NLRP4 negatively regulates type I interferon response and influences the outcome in anti-programmed cell death protein (PD)-1/PD-ligand 1 therapy.

The challenge to improve the clinical efficacy and enlarge the population that benefits from immune checkpoint inhibitors (ICIs) for non-small-cell lung cancer (NSCLC) is significant. Based on whole-exosome sequencing analysis of biopsies from NSCLC patients before anti-programmed cell death protein-2 (PD-1) treatment, we identified NLRP4 mutations in the responders with a longer progression-free survival (PFS). Knockdown of NLRP4 in mouse Lewis lung cancer cell line enhanced interferon (IFN)-α/ß production through the cGAS-STING-IRF3/IRF7 axis and promoted the accumulation of intratumoral CD8+ T cells, leading to tumor growth retardation in vivo and a synergistic effect with anti-PD-ligand 1 therapy. This was consistent with clinical observations that more tumor-infiltrating CD8+ T cells and elevated peripheral IFN-α before receiving nivolumab treatment were associated with a longer PFS in NSCLC patients. Our study highlights the roles of tumor-intrinsic NLRP4 in remodeling the immune contextures in the tumor microenvironment, making regional type I IFN beneficial for ICI treatment.

Exosomal PD-L1 predicts response with immunotherapy in NSCLC patients.

Immune Check-Point Inhibitors (ICIs) have shown remarkable promise in treating tumors, including non-small cell lung cancer (NSCLC). Nevertheless, the treatment response rate is low. Studies have found that the high expression of exosomal PD-L1 is one of the reasons for the low treatment response. Therefore, this study focused on the relationship between the exosomal PD-L1 and the clinical response to immunotherapy in NSCLC patients to evaluate whether it could be used as a biomarker to predict the efficacy of ICIs. In this study, clinical information and blood samples of 149 NSCLC patients receiving ICIs were collected. The expression level of exosomal PD-L1 was detected by enzyme-linked immunosorbent assay method, and the relationship between exosomal PD-L1 and the efficacy of ICIs was explored. Overall, our study found that the expression level of exosomal PD-L1 was lower at pre-treatment, or the max fold increasing change higher at 3-6 weeks had a higher disease control rate and longer progression-free survival. It revealed that the exosomal PD-L1 was associated with the treatment response of patients using ICIs and provided a new tool for the evaluation of clinical efficacy of lung cancer immunotherapy.

PD-1/PD-L1 Blockade Therapy in Advanced Non-Small-Cell Lung Cancer: Current Status and Future Directions.

The use of immune checkpoint inhibitors (ICIs) has become one of the most promising approaches in the field of cancer therapy. Unlike the current therapies that target tumor cells, such as chemotherapy, radiotherapy, or targeted therapy, ICIs directly restore the exhausted host antitumor immune responses mediated by the tumors. Among multiple immune modulators identified, the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis leading to the exhaustion of T-cell immunity in chronic infections and tumors has been widely investigated. Therefore, blocking antibodies targeting PD-1 or PD-L1 have been developed and approved for the treatment of various advanced cancers, including non-small-cell lung cancer (NSCLC), making them the most successful ICIs. Compared with chemotherapy or radiotherapy, PD-1/PD-L1 blockade therapy significantly improves the durable response rate and prolongs long-term survival with limited adverse effects in both monotherapy and combination therapy for advanced NSCLC. However, extensive challenges exist for further clinical applications, such as a small fraction of benefit population, primary and acquired resistance, the lack of predictive and prognostic biomarkers, and treatment-related adverse effects. In this article, we summarize the latest clinical applications of PD-1/PD-L1 blockade therapy in advanced NSCLC worldwide, as well as in China, and discuss the bottlenecks related to the use of this therapy in clinical practice. An exploration of the underlying mechanism of PD-1/PD-L1 blockade therapy and biomarker identification will maximize the application of ICIs in advanced NSCLC and facilitate bedside-to-bench studies in cancer immunotherapy as well. IMPLICATIONS FOR PRACTICE: Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) and programmed cell death protein ligand 1 (PD-L1) display apparent benefits for the treatment of advanced non-small-cell lung cancer (NSCLC). However, the clinical applications of these therapies are challenged by the limited benefit population with additional high economic burden and adverse events. This review discusses the bottlenecks of ICI therapy in clinical practice and provides appropriate guidance in the development of predictive biomarkers, the establishment of the criteria for combining PD-1/PD-L1 blockade therapy with the existing therapies, and the management of adverse events observed both in monotherapy and combination therapy, which will help maximize the applications of ICIs in advanced NSCLC.

