IGSF8 is an innate immune checkpoint and cancer immunotherapy target.

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival in vitro or in vivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells in vitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and T cell signaling in vivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Protocol for fast clonal family inference and analysis from large-scale B cell receptor repertoire sequencing data.

The expeditious identification and comprehensive analysis of clonal families from extensive B cell receptor (BCR) repertoire sequencing data are imperative for elucidating the intricacies of B cell immune responses. Here, we introduce a computational pipeline designed to swiftly deduce clonal families from bulk BCR heavy-chain sequencing data, accompanied by a suite of functional modules tailored to streamline post-clustering analysis. The outlined methodology encompasses guidelines for software installation, meticulous data preparation, and the systematic inference and analysis of clonal families. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Fast clonal family inference from large-scale B cell repertoire sequencing data.

Advances in high-throughput sequencing technologies have facilitated the large-scale characterization of B cell receptor (BCR) repertoires. However, the vast amount and high diversity of the BCR sequences pose challenges for efficient and biologically meaningful analysis. Here, we introduce fastBCR, an efficient computational approach for inferring B cell clonal families from massive BCR heavy chain sequences. We demonstrate that fastBCR substantially reduces the running time while ensuring high accuracy on simulated datasets with diverse numbers of B cell lineages and varying mutation rates. We apply fastBCR to real BCR sequencing data from peripheral blood samples of COVID-19 patients, showing that the inferred clonal families display disease-associated features, as well as corresponding antigen-binding specificity and affinity. Overall, our results demonstrate the advantages of fastBCR for analyzing BCR repertoire data, which will facilitate the identification of disease-associated antibodies and improve our understanding of the B cell immune response.

Fluorescent detection of brown spot of tobacco caused by Alternaria alternata based on lambda exonuclease-induced DNAzyme amplification.

A rapid, simple, and sensitive fluorescent detection method for brown spot of tobacco is established by lambda exonuclease-induced Mg2+-dependent DNAzyme amplification. It contains hybridization of the Alternaria alternata genome and HP1, digestion of the 5'-phosphorylated strand of the hybrid dsDNA by lambda exonuclease, acquisition of complete Mg2+-dependent DNAzyme, cleavage of the substrate modified with FAM and BHQ-1, and fluorescent detection. The proposed assay exhibits good sensitivity (10 pg L-1), selectivity and reproducibility. The method does not require pure DNA and expensive instruments, and can be performed within 2.5 hours. To the best of our knowledge, this is the first report of fluorescent detection of Alternaria alternata and its tobacco field samples. This method can be applied to the rapid and sensitive detection of Alternaria alternata in tobacco and its seedlings, and is particularly important for the green prevention and control of tobacco brown spot disease.

Subperiosteal orbital hematoma concomitant with abscess in a patient with sinusitis: A case report.

BACKGROUND: Sinusitis-derived subperiosteal orbital collection is uncommon and is usually diagnosed as subperiosteal orbital abscess or, rarely, as hematoma. We report a unique and even rarer case of subperiosteal orbital collection, which is actually a complication of hematoma and abscess. CASE SUMMARY: A 26-year-old female presented with left eyeball pain and ipsilateral chemosis. She had no history of head trauma or upper respiratory infection. Her blood cell count showed an increase in leukocytes. Fiberoptic rhinolaryngoscopy revealed only mucosal edema of the left olfactory crest without apparent discharge. The computed tomography scan results showed an opaque left posterior ethmoid cell with a thickened bony shell and fusiform changes in the periosteal elevation of the medial wall of the left orbit. Emergent surgery revealed an ethmoid mucocele complicated with subperiosteal orbital hematoma and abscess. The pathology of the lamina papyracea between the mucocele and subperiosteal collection was necrotic, and the overlying mucosa was de-epithelialized. CONCLUSION: Subperiosteal orbital hematoma with abscess in a patient with sinusitis adds to the current knowledge of orbital complications of sinusitis.

Comprehensive Characterizations of Immune Receptor Repertoire in Tumors and Cancer Immunotherapy Studies.

