Antigen Processing Pathway Research Articles

Selected Articles from This Issue

BRAF mutation is a common driver event in melanoma, rendering the tumors reliant on constitutive signaling through the MAp. pathway and the extracellular signal-regulated kinases (ERK) 1 and 2. Thus, MAPK pathway inhibitors that act through both ERK1 and ERK2 have become a key component of the therapeutic arsenal against melanoma, but their use is associated with dose-limiting toxicities. Here, Crowe and colleagues demonstrate that BRAF-mutant melanoma is specifically reliant on ERK2 signaling. Targeted ERK2 ablation resulted in transcriptomic and proteomic alterations that aligned with those effected by full MAPK pathway inhibitors, whereas targeted ERK1 ablation produced a distinct set of responses. Both isoforms promoted cell viability, but ERK2 loss suppressed MAPK pathway activity and cell proliferation more effectively than ERK1 loss. Moreover, ERK2 was shown to compensate for ERK1 depletion in the context of BRAF mutation, whereas ERK1 was not able to compensate for loss of ERK2 activity. The authors argue that these findings support the development of ERK2-selective inhibitors, which could potentially be achieved through the targeting of protein-protein interaction domains that are differentially utilized between the two isoforms.Mutation of DNA damage-responsive Fanconi Anemia family members (FANC) is associated with hereditary cancer syndromes and familial cancers, particularly breast and ovarian cancer, due to their role in mediating homologous recombination repair. In particular, FANCJ has been nominated as a key tumor suppressor in several cancer types, though functional data on the many clinically identified FANCJ mutations are lacking. Here, Calvo and colleagues employ a mutation library to screen for loss-of-function (LOF) mutations that confer sensitivity to interstrand crosslinking (ICL) agents. They find that missense and nonsense mutations in the helicase domain are associated with FANCJ LOF, whereas mutations outside this domain did not affect the response to ICL chemotherapeutics. Notably, mutations in the carboxy-terminal domain, which mediates protein-protein interactions with other homologous recombination mediators such as BRCA1, did not significantly affect FANCJ function in response to ICL agents. Finally, while only a minority of FANCJ mutations (∼12%) exhibited LOF in response to ICL, the authors note that the comprehensive mutation library developed here will continue to be useful in characterizing FANCJ mutants and their functionality in response to other stimuli and other types of genomic lesions.Circulating tumor cells (CTC) provide important prognostic information in metastatic castration-resistant prostate cancer (mCRPC), in which the expression of androgen receptor (AR) splice variants is a key prognostic indicator of patient response to enzalutamide and/or abiraterone (enza/abi). However, most patients do not express AR variants, and therefore additional markers are needed. In this study, Gupta et al. leverage CTC exome sequencing and cop. number alteration profiles from men with mCRPC undergoing therapy with enza/abi to identify genomic events associated with therapy resistance. The authors identify a subset of patients who progressed rapidly on enza/abi and bore genomic lesions that were detectable in pooled CTC DNA, including gain of MYCN, BRCA2, and AR, and loss of CHD1, NCOR2, and PTEN. In patients who initially responded and later acquired resistance, CTC sampling uncovered mutations and copy number alterations which converged on genes linked to proliferation, hormone signaling, lineage plasticity and epigenetic signaling, and resistance to cell death. Taken together, these data could form the molecular basis for more effective therapeutic decision making in mCRPC.Tumors commonly down-regulate antigen processing and presentation pathways in order to evade the anti-tumor immune response, which can undermine the efficacy of immune checkpoint blockade. Here, Zhang and colleagues show that Qa-1b—the mouse homolog of the human major histocompatibility complex class I molecule HLA-E—is a key player in how tumors and the immune system interact when antigen presentation is defective. Loss of TAP1 resulted in the establishment of an immunosuppressive tumor microenvironment, but loss of both TAP1 and Qa-1b restored sensitivity to immunotherapy. This effect was mediated at least in part through direct interactions between Qa-1b and natural killer cells, likely through the inhibitory NKG2A receptor through which Qa-1b and HLA-E normally act. Concurrent disruption of TAP1 and Qa-1b reversed immunoresistance by causing increased CD8+ T cell to Treg ratios, increased natural killer cell infiltration and activity, and sensitization to immune checkpoint blockade with anti-PD-1 antibodies. The data suggest that Qa-1b and HLA-E engage in crosstalk with the antigen presentation machinery and represent a key control node that governs the immune response to antigen presentation by tumor cells.