EGFR-mutant NSCLC may remodel TME from non-inflamed to inflamed through acquiring resistance to EGFR-TKI treatment.

BACKGROUND: EGFR-TKI represent the standard first-line therapy for advanced NSCLC harboring EGFR mutations. However, resistance to EGFR-TKI inevitably develops in nearly all patients. Previous clinical study have demonstrated that, some patients that failed EGFR-TKI therapy show a benefit outcome from immunotherapy. Our objective is to explore the immune microenviroment remodeling induced by EGFR-TKI treatment in EGFR mutant lung cancer patients and to investigate the immune cell types and potential molecular signatures involved. METHODS: A cohort of 37 EGFR mutant advanced-stage NSCLC patients, who are resistant to at least one type of TKI treatment, was retrospectively established. Both pre-treatment and TKI resistance tumor FFPE samples of each pairs were collected. Transcriptional profiling and bioinformatics analysis were employed to evaluate the change of immune associated hallmarks before and after EGFR-TKI therapy. RESULTS: Tumor samples after EGFR-TKI treatment displayed enrichment of proinflammatory signaling like interferon-γ, allograft rejection and inflammatory response. Of note, cytotoxic factor granzyme A as well as PD-L1 were found to be more expressed in EGFR-TKI resistance samples. Approximately 33.3 % (11/33) of EGFR-TKI treated samples were classified as "hot" tumor, especially for EGFR L858R mutated NSCLC patients (46.7 %,7/15). Effector cells were significantly overexpressed in 'hot' tumors feature following TKI resistance. In addition, we found that four effector genes (CD8A, CDB8, GZMB, GZMK) showed higher expression in 'hot' tumors post-TKI resistance, and its 4-gene effector cell signature was found to have a good correlation with survival benefit in external immunotherapy database. CONCLUSIONS: TKI treatment may initiate immune activation in EGFR mutant NSCLC, leading to changes in immune cell infiltration following TKI resistance. We mechanistically explored that this might be due to an increased immune response caused by the rise in effector cells post-TKI resistance.

Cisplatin-induced Pyroptosis Enhances the Efficacy of PD-L1 Inhibitor in Small-Cell Lung Cancer via GSDME/IL12/CD4Tem Axis.

The combination therapy of platinum-based chemotherapy and PD-L1 inhibitors but not the single anti-PD-L1 therapy has significantly improved the prognosis of patients with small-cell lung cancer (SCLC). However, the synergistic mechanism of combination therapy has not been fully elucidated. In this work, we identified a positive correlation between the expression of pyroptosis-related proteins Gasdermin E (GSDME) and the survival rates of patients with SCLC. Importantly, it was shown that human SCLC cell lines with high expression of GSDME showed more sensitivity to cisplatin, as well as cisplatin plus anti-PD-L1 treatment both in vitro and in vivo. Mechanically, cisplatin induced the activation of GSDME and the release of cytokines including IL-12, which enhance the expression of IFN-γ in T cells in the tumor immune microenvironment (TME) and subsequently improve anti-PD-L1 response. Altogether, our work demonstrates that cisplatin could induce GSDME-dependent cell pyroptosis to improve the response of anti-PD-L1 therapy though switching the TME from "cold" to "hot" in SCLC, indicating GSDME as a response biomarker for combination therapy of anti-PD-L1 and chemotherapy, as well as a potential target to sensitize the response to PD-L1 inhibitor therapy in future.

A 3D Ex Vivo Tumor-Immune Coculture System Mimicking In Vivo Tumor Environmental Stress on CD8+ T Cells Exhaustion.