We applied our computational algorithm TRUST4 to assemble immune receptor (T-cell receptor/B-cell receptor) repertoires from approximately 12,000 RNA sequencing samples from The Cancer Genome Atlas and seven immunotherapy studies. From over 35 million assembled complete complementary-determining region 3 sequences, we observed that the expression of CCL5 and MZB1 is the most positively correlated genes with T-cell clonal expansion and B-cell clonal expansion, respectively. We analyzed amino acid evolution during B-cell receptor somatic hypermutation and identified tyrosine as the preferred residue. We found that IgG1+IgG3 antibodies together with FcRn were associated with complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity or phagocytosis. In addition to B-cell infiltration, we discovered that B-cell clonal expansion and IgG1+IgG3 antibodies are also correlated with better patient outcomes. Finally, we created a website, VisualizIRR, for users to interactively explore and visualize the immune repertoires in this study. See related Spotlight by Liu and Han, p. 786.

A high-resolution view of intra-tumoral B cell immunity.

In this issue of Immunity, Meylan et al. (2022) uses spatial transcriptomics to examine B cell immunity within intratumoral tertiary lymphoid structures (TLSs). They find that B cells expand and mature into plasma cells (PCs) within the TLS, migrate along fibroblastic tracks to tumor beds, and produce IgG antibodies that target cancer cells.

TRUST4: immune repertoire reconstruction from bulk and single-cell RNA-seq data.

We introduce the TRUST4 open-source algorithm for reconstruction of immune receptor repertoires in αß/γδ T cells and B cells from RNA-sequencing (RNA-seq) data. Compared with competing methods, TRUST4 supports both FASTQ and BAM format and is faster and more sensitive in assembling longer-even full-length-receptor repertoires. TRUST4 can also call repertoire sequences from single-cell RNA-seq (scRNA-seq) data without V(D)J enrichment, and is compatible with both SMART-seq and 5' 10x Genomics platforms.

Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments.

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics.

Clonal tracing reveals diverse patterns of response to immune checkpoint blockade.

BACKGROUND: Immune checkpoint blockade (ICB) therapy has improved patient survival in a variety of cancers, but only a minority of cancer patients respond. Multiple studies have sought to identify general biomarkers of ICB response, but elucidating the molecular and cellular drivers of resistance for individual tumors remains challenging. We sought to determine whether a tumor with defined genetic background exhibits a stereotypic or heterogeneous response to ICB treatment. RESULTS: We establish a unique mouse system that utilizes clonal tracing and mathematical modeling to monitor the growth of each cancer clone, as well as the bulk tumor, in response to ICB. We find that tumors derived from the same clonal populations showed heterogeneous ICB response and diverse response patterns. Primary response is associated with higher immune infiltration and leads to enrichment of pre-existing ICB-resistant cancer clones. We further identify several cancer cell-intrinsic gene expression signatures associated with ICB resistance, including increased interferon response genes and glucocorticoid response genes. These findings are supported by clinical data from ICB treatment cohorts. CONCLUSIONS: Our study demonstrates diverse response patterns from the same ancestor cancer cells in response to ICB. This suggests the value of monitoring clonal constitution and tumor microenvironment over time to optimize ICB response and to design new combination therapies. Furthermore, as ICB response may enrich for cancer cell-intrinsic resistance signatures, this can affect interpretations of tumor RNA-seq data for response-signature association studies.

A peripheral immune signature of responsiveness to PD-1 blockade in patients with classical Hodgkin lymphoma.

PD-1 blockade is highly effective in classical Hodgkin lymphomas (cHLs), which exhibit frequent copy-number gains of CD274 (PD-L1) and PDC1LG2 (PD-L2) on chromosome 9p24.1. However, in this largely MHC-class-I-negative tumor, the mechanism of action of anti-PD-1 therapy remains undefined. We utilized the complementary approaches of T cell receptor (TCR) sequencing and cytometry by time-of-flight analysis to obtain a peripheral immune signature of responsiveness to PD-1 blockade in 56 patients treated in the CheckMate 205 phase II clinical trial (NCT02181738). Anti-PD-1 therapy was most effective in patients with a diverse baseline TCR repertoire and an associated expansion of singleton clones during treatment. CD4+, but not CD8+, TCR diversity significantly increased during therapy, most strikingly in patients who had achieved complete responses. Additionally, patients who responded to therapy had an increased abundance of activated natural killer cells and a newly identified CD3-CD68+CD4+GrB+ subset. These studies highlight the roles of recently expanded, clonally diverse CD4+ T cells and innate effectors in the efficacy of PD-1 blockade in cHL.