Why TAPBPR? Implications of an additional player in MHC class I peptide presentation.

The peptide editor TAPBPR is the newest member of the major histocompatibility complex class I (MHC-I) antigen processing and presentation pathway. Since 2013, studies have explored the functions and mechanisms of action of this tapasin homolog. Here, we review the key insights gained from structural studies of the TAPBPR:MHC-I complex and the involvement of the TAPBPR loop in peptide exchange. However, despite recent advances, the question still remains: why do we need TAPBPR? The recent appreciation that different MHC-I allotypes vary in their ability to interact with TAPBPR, together with a role for TAPBPR in alternative presentation pathways highlights that much remains unknown concerning the biological need for TAPBPR.

Cancer pathways analysis and correlation with survival in patients with advanced stage non-small cell lung cancer treated with PD-1 inhibitor.

e21007 Background: Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein-1 (PD-1) have demonstrated activity in the first-line setting of advanced stage non-small cell lung cancer (NSCLC); however, a proportion of patients progresses to immunotherapy, hence the definition of prognostic and predictive factors is of interest. Potentially, the expression pattern of gene sets can serve as clinical indicator. We hereby present data from a pilot study of a larger project of gene expression analysis in patients with advanced NSCLC treated with ICIs, to explore the baseline expression level of single and grouped-in-pathways genes and to draw correlations with the overall survival (OS). Methods: We performed a retrospective transcriptome analysis of diagnostic cancer tissue from 14 consecutive patients with advanced stage NSCLC with PD-L1 expression > 50% treated with anti-PD-1 pembrolizumab as first-line. Two hundred ng of RNA was profiled by Nanostring technology using the human nCounter® PanCancer IO 360™ Panel, that includes 770 genes from 14 different immune cell types, common checkpoint inhibitors, Cancer-Testis antigens, and genes covering both the adaptive and innate immune response. Data analysis was performed by nCounter Advanced Analysis (version 2.0.134) plug in for nSolver Software and R package. Results: The median age of patients was 71 years (range 59-88), most were males (78%) with an ECOG performance status ranging from 0 to 1 (86%). All patients were smokers or former smokers. Seven out of 14 patients (50%) had a partial/complete response as best response, whereas the remaining population reported disease progression, 3 of whom died before the first CT-scan evaluation. The median OS was 12 months (range 1–40). The 770-tumor/immune gene expression profiling reported 11 genes (6 overexpressed and 5 down-modulated in patients with a survival under the median), which were significantly associated with poor outcome ( p-value < 0.01). Notably, when we adjusted the expression data to the site of biopsy (tumor tissue vs. neoplastic lymph node), we identified a 27-gene signature ( ATG5, BMI1, CASP1, CCL11, CCL8, CD164, CD86, CD97, EGR1, FADD, FOS, HLA-DQA1, HLA-DQB1, IFITM1, IRF3, LY96, MCAM, NFKBIA, RRAD, SMAD2, STAT6, TLR3, TNFAIP3, TNFRSF1A, TOLLIP, VEGFC, and YTHDF2) able to separate patients with a survival below or above the median. The in-silico prediction of this signature showed that the antigen processing pathway (HLA-DQA1, HLA-DQB1), already described as an immune checkpoint resistance mechanism, was significantly down-modulated in long survivors. Conclusions: By a multiplexed analysis of gene expression through messenger RNA digital detection, we identified a gene set that, if confirmed, might have a prognostic value in patients with advanced stage NSCLC with PD-L1 expression >50% treated with pembrolizumab as first-line.