Dissection of exhaustion trajectories of immune cells under tumor selection pressure in the tumor microenvironment (TME) elucidates the underlying machinery in anti-tumor immunity, which still lacks easy-to-use models to decipher. Herein, gelatin methacryloyl (GelMA)-poly (ethylene oxide) (PEO) based 3D hydrogel microspheroids are constructed with non-immunogenicity and controllable macroporous structure to establish a tumor-immune cell coculture (3D-HyGTIC) system. In 3D-HyGTIC system, when immune cells embarked, stepwise up-regulation of main immune checkpoints (ICs) molecules is observed with compromised cytokine production in CD8+ T cells, the trajectory of which is in lineage correlation with in vivo grafted tumors. Reinvigoration of CD8+ T cells is more obvious with the addition of an anti-PD-1 regimen at the early time point, which is recapitulated during the coculture of patient-derived tumor fragments (PDTF) and autologous T cells. Moreover, the upregulation of LAG-3 on CD8+ T cells after anti-PD-1 treatment is uncovered. Sequential addition of anti-LAG-3 successfully rescues the otherwise failed reactivation of CD8+ T cells. Therefore, the 3D-HyGTIC system is not only inclined to mimic the early differentiation trajectories of tumor-infiltrating CD8+ T cells but also may facilitate an evaluation of the efficacy of IC blockades and guide the designing of combination immunotherapy.

Accumulation of branched-chain amino acids reprograms glucose metabolism in CD8+ T cells with enhanced effector function and anti-tumor response.

Branched-chain amino acids (BCAAs) provide nutrient signals for cell survival and growth. How BCAAs affect CD8+ T cell functions remains unexplored. Herein, we report that accumulation of BCAAs in CD8+ T cells due to the impairment of BCAA degradation in 2C-type serine/threonine protein phosphatase (PP2Cm)-deficient mice leads to hyper-activity of CD8+ T cells and enhanced anti-tumor immunity. CD8+ T cells from PP2Cm-/- mice upregulate glucose transporter Glut1 expression in a FoxO1-dependent manner with more glucose uptake, as well as increased glycolysis and oxidative phosphorylation. Moreover, BCAA supplementation recapitulates CD8+ T cell hyper-functions and synergizes with anti-PD-1, in line with a better prognosis in NSCLC patients containing high BCAAs when receiving anti-PD-1 therapy. Our finding thus reveals that accumulation of BCAAs promotes effector function and anti-tumor immunity of CD8+ T cells through reprogramming glucose metabolism, making BCAAs alternative supplementary components to increase the clinical efficacy of anti-PD-1 immunotherapy against tumors.

Impact of KRAS Mutation Subtypes and Co-Occurring Mutations on Response and Outcome in Advanced NSCLC Patients following First-Line Treatment.

(1) Background: The purpose was to systematically assess the impact of KRAS subtypes and co-mutations on responses of first-line treatment and outcomes by genetic classification in advanced KRAS mutant NSCLC. (2) Methods: Molecular pathology was confirmed with NGS; Kaplan−Meier analysis and Cox multivariate model were used to analyze the efficacy of first-line treatment and prognosis in KRAS subgroups. (3) Results: Advanced KRAS mutant NSCLC was confirmed among 183 patients, who received first-line therapy. The most common KRAS subtype and co-mutation were G12C (29.5%) and TP53 (59.6%). ICIs/CHE group prolonged PFS to 16.9 m, vs. (CHE)4.6 m vs. (CHE/BEV)7.0 m (p < 0.0001); mOS (ICIs/CHE)37.1 m vs. (CHE)19.8 m vs. [CHE/BEV] 20.7 m (p = 0.024). PFS benefited to different degrees after first-line ICI-based treatment in each genetic classification. KRAS G12D even benefited from OS (p = 0.045). CHE/BEV prolonged mPFS of KRAS/STK11 co-mutation (p = 0.043), but decreased mPFS in G12A subtype (p = 0.026). Multivariate analysis indicated that heavy smoking history (≥20 pack-years) (HR = 0.45, p = 0.039) predicts optimistic prognosis; PS score 1 (HR = 3.604, p = 0.002) and KRAS/SMAD4 co-mutation (HR = 4.293, p = 0.027) remained as independent predictors of shorter OS. (4) Conclusions: First-line treatment with ICI benefited KRAS-mutant-NSCLC patients and resulted in non-negative predictive value for any genetic classification. Bevacizumab should be cautiously chosen for patients with KRAS G12A subtype but is recommended for KRAS/STK11 patients. KRAS/SMAD4 is a new co-mutation genotype that displayed independent risk prognostic factors in patients with advanced KRAS-mutant NSCLC.