Immune receptor repertoires in pediatric and adult acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML), caused by the abnormal proliferation of immature myeloid cells in the blood or bone marrow, is one of the most common hematologic malignancies. Currently, the interactions between malignant myeloid cells and the immune microenvironment, especially T cells and B cells, remain poorly characterized. METHODS: In this study, we systematically analyzed the T cell receptor and B cell receptor (TCR and BCR) repertoires from the RNA-seq data of 145 pediatric and 151 adult AML samples as well as 73 non-tumor peripheral blood samples. RESULTS: We inferred over 225,000 complementarity-determining region 3 (CDR3) sequences in TCR α, ß, γ, and δ chains and 1,210,000 CDR3 sequences in B cell immunoglobulin (Ig) heavy and light chains. We found higher clonal expansion of both T cells and B cells in the AML microenvironment and observed many differences between pediatric and adult AML. Most notably, adult AML samples have significantly higher level of B cell activation and more secondary Ig class switch events than pediatric AML or non-tumor samples. Furthermore, adult AML with highly expanded IgA2 B cells, which might represent an immunosuppressive microenvironment, are associated with regulatory T cells and worse overall survival. CONCLUSIONS: Our comprehensive characterization of the AML immune receptor repertoires improved our understanding of T cell and B cell immunity in AML, which may provide insights into immunotherapies in hematological malignancies.

Publisher Correction: Landscape of B cell immunity and related immune evasion in human cancers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Landscape of B cell immunity and related immune evasion in human cancers.

Tumor-infiltrating B cells are an important component in the microenvironment but have unclear anti-tumor effects. We enhanced our previous computational algorithm TRUST to extract the B cell immunoglobulin hypervariable regions from bulk tumor RNA-sequencing data. TRUST assembled more than 30 million complementarity-determining region 3 sequences of the B cell heavy chain (IgH) from The Cancer Genome Atlas. Widespread B cell clonal expansions and immunoglobulin subclass switch events were observed in diverse human cancers. Prevalent somatic copy number alterations in the MICA and MICB genes related to antibody-dependent cell-mediated cytotoxicity were identified in tumors with elevated B cell activity. The IgG3-1 subclass switch interacts with B cell-receptor affinity maturation and defects in the antibody-dependent cell-mediated cytotoxicity pathway. Comprehensive pancancer analyses of tumor-infiltrating B cell-receptor repertoires identified novel tumor immune evasion mechanisms through genetic alterations. The IgH sequences identified here are potentially useful resources for future development of immunotherapies.

RNA-binding protein DDX1 is responsible for fatty acid-mediated repression of insulin translation.

The molecular mechanism in pancreatic ß cells underlying hyperlipidemia and insulin insufficiency remains unclear. Here, we find that the fatty acid-induced decrease in insulin levels occurs due to a decrease in insulin translation. Since regulation at the translational level is generally mediated through RNA-binding proteins, using RNA antisense purification coupled with mass spectrometry, we identify a novel insulin mRNA-binding protein, namely, DDX1, that is sensitive to palmitate treatment. Notably, the knockdown or overexpression of DDX1 affects insulin translation, and the knockdown of DDX1 eliminates the palmitate-induced repression of insulin translation. Molecular mechanism studies show that palmitate treatment causes DDX1 phosphorylation at S295 and dissociates DDX1 from insulin mRNA, thereby leading to the suppression of insulin translation. In addition, DDX1 may interact with the translation initiation factors eIF3A and eIF4B to regulate translation. In high-fat diet mice, the inhibition of insulin translation happens at an early prediabetic stage before the elevation of glucose levels. We speculate that the DDX1-mediated repression of insulin translation worsens the situation of insulin resistance and contributes to the elevation of blood glucose levels in obese animals.

The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes.