The genomic and transcriptomic characteristics of early recurrence patients in stage IA non-small cell lung cancer.

e20547 Background: Lung cancer is the leading cause of cancer-related death worldwide. The mortality risk is still high in early stage because of the high relapse rates about 30–45% within 5 years after surgery. The main purpose of this study was to depict the genomic and transcriptomic characteristics in stage IA non-small cell lung cancer (NSCLC) patients and elucidate the recurrence risk factors for providing more postoperative intervention management and treatment. Methods: We prospectively enrolled 60 patients with stage IA NSCLC from 2012 to 2018 including 29 non-relapse patients and 31 early-relapse patients within 5 years. A total of 60 primary tumor specimens and 10 relapse tumor specimens were obtained. The paired adjacent non-tumor tissues for each patient were collected as negative control. DNA and RNA were co-extracted and performed with whole exon and transcriptome sequencing. The immune cell infiltration scores were estimated using ssGSEA algorithm. Results: The most frequently mutated genes in our cohort were EGFR (46.4%), TP53 (38.0%), TTN (35.2%) and USH2A (21.1%). Notably, USHA was enriched in relapse patients (32.3% vs 3.4%, p = 0.006). The median tumor mutation burden of the post-relapse group was 6.14/Mb, significantly higher than the pre-relapse group (Median: 2.63/Mb, p = 0.047) and the non-relapse group (1.91/Mb, p = 0.025). Among the early-relapse patients, copy number amplification at 2q31.1 was prevalent in the pre-relapse group (p < 0.05). Two patterns of clonal evolution were identified during the recurrence, including autonomous clonal evolution and acquired clonal evolution. Compared with non-tumor tissues, differentially expressed genes in pre-relapse affected the metabolism-associated pathways. Furthermore, post-relapse samples down-regulated antigen processing and presentation signaling pathway compared to non-relapse group. Interestingly, lower plasma dendritic cell infiltration was detected in post-relapse (p = 0.0078), indicating the defect of antigen presentation in recurrence. Concentrating on the primary tumor specimens, samples from relapse patients showed higher infiltration of CD8+T cell (p = 0.019), Helper T cells (p = 0.0036) and Treg cells (p = 0.0071) than that from non-relapse patients. Conclusions: This study elucidated the recurrence risk molecular and immune microenvironment in stage IA NSCLC patients. Findings in our research may help find out the subset of patients with early stage at high risk for recurrence who have the urgent need for postoperative intervention and guide therapeutic strategies.

Seq-ing the source of radiation-induced anti-tumor immunogenicity in patient tumors

Abstract Translational studies in a small number of patients have demonstrated a clinical benefit when combining radiation and immunotherapy. These findings are supported by correlative data showing that transcriptional changes to the tumor microenvironment following radiotherapy are associated with responses. Yet, given limited access to patient specimens following treatment, few studies have addressed how radiation primes anti-tumor immunity at single cell resolution, thus limiting our understanding of the source of radiation-induced changes within tumors. Here, we acquired renal cell carcinoma (RCC) tumors from patients treated with stereotactic body radiation therapy (SBRT) followed by nephrectomy in a pilot study (NCT01892930), to evaluate radiation-induced transcriptional changes to immune and RCC cells by single-cell RNA sequencing. We hypothesized that SBRT-induced interferon (IFN) and immune-activating signals, previously detected by bulk sequencing and array platforms, would localize to the dense lymphocyte infiltrate characterized in RCC and that the transcriptional signatures in SBRT-treated RCC would reflect increased immune recognition. Single-cell transcriptomics uncovered increased frequency of mature T cells in irradiated tumors, including increased expression of IFN-gamma and granzymes, as well as PD1 and other exhaustion markers. Additionally, irradiated-RCC cells expressed more IFN-gamma receptor and were enriched for IFN signaling and antigen processing pathways. Collectively, these results indicate radiation may improve anti-tumor immunity by increasing both T cell activity and tumor cell sensitivity to pro-inflammatory cytokines and cytolysis in human malignancies.