Landscape and Predictive Significance of the Structural Classification of EGFR Mutations in Chinese NSCLCs: A Real-World Study.

Background: Non-classical EGFR mutations demonstrate heterogeneous and attenuated responsiveness to EGFR TKIs. Non-small cell lung cancer (NSCLC) patients with atypical EGFR mutations have limited therapeutic options. A recent study established a novel structural-based classification of EGFR mutations and showed its value in predicting the response to TKI. We sought to interrogate the distribution of different structural types and to validate the predictive value in Chinese NSCLCs. Methods: A total of 837 tumor samples were retrospectively recruited from 522 patients with unresectable EGFR-mutant NSCLC. EGFR mutations were classified into four groups: classical-like, T790M-like, Ex20ins-L, and PACC. Treatment information and clinical outcomes were obtained from 436 patients. The time to treatment failure (TTF) was determined on a per-sample basis. Results: Of the 837 EGFR-mutant samples, 67.9%, 18.5%, 9.0%, and 3.1% harbored classical-like, T790M-like, PACC, and Ex20ins-L mutations, respectively. Thirteen (1.6%) samples carried mutations beyond the four types. Among the 204 samples with atypical mutations, 33.8%, 36.7%, 12.7%, and 10.3% were classical-like, PACC, Ex20ins-L, and T790M-like, respectively. In patients with PACC mutations, second-generation TKIs demonstrated a significantly longer TTF than first-generation TKIs (first-line: 15.3 vs. 6.2 months, p = 0.009; all-line: 14.7 vs. 7.1 months, p = 0.003), and a trend of longer TTF than third-generation TKIs (all-line: 14.7 vs. 5.1 months, p = 0.135). Conclusions: Our study depicted the landscape of structural types of EGFR mutations in Chinese NSCLC patients. Our results also suggest that the structural classification can serve as a predictive marker for the efficacy of various EGFR TKIs, which would guide therapeutic decision making.

Early-like differentiation status of systemic PD-1+CD8+ T cells predicts PD-1 blockade outcome in non-small cell lung cancer.

Objectives: Despite remarkable advances in the treatment of non-small cell lung cancer (NSCLC) with anti-programmed death (PD)-1 therapy; only a fraction of patients derives durable clinical benefit. In this study, we investigated whether the differentiation status of systemic CD8+ T cells predicts the outcome of PD-1 blockade in NSCLC. Methods: We carried out a prospective study on a total of 77 NSCLC patients receiving anti-PD-1 blockers, among which 47 patients were assigned as a discovery cohort and 30 patients as a validation cohort. Peripheral blood samples were obtained at baseline and upon multiple therapy cycles and analyzed by multi-parameter flow cytometry. Results: We found that a higher baseline ratio of PD-1+ early effector memory CD8+ T cells (CD28+CD27-CD45RO+, TEEM) to PD-1+ effector CD8+ T cells (CD28-CD27-CD45RO-, TE) delineated responders to PD-1 blockade from progressors and was associated with prolonged progression-free survival (PFS) and durable clinical benefit. Moreover, PD-1+CD8 TEEM cells exhibited early responses after anti-PD-1 therapy and was the major fraction of cycling PD-1+Ki67+CD8+ T cells to expand specifically with positive impact on PFS. Conclusion: These findings provide insights into how the baseline differentiation status of the peripheral immune system determines responses to PD-1-targeted therapies.

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma.

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3+, CD4+ and CD8+ T cells in spleens and lower PD-1 expression in CD4+ and CD8+ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.

A multi-omics-based serial deep learning approach to predict clinical outcomes of single-agent anti-PD-1/PD-L1 immunotherapy in advanced stage non-small-cell lung cancer.

