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 NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and ␥-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require ␥-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n ‫؍‬ 3) or in the presence of urea (n ‫؍‬ 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.The development of multicellular organisms is orchestrated by a limited number of highly conserved signaling pathways. One such pathway involves NOTCH receptors and downstream mediators, which can variously regulate the specification of cell fate, proliferation, self-renewal, survival, and apoptosis in a dose-and context-dependent fashion (3,47).Like other members of the NOTCH receptor family, mammalian NOTCH1 is a large multimodular type I transmembrane glycoprotein (Fig. 1A). During maturation, NOTCH1 undergoes proteolytic processing by furin at a site termed S1 that lies ϳ70 amino acids external to the transmembrane domain (25), yielding two noncovalently associated extracellular (N EC ) and transmembrane (N TM ) subunits (6,25,37). N EC contains 36 N-terminal epidermal growth factor (EGF)-like repeats that participate in binding to ligands (23, 39, 51) and three iterated LIN-12/NOTCH repeats (LNR), which help to maintain NOTCH receptors in the "off" state prior to ligand binding (13,24,40). The association of N EC and N TM is mediated by sequences lying immediately N terminal (HD-N) and C terminal (HD-C) of site S1; together, these sequences constitute the NOTCH subunit association, or "heterodimerization" (HD) domain (40).Binding of ligands to N EC triggers two sequential proteolytic events within the N TM subunit at sites S2 and S3. S2 cleavage occurs just...

Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2–5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

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