Unusual distribution of a non-classical MHC I restricted CD8+ T population

Abstract A broad repertoire of peptides is constitutively presented through the MHC I molecules on the cell surface. ER aminopeptidase associated with Ag processing (ERAAP), a critical molecule in the antigen processing and presentation pathway, has profound effects on the generation of this peptide repertoire. Interestingly, the ERAAP deficient cells can be effectively detected by a unique population of the cytotoxic CD8+ T cell, named QFL-T cells which are specific for the ligand presented by the non-classical MHC Ib molecule, Qa-1b. The QFL-T cells showed similar characteristics with the other non-classical MHC Ib restricted unconventional T cells, such as the NKT or MAIT cells in their semi-invariant αβ TCRs, but their function(s) remains unknown. To study the potential function of the QFL-T cells, we assessed a variety of tissues for the ligand expression and the presence of the QFL-T cells. We observed high expression of this ERAAP dependent Qa-1b restricted ligand and relatively high frequency of the QFL-T cells in the small intestine. Remarkably, the number and the frequency of QFL-T cells among the intraepithelial lymphocytes (IELs) was tightly associated with the age and the gut microbiota of the mice. These findings suggest a role for gut microbiota in the homing/maintenance of the QFL-T cells in the small intestine.

Distinguishing Signal From Noise in Immunopeptidome Studies of Limiting-Abundance Biological Samples: Peptides Presented by I-Ab in C57BL/6 Mouse Thymus.

Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.

Analysis of transcripts and splice isoforms in red claw crayfish (Cherax quadricarinatus) using single-molecule long-read sequencing

Red claw crayfish (Cherax quadricarinatus) has recently attracted widespread attention as an emerging candidate for sustainable aquaculture production in Australia and abroad. Although they are of great economic importance, few full-length transcriptomes are available. Here, transcripts of C. quadricarinatus were generated by using Pacific Biosciences single-molecule real-time (SMRT) long-read sequencing technology. With SMRT, 289,232 full-length nonchimeric reads were identified, resulting in 14,089 unigene, with an N50 length of 4363 bp and a mean read length of 3913 bp. The error rate of SMRT sequences by comparison with Illumina-produced short reads was only 0.011%. A total of 13,449 transcripts (95.4%) were annotated against the protein database. Across all transcripts, 4952 long noncoding RNAs (lncRNAs), 1090 putative TF members and 21,108 simple sequence repeats (SSRs) were identified. After Vibrio alginolyticus infection, a total of 252 DEGs were screened out after a comparative analysis between samples. Of the 252 DEGs, Hsps, IAPs and Gpx were upregulated during V. alginolyticus infection, many genes were associated with V. cholerae infection, the NF-kappa B signalling pathway, apoptosis-multiple species, the TNF signalling pathway and antigen processing and presentation pathways. Our study provides a rich set of full-length cDNA sequences for C. quadricarinatus, which will greatly facilitate crayfish transcriptome research.

Abstract PO-038: Hide and Seq: Radiation-induced immunogenicity of patient tumors revealed by single cell analysis

Abstract Robust preclinical investigations into radiation-induced immunogenicity has led to increased enthusiasm for combining radiation with immunotherapy. Promising results in a limited number of patients treated with radiotherapy and immune-activating strategies has supported the initiation of over 150 ongoing clinical trials. Yet, due to reduced availability of patient samples following treatment, tumor tissue in particular, there has been sparse investigation of the impact of radiation on immunogenicity in human malignancies. While murine models have revealed important insights into understanding radiation-induced immunogenic mechanisms, inherent differences in radiation sensitivity between mice and humans limit translation to patients. To determine the impact of radiation on patient tumor cells we analyzed samples from renal cell carcinoma (RCC) patients treated with stereotactic body radiation therapy (SBRT) in a pilot study (NCT01892930). Previous data showed radiation induced surface presentation of tumor-associated antigens in resected irradiated tumors. Bulk transcriptomic data of in situ-irradiated tumors showed evidence of immune activation, including increased interferon-gamma (IFNG) signaling, as well as increased T cell clonality and higher abundance of T cells clones with similar binding affinities predicted by dominant motif analysis. We hypothesized that the tumor response to in situ irradiation included increased transcript abundance for tumor antigen genes and antigen-presentation machinery, as well as a response to IFNG signaling. Tumor cell comparisons following single-cell RNA sequencing uncovered RCC cell-specific responses to in situ irradiation. Irradiated RCC tumor cells expressed higher levels of genes encoding MUC-1 and carbonic anhydrase 9, as well as higher expression of calreticulin and genes encoding MHC class 1 molecules: HLA-A, HLA-B, and HLA-C; furthermore, gene set enrichment identified enhancement of antigen processing and presentation pathways. Additionally, irradiated tumor cells expressed higher levels of the IFNG receptor, its downstream targets, and the enrichment of several interferon response gene sets. Collectively, these results indicate radiation may improve tumor recognition through both increased antigen expression and surface presentation as well as sensitize tumor cells to immunogenic cytokines and T cell-mediated cytolysis in human malignancies, such as RCC. Citation Format: Jacky L. Chow, Nicholas Hoffend, Madeline Gaudieri, Scott Abrams, Thomas Schwaab, Anurag Singh, Jason Muhitch. Hide and Seq: Radiation-induced immunogenicity of patient tumors revealed by single cell analysis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-038.