Only 20% NSCLC patients benefit from immunotherapy with a durable response. Current biomarkers are limited by the availability of samples and do not accurately predict who will benefit from immunotherapy. To develop a unified deep learning model to integrate multimodal serial information from CT with laboratory and baseline clinical information. We retrospectively analyzed 1633 CT scans and 3414 blood samples from 200 advanced stage NSCLC patients who received single anti-PD-1/PD-L1 agent between April 2016 and December 2019. Multidimensional information, including serial radiomics, laboratory data and baseline clinical data, was used to develop and validate deep learning models to identify immunotherapy responders and nonresponders. A Simple Temporal Attention (SimTA) module was developed to process asynchronous time-series imaging and laboratory data. Using cross-validation, the 90-day deep learning-based predicting model showed a good performance in distinguishing responders from nonresponders, with an area under the curve (AUC) of 0.80 (95% CI: 0.74-0.86). Before immunotherapy, we stratified the patients into high- and low-risk nonresponders using the model. The low-risk group had significantly longer progression-free survival (PFS) (8.4 months, 95% CI: 5.49-11.31 vs. 1.5 months, 95% CI: 1.29-1.71; HR 3.14, 95% CI: 2.27-4.33; log-rank test, P<0.01) and overall survival (OS) (26.7 months, 95% CI: 18.76-34.64 vs. 8.6 months, 95% CI: 4.55-12.65; HR 2.46, 95% CI: 1.73-3.51; log-rank test, P<0.01) than the high-risk group. An exploratory analysis of 93 patients with stable disease (SD) [after first efficacy assessment according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1] also showed that the 90-day model had a good prediction of survival and low-risk patients had significantly longer PFS (11.1 months, 95% CI: 10.24-11.96 vs. 3.3 months, 95% CI: 0.34-6.26; HR 2.93, 95% CI: 1.69-5.10; log-rank test, P<0.01) and OS (31.7 months, 95% CI: 23.64-39.76 vs. 17.2 months, 95% CI: 7.22-27.18; HR 2.22, 95% CI: 1.17-4.20; log-rank test, P=0.01) than high-risk patients. In conclusion, the SimTA-based multi-omics serial deep learning provides a promising methodology for predicting response of advanced NSCLC patients to anti-PD-1/PD-L1 monotherapy. Moreover, our model could better differentiate survival benefit among SD patients than the traditional RECIST evaluation method.

Peripheral CD4+ T cell signatures in predicting the responses to anti-PD-1/PD-L1 monotherapy for Chinese advanced non-small cell lung cancer.

Limited benefit population of immune checkpoint inhibitors makes it urgent to screen predictive biomarkers for stratifying the patients. Herein, we have investigated peripheral CD4+ T cell signatures in advanced non-small cell lung cancer (NSCLC) patients receiving anti-PD-1/PD-L1 treatments. It was found that the percentages of IFN-γ and IL-17A secreting naïve CD4+ T cells (Tn), and memory CD4+ T cells (Tm) expressing PD-1, PD-L1 and CTLA-4 were significantly higher in responder (R) than non-responder (NonR) NSCLC patients associated with a longer progression free survival (PFS). Logistic regression analysis revealed that the baseline IFN-γ-producing CD4+ Tn cells and PD-1+CD4+ Tm cells were the most significant signatures with the area under curve (AUC) value reaching 0.849. This was further validated in another anti-PD-1 monotherapy cohort. Conversely, high percentage of CTLA-4+CD4+ Tm cells was associated with a shorter PFS in patients receiving anti-PD-L1 monotherapy. Our study therefore elucidates the significance of functional CD4+ Tn and Tm subpopulations before the treatment in predicting the responses to anti-PD-1 treatment in Chinese NSCLC patients. The fact that there display distinct CD4+ T cell signatures in the prediction to anti-PD-1 and anti-PD-L1 monotherapy from our study provides preliminary evidence on the feasibility of anti-PD-1 and anti-PD-L1 combination therapy for advanced NSCLC patients.

Predictable Roles of Peripheral IgM Memory B Cells for the Responses to Anti-PD-1 Monotherapy Against Advanced Non-Small Cell Lung Cancer.

