L1-directed therapies, and validate a method to detect PD-L1 in FFPE tissue biopsies.

The full neutrophil heterogeneity and differentiation landscape remains incompletely characterized. Here we profiled >25,000 differentiating and mature mouse neutrophils using single-cell RNA sequencing to provide a comprehensive transcriptional landscape of neutrophil maturation, function, and fate decision in their steady state and during bacterial infection. Eight neutrophil populations were defined by distinct molecular signatures. The three mature peripheral blood neutrophil subsets arise from distinct maturing bone marrow neutrophil subsets. Driven by both known and uncharacterized transcription factors, neutrophils gradually acquire microbicidal capability as they traverse the transcriptional landscape, representing an evolved mechanism for fine-tuned regulation of an effective but balanced neutrophil response. Bacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transition between each subpopulation, and primes neutrophils for augmented functionality without affecting overall heterogeneity. In summary, these data establish a reference model and general framework for studying neutrophil-related disease mechanisms, biomarkers, and therapeutic targets at single-cell resolution.

SUMMARY Gasdermin D (GSDMD) is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT) to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE), released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT) that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies.

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein-Barr virus (EBV), unlike sporadic Burkitt Lymphoma (sBL) that occurs with a lower incidence in developed countries. Given these differences and the variable clinical presentation and outcomes, we sought to further understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from multiple primary eBL tumors compared to sBL tumors. Within eBL tumors, minimal expression differences were found based on: anatomical presentation site, in-hospital survival rates, and EBV genome type; suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex (PSMB9/β1i, PSMB10/β2i, PSMB8/β5i, and PSME2/PA28β) in eBL tumors carrying type 2 EBV compared to type 1 EBV. Secondly, in comparison to previously published pediatric sBL specimens, the majority of the expression and pathway differences was related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than geographic designation. Third, common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, a set of new genes mutated in BL including TFAP4, MSH6, PRRC2C, BCL7A, FOXO1, PLCG2, PRKDC, RAD50, and RPRD2 were identified. Overall, these data establish that EBV, particularly EBV type 1, supports BL oncogenesis alleviating the need for certain driver mutations in the human genome.

Gene expression profiling identified genes uniquely expressed by human germinal-center T-helper (GCTh) cells, including programmed death-1 (PD-1) and CXCL13. Recently, we demonstrated that PD-1 is an immunophenotypic marker of GCTh cells and angioimmunoblastic T-cell lymphoma (AITL). The goal of this study was to investigate the expression pattern of CXCL13 in comparison with PD-1. We studied 63 cases of T-cell lymphoproliferative disorders, including 22 cases of AITL. In cases of AITL, PD-1+ and CXCL13+ neoplastic cells were seen at foci of expanded CD21+ follicular dendritic cell networks. CXCL13 expression was limited in other peripheral T-cell lymphomas. PD-1 and CXCL13 identified germinal-center T-helper cells, showed a similar pattern of expression in AITL, and should serve as useful new markers for AITL. The similar pattern of expression of CXCL13 and PD-1 in AITL provides further evidence that AITL is a neoplasm derived from germinal-center T-helper cells.

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