Circular RNA expression alteration identifies a novel circulating biomarker in serum exosomal for detection of alcohol dependence.

Alcohol dependence (AD) is one of the most common and detrimental neuropsychological disorders. Recently, more and more studies have focused on circular RNA as markers for central nervous system (CNS) diseases. The present study was conducted to evaluate the circular RNA expression alteration in serum exosomal and to identify a novel circulating biomarker for the detection of AD. We first isolated exosomes from serum and then investigated the circRNA expression alterations by high throughput whole transcriptome sequencing. The data were then analyzed using bioinformatics methods. Moreover, we verified the circRNA-seq by qRT-PCR. Furthermore, we analyzed the correlations between the levels of hsa_circ_0004771 and both Severity of Alcohol Dependence Questionnaire (SADQ) and Alcohol Dependence Scale (ADS). The diagnostic value of hsa_circ_0004771 in AD patients was evaluated by receiver operating characteristic (ROC). In this study, 254 differentially expressed circRNAs were identified, with 149 upregulated and 105 downregulated. GO analysis showed that these differentially expressed circRNAs from exosomes might be associated with the regulation of neuron projection and axon regeneration. KEGG analysis revealed that T cell receptor signaling and antigen processing and presentation pathway had a regulating effect on upstream levels. We found that hsa_circ_0004771 was related to the severity of AD. The AUC for the diagnostic value of hsa_circ_0004771 in AD patients was 0.874. These findings indicated that circRNA in serum exosomes provide novel targets for further research on molecular mechanisms of AD. Among these, hsa_circ_0004771 may be a sensitive biomarker that was related to the severity of AD.

Making Insulin and Staying Out of Autoimmune Trouble: The Beta-Cell Conundrum

Autoimmune type 1 diabetes (T1D) results from the intricate crosstalk of various immune cell types. CD8+ T cells dominate the pro-inflammatory milieu of islet infiltration (insulitis), and are considered as key effectors of beta-cell destruction, through the recognition of MHC Class I-peptide complexes. The pathways generating MHC Class I-restricted antigens in beta cells are poorly documented. Given their specialized insulin secretory function, the associated granule processing and degradation pathways, basal endoplasmic reticulum stress and susceptibility to additional stressors, alternative antigen processing and presentation (APP) pathways are likely to play a significant role in the generation of the beta-cell immunopeptidome. As direct evidence is missing, we here intersect the specificities of beta-cell function and the literature about APP in other cellular models to generate some hypotheses on APPs relevant to beta cells. We further elaborate on the potential role of these pathways in T1D pathogenesis, based on the current knowledge of antigens presented by beta cells. A better understanding of these pathways may pinpoint novel mechanisms amenable to therapeutic targeting to modulate the immunogenicity of beta cells.

Immune gene expression networks in sepsis: A network biology approach.