Tumor-infiltrating B cells and tertiary lymphoid structures have been identified to predict the responses to immune checkpoint inhibitors (ICIs) in cancer immunotherapy. Considering the feasibility of sample collection, whether peripheral B cell signatures are associated with the responses to ICI therapy remains unclear. Herein, we have defined peripheral B cell signatures in advanced non-small cell lung cancer (NSCLC) patients receiving anti-PD-1 monotherapy and investigated their associations with clinical efficacy. It was found that the percentages of B cells before the treatment (baseline) were significantly higher (P = 0.004) in responder (R, n = 17) than those in non-responder (NonR, n = 33) NSCLC patients in a discovery cohort. Moreover, the percentages of baseline IgM+ memory B cells were higher (P < 0.001) in R group than those in NonR group, and associated with a longer progression free survival (PFS) (P = 0.003). By logistic regression analysis peripheral baseline IgM+ memory B cells were identified as an independent prognostic factor (P = 0.002) for the prediction of the responses to anti-PD-1 monotherapy with the AUC value of 0.791, which was further validated in another anti-PD-1 monotherapy cohort (P = 0.011, n = 70) whereas no significance was observed in patients receiving anti-PD-L1 monotherapy (P = 0.135, n = 30). Therefore, our data suggest the roles of peripheral IgM+ memory B cells in predicting the responses to anti-PD-1 treatment in Chinese advanced NSCLC patients.

Serum Metabolite Biomarkers Predictive of Response to PD-1 Blockade Therapy in Non-Small Cell Lung Cancer.

Background: Despite remarkable success of immunotherapies with checkpoint blockade antibodies targeting programmed cell death protein 1 (PD-1), the majority of patients with non-small-cell lung cancer (NSCLC) have yet to receive durable benefits. We used the metabolomic profiling of early on-treatment serum to explore predictors of clinical outcomes of anti-PD-1 treatment in patients with advanced NSCLC. Methods: We recruited 74 Chinese patients who had stage IIIB/IV NSCLC-proven tumor progression and were treated with PD-1 inhibitor. The study was comprised of a discovery cohort of patients treated with nivolumab and two validation cohorts of patients receiving tislelizumab or nivolumab. Serum samples were collected 2-3 weeks after the first infusion of PD-1 inhibitor. Metabolomic profiling of serum was performed using ultrahigh performance lipid chromatograph-mass spectrometry. The serum metabolite biomarkers were identified using an integral workflow of nontargeted metabolomic data analysis. Results: A serum metabolite panel consisting of hypoxanthine and histidine was identified and validated as a predictor of response to PD-1 blockade treatment in patients with advanced NSCLC. High levels of both hypoxanthine and histidine in early on-treatment serum were associated with improved progression-free survival [hazard ratio (HR) = 0.078, 95% confidence interval (CI), 0.027-0.221, p < 0.001] and overall survival (HR = 0.124, 95% CI, 0.039-0.397, p < 0.001) in the discovery cohort. The serum metabolite panel showed a high sensitivity and specificity in distinguishing responders and non-responders in the validation cohorts 1 and 2, with an area under the receiver-operating characteristic curve of 0.933 and 1.000, respectively. High levels of serum hypoxanthine and histidine were correlated with improved progression-free survival in the validation cohort 1 (HR = 0.137, 95% CI, 0.040-0.467, p = 0.001) and in the validation cohort 2 (HR = 0.084, 95% CI, 0.009-0.762, p = 0.028). Conclusion: Our results revealed that hypoxanthine and histidine in early on-treatment serum are predictive biomarkers of response to PD-1 blockade therapy in patients with advanced NSCLC. The serum biomarker panel would enable early identification of NSCLC patients who may benefit from PD-1 blockade therapy.

Identification of Mutated Peptides in Bladder Cancer From Exomic Sequencing Data Reveals Negative Correlation Between Mutation-Specific Immunoreactivity and Inflammation.

