Combination anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1) therapy promotes antitumor immunity and provides superior benefit to patients with advanced-stage melanoma compared with either therapy alone. T cell immunity requires recognition of antigens in the context of major histocompatibility complex (MHC) class I and class II proteins by CD8 and CD4 T cells, respectively. We examined MHC class I and class II protein expression on tumor cells from previously untreated melanoma patients and correlated the results with transcriptional and genomic analyses and with clinical response to anti-CTLA-4, anti-PD-1, or combination therapy. Most (>50% of cells) or complete loss of melanoma MHC class I membrane expression was observed in 78 of 181 cases (43%), was associated with transcriptional repression of ,, , and, and predicted primary resistance to anti-CTLA-4, but not anti-PD-1, therapy. Melanoma MHC class II membrane expression on >1% cells was observed in 55 of 181 cases (30%), was associated with interferon-γ (IFN-γ) and IFN-γ-mediated gene signatures, and predicted response to anti-PD-1, but not anti-CTLA-4, therapy. We conclude that primary response to anti-CTLA-4 requires robust melanoma MHC class I expression. In contrast, primary response to anti-PD-1 is associated with preexisting IFN-γ-mediated immune activation that includes tumor-specific MHC class II expression and components of innate immunity when MHC class I is compromised. The benefits of combined checkpoint blockade may be attributable, in part, to distinct requirements for melanoma-specific antigen presentation to initiate antitumor immunity.

Background: Given the demonstrated utility of Third Generation Sequencing [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand. Methods: Based on the transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of size-selected PacBio, non-size-selected ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina). Results: PacBio shows overall better data quality, while ONT provides a higher yield. As with data quality, PacBio performs marginally better than ONT in most aspects for both long reads only and Hybrid-Seq strategies in transcriptome analysis. In addition, Hybrid-Seq shows superior performance over long reads only in most transcriptome analyses. Conclusions: Both PacBio and ONT sequencing are suitable for full-length single-molecule transcriptome analysis. As this first use of ONT reads in a Hybrid-Seq analysis has shown, both PacBio and ONT can benefit from a combined Illumina strategy. The tools and analytical methods developed here provide a resource for future applications and evaluations of these rapidly-changing technologies.

These authors contributed equally to this work. SUMMARYDanshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short-read next-generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full-length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single-molecule real-time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long-read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.

IMPORTANCE Novel approaches are needed to improve outcomes in patients with squamous cell carcinoma of the oral cavity. Neoadjuvant immunotherapy given prior to surgery and combining programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) immune checkpoint inhibitors are 2 strategies to enhance antitumor immune responses that could be of benefit. DESIGN, SETTING, AND PARTICIPANTSIn this randomized phase 2 clinical trial conducted at 1 academic center, 29 patients with untreated squamous cell carcinoma of the oral cavity (ՆT2, or clinically node positive) were enrolled between 2016 to 2019.INTERVENTIONS Treatment was administered with nivolumab, 3 mg/kg, weeks 1 and 3, or nivolumab and ipilimumab (ipilimumab, 1 mg/kg, given week 1 only). Patients had surgery 3 to 7 days following cycle 2.MAIN OUTCOMES AND MEASURES Safety and volumetric response determined using bidirectional measurements. Secondary end points included pathologic and objective response, progression-free survival (PFS), and overall survival. Multiplex immunofluorescence was used to evaluate primary tumor immune markers.RESULTS Fourteen patients were randomized to nivolumab (N) and 15 patients to nivolumab/ipilimumab (N+I) (mean [SD] age, 62 [12] years; 18 men [62%] and 11 women [38%]). The most common subsite was oral tongue (n = 16). Baseline clinical staging included patients with T2 (n = 20) or greater (n = 9) T stage and 17 patients (59%) with node-positive disease. Median time from cycle 1 to surgery was 19 days (range, 7-21 days); there were no surgical delays. There were toxic effects at least possibly related to study treatment in 21 patients, including grade 3 to 4 events in 2 (N), and 5 (N+I) patients. One patient died of conditions thought unrelated to study treatment (postoperative flap failure, stroke). There was evidence of response in both the N and N+I arms (volumetric response 50%, 53%; pathologic downstaging 53%, 69%; RECIST response 13%, 38%; and pathologic response 54%, 73%, respectively). Four patients had major/complete pathologic response greater than 90% (N, n = 1; N+I, n = 3). With 14.2 months median follow-up, 1-year progression-free survival was 85% and overall survival was 89%.CONCLUSIONS AND RELEVANCE Treatment with N and N+I was feasible prior to surgical resection. We observed promising rates of response in both arms, supporting further neoadjuvant studies with these agents.

The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi-or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.

Key Points CTLA-4 is enriched in the microenvironmental niche in cHL, and identifies a T-cell population largely distinct from PD-1 CD86, the natural ligand for CTLA-4, is enriched in the niche and expressed on HRS cells and a subset of TAMs that also express PD-L1

Given the demonstrated utility of Third Generation Sequencing Background: [Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)] long reads in many studies, a comprehensive analysis and comparison of their data quality and applications is in high demand.Based on the Methods: transcriptome sequencing data from human embryonic stem cells, we analyzed multiple data features of PacBio and ONT, including error pattern, length, mappability and technical improvements over previous platforms. We also evaluated their application to transcriptome analyses, such as isoform identification and quantification and characterization of transcriptome complexity, by comparing the performance of PacBio, ONT and their corresponding Hybrid-Seq strategies (PacBio+Illumina and ONT+Illumina).PacBio shows overall better data quality, while ONT provides a higher Results: yield. As with data quality, PacBio performs marginally better than ONT in most aspects for both long reads only and Hybrid-Seq strategies in transcriptome analysis. In addition, Hybrid-Seq shows superior performance over long reads only in most transcriptome analyses.Both PacBio and ONT Conclusions: sequencing are suitable for full-length single-molecule transcriptome analysis. As this first use of ONT reads in a Hybrid-Seq analysis has shown, both PacBio and ONT can benefit from a combined Illumina strategy. The tools and analytical methods developed here provide a resource for future applications and evaluations of these rapidly-changing technologies.

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell–dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. Significance: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell–based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials. This article is highlighted in the In This Issue feature, p. 1307