To study the dysregulated host immune response to infection in sepsis, gene expression profiles from the Gene Expression Omnibus (GEO) datasets GSE54514, GSE57065, GSE64456, GSE95233, GSE66099 and GSE72829 were selected. From the Kyoto Encyclopedia of Genes and Genomes (KEGG) immune system pathways, 998 unique genes were selected, and genes were classified as follows based on gene annotation from KEGG, Gene Ontology, and Reactome: adaptive immunity, antigen presentation, cytokines and chemokines, complement, hematopoiesis, innate immunity, leukocyte migration, NK cell activity, platelet activity, and signaling. After correlation matrix formation, correlation coefficient of 0.8 was selected for network generation and network analysis. Total transcriptome was analyzed for differentially expressed genes (DEG), followed by gene set enrichment analysis. The network topological structure revealed that adaptive immunity tended to form a prominent and isolated cluster in sepsis. Common genes within the cluster from the 6 datasets included CD247, CD8A, ITK, LAT, and LCK. The clustering coefficient and modularity parameters were increased in 5/6 and 4/6 datasets in the sepsis group that seemed to be associated with functional aspect of the network. GSE95233 revealed that the nonsurvivor group showed a prominent and isolated adaptive immunity cluster, whereas the survivor group had isolated complement-coagulation and platelet-related clusters. T cell receptor signaling (TCR) pathway and antigen processing and presentation pathway were down-regulated in 5/6 and 4/6 datasets, respectively. Complement and coagulation, Fc gamma, epsilon related signaling pathways were up-regulated in 5/6 datasets. Altogether, network and gene set enrichment analysis showed that adaptive-immunity-related genes along with TCR pathway were down-regulated and isolated from immune the network that seemed to be associated with unfavorable prognosis. Prominence of platelet and complement-coagulation-related genes in the immune network was associated with survival in sepsis. Complement-coagulation pathway was up-regulated in the sepsis group that was associated with favorable prognosis. Network and gene set enrichment analysis supported elucidation of sepsis pathogenesis.

Correlations Between Tumor Mutation Burden and Immunocyte Infiltration and Their Prognostic Value in Colon Cancer.

BackgroundColon cancer has a huge incidence and mortality worldwide every year. Immunotherapy could be a new therapeutic option for patients with advanced colon cancer. Tumor mutation burden (TMB) and immune infiltration are considered critical in immunotherapy but their characteristics in colon cancer are still controversial.MethodsThe somatic mutation, transcriptome, and clinical data of patients with colon cancer were obtained from the TCGA database. Patients were divided into low or high TMB groups using the median TMB value. Somatic mutation landscape, differentially expressed genes, and immune-related hub genes, Gene Ontology and KEGG, gene set enrichment, and immune infiltration analyses were investigated between the two TMB groups. Univariate and multivariate Cox analyses were utilized to construct a prognostic gene signature. The differences in immune infiltration, and the expression of HLA-related genes and checkpoint genes were investigated between the two immunity groups based on single sample gene set enrichment analysis. Finally, a nomogram of the prognostic prediction model integrating TMB, immune infiltration, and clinical parameters was established. Calibration plots and receiver operating characteristic curves (ROC) were drawn, and the C-index was calculated to assess the predictive ability.ResultsMissense mutations and single nucleotide polymorphisms were the major variant characteristics in colon cancer. The TMB level showed significant differences in N stage, M stage, pathological stage, and immune infiltration. CD8+ T cells, activated memory CD4+ T cells, activated NK cells, and M1 macrophages infiltrated more in the high-TMB group. The antigen processing and presentation signaling pathway was enriched in the high-TMB group. Two immune related genes (CHGB and SCT) were identified to be correlated with colon cancer survival (HR = 1.39, P = 0.01; HR = 1.26, P = 0.02, respectively). Notably, the expression of SCT was identified as a risk factor in the immune risk model, in which high risk patients showed poorer survival (P = 0.04). High immunity status exhibited significant correlations with immune response pathways, HLA-related genes, and immune checkpoint genes. Finally, including nine factors, our nomogram prediction model showed better calibration (C-index = 0.764) and had an AUC of 0.737.ConclusionIn this study, we investigated the patterns and prognostic roles of TMB and immune infiltration in colon cancer, which provided new insights into the tumor microenvironment and immunotherapies and the development of a novel nomogram prognostic prediction model for patients with colon cancer.