Introduction and Objective: Neoantigen-based immunotherapy is one of the breakthroughs in cancer immunotherapy. Benefit from the Cancer Genome Atlas database, we intended to identify mutant peptides with neoantigen property in bladder cancer (BC). Correlations between the immunoreactivity of candidate neoantigens and clinical manifestations were further analyzed. Methods: HLA-A*02:01 restricted mutant (MT) and wildtype (WT) peptides were predicted by using whole exome sequencing data of 412 BC patients in the TCGA database. Binding affinity to HLA-A2 molecules was determined by using T2 cell-based binding assay. The immunoreactivity to WT and MT peptides in HLA-A2+ BC patients was determined by using an ELISPOT assay upon in vitro stimulation with MT and WT peptides individually. Clinical relevance to peptide-specific immunoreactivity was analyzed by Pearson correlation analysis. The disease free survival (DFS) curves were plotted using the Kaplan-Meier method in BC patients with or without mutations and compared using the log-rank test online. Results: Fifty-seven HLA-A*02:01 restricted WT and MT peptides were selected based on predicted high affinity and expression frequency, among which 12 MT peptides from 12 individual genes exhibited strong affinity to HLA-A2 molecules when compared to WT counterparts. MT peptides induced more peptide-specific IFNγ spot forming units (SFUs) than WT counterparts in HLA-A2+ BC patients upon in vitro stimulation. They were negatively correlated to the counts of peripheral leukocytes and platelets. Patients with higher C-reactive protein level exhibited lower immunoreactivity to MT peptides. Combination of MT peptides from 6 genes, including CDKN1AG61V , RHOBP75L , DDB1S25L , AHNAKD4855Y , ANP32AS56L and MKI67H84L covered 47.5% of the patients under investigation. Patients harboring combinational mutations in these genes were associated with a longer DFS according to the cBioportal online analysis. Conclusion: Twelve HLA-A*02:01 restricted MT peptides have been identified exhibiting higher binding affinity to HLA-A2 molecules and stronger immunoreactivity than WT counterparts in BC patients. Combination of MT peptides from six genes might be potential as neoantigen candidates in cancer immunotherapy against BC in the future. Inflammatory modulation is inclined to be a strategy to enhance the efficacy of neoantigen-based immunotherapy.

The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC.

INTRODUCTION: Gut microbiome affecting the responses to immune checkpoint inhibitors against advanced NSCLC has been investigated in the Western population. However, considering pre-existing genetic and gut microbiota variation, the relevance remains unknown in the East-Asian NSCLC population. This study is designed to explore the relationship between gut microbiome and clinical outcomes in Chinese patients with NSCLC who have received treatment using an anti-programmed death 1 (PD-1) blockade. METHODS: Thirty-seven patients with advanced NSCLC receiving treatment with nivolumab were enrolled in CheckMate 078 (NCT02613507) and CheckMate 870 (NCT03195491). Fecal samples were collected at the starting point, when patients received nivolumab, at clinical evaluation, and when disease progression was noted. 16S ribosome RNA gene sequencing was applied to assess gut microbiota profiles. Peripheral immune signatures were determined by multicolor flow cytometry in parallel. RESULTS: When subgrouping patients into responder (R) and nonresponder according to the clinical response assessed using Response Evaluation Criteria in Solid Tumor version 1.1, R patients harbored higher diversity of gut microbiome at the starting point with stable composition during the treatment. Patients with high microbiome diversity had significantly prolonged progression-free survival when compared to those with low diversity. Compositional difference was observed between the two groups as well with the enrichment of Alistipes putredinis, Bifidobacterium longum, and Prevotella copri in R whereas Ruminococcus_unclassified enriched in nonresponding patients. Analysis of systemic immune responses using multicolor flow cytometry revealed that patients with a high abundance of microbiome diversity in the gut had a greater frequency of unique memory CD8+ T cell and natural killer cell subsets in the periphery in response to anti-PD-1 therapy. CONCLUSIONS: Our results reveal strong correlation between gut microbiome diversity and the responses to anti-PD-1 immunotherapy in Chinese patients with advanced NSCLC. Patients with favorable gut microbiome (such as those with high diversity) exhibit enhanced memory T cell and natural killer cell signatures in the periphery. These findings provide important implications for the prediction and the evaluation of anti-PD-1 immunotherapy against NSCLC in the Chinese population.

PD-1 blockade augments humoral immunity through ICOS-mediated CD4+ T cell instruction.