Activation-induced cytidine deaminase (AID) mediates class switching by binding to a small fraction of single-stranded DNA (ssDNA) to diversify the antibody repertoire. The precise mechanism for highly selective AID targeting in the genome has remained elusive. Here, we report an RNA-binding protein, ROD1 (also known as PTBP3), that is both required and sufficient to define AID-binding sites genome-wide in activated B cells. ROD1 interacts with AID via an ultraconserved loop, which proves to be critical for the recruitment of AID to ssDNA using bi-directionally transcribed nascent RNAs as stepping stones. Strikingly, AID-specific mutations identified in human patients with hyper-IgM syndrome type 2 (HIGM2) completely disrupt the AID interacting surface with ROD1, thereby abolishing the recruitment of AID to immunoglobulin (Ig) loci. Together, our results suggest that bi-directionally transcribed RNA traps the RNA-binding protein ROD1, which serves as a guiding system for AID to load onto specific genomic loci to induce DNA rearrangement during immune responses.

Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response.

Cancer treatment by immune checkpoint blockade (ICB) can bring long-lasting clinical benefits, but only a fraction of patients respond to treatment. To predict ICB response, we developed TIDE, a computational method to model two primary mechanisms of tumor immune evasion: the induction of T cell dysfunction in tumors with high infiltration of cytotoxic T lymphocytes (CTL) and the prevention of T cell infiltration in tumors with low CTL level. We identified signatures of T cell dysfunction from large tumor cohorts by testing how the expression of each gene in tumors interacts with the CTL infiltration level to influence patient survival. We also modeled factors that exclude T cell infiltration into tumors using expression signatures from immunosuppressive cells. Using this framework and pre-treatment RNA-Seq or NanoString tumor expression profiles, TIDE predicted the outcome of melanoma patients treated with first-line anti-PD1 or anti-CTLA4 more accurately than other biomarkers such as PD-L1 level and mutation load. TIDE also revealed new candidate ICB resistance regulators, such as SERPINB9, demonstrating utility for immunotherapy research.

Mass cytometry of Hodgkin lymphoma reveals a CD4+ regulatory T-cell-rich and exhausted T-effector microenvironment.

In classical Hodgkin lymphoma (cHL), the host antitumor immune response is ineffective. Hodgkin Reed-Sternberg (HRS) cells have multifaceted mechanisms to evade the immune system, including 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) genetic alterations, overexpression of PD-1 ligands, and associated T-cell exhaustion and additional structural bases of aberrant antigen presentation. The clinical success of PD-1 blockade in cHL suggests that the tumor microenvironment (TME) contains reversibly exhausted T effector cells (Teffs). However, durable responses are observed in patients with ß2-microglobulin/major histocompatibility complex (MHC) class I loss on HRS cells, raising the possibility of non-CD8+ T cell-mediated mechanisms of efficacy of PD-1 blockade. These observations highlight the need for a detailed analysis of the cHL TME. Using a customized time-of-flight mass cytometry panel, we simultaneously assessed cell suspensions from diagnostic cHL biopsies and control reactive lymph node/tonsil (RLNT) samples. Precise phenotyping of immune cell subsets revealed salient differences between cHLs and RLNTs. The TME in cHL is CD4+ T-cell rich, with frequent loss of MHC class I expression on HRS cells. In cHLs, we found concomitant expansion of T helper 1 (Th1)-polarized Teffs and regulatory T cells (Tregs). The cHL Th1 Tregs expressed little or no PD-1, whereas the Th1 Teffs were PD-1+ The differential PD-1 expression and likely functional Th1-polarized CD4+ Tregs and exhausted Teffs may represent complementary mechanisms of immunosuppression in cHL.

Predicting essential proteins by integrating orthology, gene expressions, and PPI networks.

Identifying essential proteins is very important for understanding the minimal requirements of cellular life and finding human disease genes as well as potential drug targets. Experimental methods for identifying essential proteins are often costly, time-consuming, and laborious. Many computational methods for such task have been proposed based on the topological properties of protein-protein interaction networks (PINs). However, most of these methods have limited prediction accuracy due to the noisy and incomplete natures of PINs and the fact that protein essentiality may relate to multiple biological factors. In this work, we proposed a new centrality measure, OGN, by integrating orthologous information, gene expressions, and PINs together. OGN determines a protein's essentiality by capturing its co-clustering and co-expression properties, as well as its conservation in the evolution process. The performance of OGN was tested on the species of Saccharomyces cerevisiae. Compared with several published centrality measures, OGN achieves higher prediction accuracy in both working alone and ensemble.