PPP2R2B downregulation is associated with immune evasion and predicts poor clinical outcomes in triple-negative breast cancer

BackgroundAlthough immune checkpoint blockade has emerged as a novel promising strategy for triple-negative breast cancer (TNBC), many patients fail response or acquire resistance to current agents. Consequently, our focus need to shift toward alternative inhibitory targets, predictor for responsiveness, and immune suppressive mechanisms.MethodsIn this study, we performed systematic bioinformatics analyses to identify PPP2R2B as a robust tumor suppressor in TNBC. Meanwhile, breast cancer progression cell line model was applied in our research. Quantitative real-time PCR assay (Q-PCR) was carried out to assess the role of PPP2R2B in the onset and progression of breast cancer. Furthermore, we validated the effect of PPP2R2B on immune activity via in vitro experiments based on macrophages. To further decipher the roles of PPP2R2B in TNBC, we investigated the transcriptome level, genomic profiles, and its clinical prognostic value.ResultsIn TNBC tissues, PPP2R2B expression was significantly downregulated compared to normal breast tissues. Kaplan‐Meier survival analysis revealed that patients with low PPP2R2B expression had shorter survival time than those with high PPP2R2B expression. Q-PCR analysis suggested that PPP2R2B downregulation could play a key role in breast-cancer initiation and progression. Additionally, our findings showed that PPP2R2B was positively related with CD8 T cells, CD4 Th1 helper cells, and M1 macrophages, but negatively related with M2 macrophages. Subsequent results identified that PPP2R2B was strongly related with immune inhibitor genes (GZMA, PRF1, and IFNG), which could improve T lymphocytes antitumor function and restrict immune evasion. Meanwhile, T cell receptor signaling pathway and antigen processing and presentation signaling pathway were significantly suppressed in low PPP2R2B expression group. Afterwards, distinct subgroups based on PPP2R2B expression exhibited several unique features in somatic mutations, copy numbers alterations, extent of copy number burden, and promoter methylation level.ConclusionOur results indicated that PPP2R2B could serve as a promising biomarker for TNBC, and help predict immunotherapeutic response and guide personalized strategies in TNBC treatment.

An experimental model of anti-PD-1 resistance exhibits activation of TGFß and Notch pathways and is sensitive to local mRNA immunotherapy

ABSTRACT Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNγ signaling pathways. However, such mutations occur in a low frequency of patients and additional mechanisms have yet to be elucidated. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or β2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. Unbiased gene expression analysis was used to further characterize potential resistance mechanisms. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFβ and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFβ and Notch in combination with PD-1 blockade decelerated tumor growth, a local mRNA-based immunotherapy potently induced regression of resistant tumors, resulting in complete tumor remission, and resensitized tumors to treatment with anti-PD-1. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade.

HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY

Human leukocyte antigen (HLA) molecules play critical roles in our adaptive immune system by signaling a cell's health status to the immune system, through presentation of small peptides. Understanding HLA biology is important because of its prominent role in autoimmune diseases and cancer immunotherapy. Although both the HLA class I and class II antigen processing and presentation pathways have been studied extensively, the fundamental rules in HLA class II antigen presentation still remain less understood. To clarify the mechanistic and adaptive differences between the HLA systems, we challenged a B lymphoblastic cell line (JY), widely used as model system in studying antigen presentation, with a high temperature treatment to mimic a “fever-like state”, representing one of the most common physiological responses to infection. In the absence of real invading pathogenic peptides to present, we could focus on delineating the intrinsic HLA pathway adaptations in response to high temperature in this particular cell line. Following a three-pronged approach, we performed quantitative analyses of the proteome, the HLA class I ligandome, as well as the HLA class II ligandome. The data reveals that elevated temperature may already prepare these cells for an immune-like response through increased HLA class II presentation capacity and specific release of, from the invariant chain originating, CLIP peptides. Interestingly, at high temperature, prominent changes in the composition of the CLIP repertoire were observed, with enrichment of peptides containing C-terminal extensions beyond the CLIP-core region. Collectively, these illustrate intriguing temperature sensitive adaptations in this B cell line.