Successful applications of PD-1/PD-L1 blockade in multiple cancers highlight the efficacy of immunotherapy mediated by enhancing CD8+ T cell immunity both in mouse and human. How PD-1 blockade affects humoral immunity remains unclear. Herein we demonstrated that treatment of anti-PD-1 antibody led to the increase in both total IgG and OVA-specific IgG in OVA-immunized mice. However, no effect was observed on Ab affinity maturation. Accumulation of germinal center (GC) and memory B cells was observed in the spleens together with elevated percentages of plasma cells in the spleens and bone marrow. More interestingly, dramatic infiltration of CD4+ T cells was apparent in GCs after PD-1 blockade with a significant increase in the expression of ICOS. When CD4+ T cells and B cells from OVA-immunized mice were co-cultured with neutralizing anti-PD-1 Ab in vitro, PD-1 blockade recapitulated the up-regulation of ICOS expression on CD4+ T cells with the activation of ERK signaling. Suppression of ERK activation not only reduced ICOS expression on CD4+ T cells but also attenuated IgG production upon PD-1 blockade. Taken together, PD-1 blockade enhances humoral immunity. This process partially relies on more accumulation of CD4+ T cells in GCs with the up-regulation of ICOS expression and the promotion of B cell terminal differentiation. The regulatory pattern of PD-1 blockade illustrated here provides a new mechanism of how immune checkpoint molecules regulating humoral immune responses.

Identification of HLA-A2-Restricted Mycobacterial Lipoprotein Z Peptides Recognized by T CellsFrom Patients With ActiveTuberculosis Infection.

Identification of HLA-restricted peptides derived from mycobacterial antigens that are endowed with high affinity and strong antigenicity is not only of interest in tuberculosis (TB) diagnostics and treatment efficacy evaluation, but might also provide potential candidates for the development of therapeutic vaccines against drug-resistant TB. Our previous work demonstrated that lipoprotein Z (LppZ) displayed high immunogenicity and antigenicity in active TB patients. In the present study, ten HLA-A2-restricted LppZ peptides (LppZp1-10) were predicted by bioinformatics, among which LppZp7 and LppZp10 were verified to possess high affinity to HLA-A2 molecules using T2 cell-based affinity binding assay. Moreover, results from ELISpot assay showed that both LppZp7 and LppZp10 peptides were able to induce more IFN-γ producing cells upon ex vivo stimulation of PBMC from HLA-A2+ active TB (ATB) patients as compared to those from healthy controls (HCs). Also, the numbers of LppZp7 and LppZp10-specific IFN-γ producing cells exhibited positive correlations with those of ESAT-6 peptide (E6p) or CFP-10 peptide (C10p) in ATB. Interestingly, stimulation with LppZp7/p10 mixture was able to induce higher intracellular expression of IFN-γ and IL-2 cytokines in CD8+ and CD4+ T cells from ATB as compared to HC, associated with lower expression of TNF-α in both CD8+ and CD4+ T cells. Taken together, HLA-A2-restricted LppZp7 and LppZp10 peptides display high immunoreactivity in HLA-matched ATB patients demonstrated by high responsiveness in both CD8+ and CD4+ T cells. With the ability to induce strong antigen-specific cellular responses, LppZp7 and LppZp10 are of potential value for the future applications in the prevention and control of TB.

DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically.

Gastric cancer (GC) is among the most malignant cancers with high incidence and poor prognoses worldwide as well as in China. dCTP pyrophosphatase 1 (DCTPP1) is overexpressed in GC with a poor prognosis. Given chemotherapeutic drugs share similar structures with pyrimidine nucleotides, the role of DCTPP1 in affecting the drug sensitivity in GC remains unclear and is worthy of investigation. In the present study, we reported that DCTPP1-knockdown GC cell line BGC-823 exhibited more sensitivity to 5-fluorouracil (5-FU), demonstrated by the retardation of cell proliferation, the increase in cell apoptosis, cell cycle arrest at S phase and more DNA damages. Multidrug resistance 1 (MDR1) expression was unexpectedly down-regulated in DCTPP1-knockdown BGC-823 cells together with more intracellular 5-FU accumulation. This was in large achieved by the elevated methylation in promoter region of MDR1 gene. The intracellular 5-methyl-dCTP level increased in DCTPP1-knockdown BGC-823 cells as well. More significantly, the strong correlation of DCTPP1 and MDR1 expression was detectable in clinical GC samples. Our results thus imply a novel mechanism of chemoresistance mediated by the overexpression of DCTPP1 in GC. It is achieved partially through decreasing the concentration of intracellular 5-methyl-dCTP, which in turn results in promoter hypomethylation and hyper-expression of drug resistant gene MDR1. Our study suggests DCTPP1 as a potential indicative biomarker for the predication of chemoresistance in GC.