The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.

Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8+ T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.

Integrative analysis identifies copy number variations and their controlled causal molecular networks in Alzheimer’s disease

AbstractBackgroundThe pathogenic mechanism of late‐onset Alzheimer’s disease (LOAD) remains elusive despite intensive studies in the past few decades. Even though ‐27 AD risk loci have been identified through genome‐wide association studies, they explain only a small fraction of the heritability of LOAD. LOAD is such a complex disease that more types of genetic variations need to be studied. Copy number variation (CNV), as one primary type of genomic variation, has been found to play a significant role in many neurological diseases such as Parkinson’s disease, schizophrenia, mental retardation, and AD. A systematic functional study of CNVs through omics‐levels can provide valuable insights into the mechanism of LOAD.MethodIn this study, we employed four complementary CNV calling approaches including CNVnator, Pindel, MetaSV and Delly2 to comprehensively identify CNVs in the WGS data from 1,662 individuals (including 827 LOAD, 330 mild cognitive impairment, and 505 control subjects) across three LOAD cohorts including the Mount Sinai/JJ Peters VA Medical Center Brain Bank (MSBB) AD cohort, the ROSMAP cohort, and the Mayo Clinic cohort. Integrative analysis of the identified CNVs and the matched transcriptomic, proteomic, clinical and pathological data was then performed to determine the functional impacts of these CNVs.ResultGenes associated with CNVs at the transcriptomic level in seven different brain regions were consistently enriched for glutathione metabolism and antigen processing and presentation pathways. The mRNA and protein expression levels of two genes (ACOT2, and GSTM1) in the frontal pole were significantly correlated with cis‐regulatory CNVs in AD cases. Rare LOAD specific CNVs were involved in axonogenesis and neurite morphogenesis. The LOAD cases had a higher disease‐specific CNV burden compared with the normal controls. Moreover, the causal networks based on the integration of CNV, transcriptomic, proteomic, and clinicopathological data demonstrated brain region‐specificity.ConclusionTo our knowledge, this is the first genomic CNV study focused on LOAD by integrating genomic, transcriptomic, proteomic, and clinicopathologic data. The identified CNVs and their downstream molecular networks provide a blueprint for studying the pathogenic mechanisms of LOAD.

Different doses of ovalbumin exposure on dendritic cells determine their genetic/epigenetic regulation and T cell differentiation.

It has been reported that allergen dosage can impact the differentiation of dendritic cells (DCs)-mediated T cells. However, the mechanisms of such dose-dependent differentiation are poorly understood. In this study, bone marrow-derived immature DCs stimulated with Ovalbumin (OVA) of different concentrations (0, 10, 100, 1000, 10000μg/ml, respectively). DCs were then co-cultured with naïve T cells. RNA-sequencing detection and DNA methylation of DCs were performed. We show that when DCs were stimulated with low-dose (10μg/ml), 77 genes were up-regulated and 87 genes down-regulated. Most activated genes were related to ribosome synthesis and ion channel inhibition. At the medium-dose (100μg/ml), 339 genes were up-regulated and 168 genes down-regulated. Most activated genes involved cytokine synthesis and regulation of immune responses. At high-dose (10000μg/ml), 2497 genes were up-regulated and 1156 genes down-regulated. TNF signaling pathway, NF-kappa B signaling pathway, antigen processing and presentation signaling pathway were mostly up-regulated. The related co-stimulators, co-inhibitory molecules, inhibitory cytokines, negative regulating enzymes were highly expressed. The monocarbate, coenzyme, fatty acid, glucolipid, starch, sucrose and other metabolism-related signaling pathways were down-regulated. The profiles of DNA methylation and RNA synthesis of DCs varied with different doses of OVA, which serves to induce T cells to differentiate in various directions.

Fidelity of human ovarian cancer patient-derived xenografts in a partially humanized mouse model for preclinical testing of immunotherapies

BackgroundImmune checkpoint blockers (ICBs) have been approved by the Food and Drug Administration to be used alone in front-line therapies or in combination with other regimens for certain advanced cancers....