Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

BACKGROUND: Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. METHODS: The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. RESULTS: The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. CONCLUSIONS: This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Silencing of genes by promoter hypermethylation shapes tumor microenvironment and resistance to immunotherapy in clear-cell renal cell carcinomas.

The efficacy of immune checkpoint inhibitors varies in clear-cell renal cell carcinoma (ccRCC), with notable primary resistance among patients. Here, we integrate epigenetic (DNA methylation) and transcriptome data to identify a ccRCC subtype characterized by cancer-specific promoter hypermethylation and epigenetic silencing of Polycomb targets. We develop and validate an index of methylation-based epigenetic silencing (iMES) that predicts primary resistance to immune checkpoint inhibition (ICI) in the BIONIKK trial. High iMES is associated with VEGF pathway silencing, endothelial cell depletion, immune activation/suppression, EZH2 activation, BAP1/SETD2 deficiency, and resistance to ICI. Combination therapy with hypomethylating agents or tyrosine kinase inhibitors may benefit patients with high iMES. Intriguingly, tumors with low iMES exhibit increased endothelial cells and improved ICI response, suggesting the importance of angiogenesis in ICI treatment. We also develop a transcriptome-based analogous system for extended applicability of iMES. Our study underscores the interplay between epigenetic alterations and tumor microenvironment in determining immunotherapy response.

Delineating the interplay between oncogenic pathways and immunity in anaplastic Wilms tumors.

Wilms tumors are highly curable in up to 90% of cases with a combination of surgery and radio-chemotherapy, but treatment-resistant types such as diffuse anaplastic Wilms tumors pose significant therapeutic challenges. Our multi-omics profiling unveils a distinct desert-like diffuse anaplastic Wilms tumor subtype marked by immune/stromal cell depletion, TP53 alterations, and cGAS-STING pathway downregulation, accounting for one-third of all diffuse anaplastic cases. This subtype, also characterized by reduced CD8 and CD3 infiltration and active oncogenic pathways involving histone deacetylase and DNA repair, correlates with poor clinical outcomes. These oncogenic pathways are found to be conserved in anaplastic Wilms tumor cell models. We identify histone deacetylase and/or WEE1 inhibitors as potential therapeutic vulnerabilities in these tumors, which might also restore tumor immunogenicity and potentially enhance the effects of immunotherapy. These insights offer a foundation for predicting outcomes and personalizing treatment strategies for aggressive pediatric Wilms tumors, tailored to individual immunological landscapes.

Development and validation of radiomics nomograms for preoperative prediction of characteristics in non-small cell lung cancer and circulating tumor cells.

To develop and validate 3 radiomics nomograms for preoperative prediction of pathological and progression diagnosis in non-small cell lung cancer (NSCLC) as well as circulating tumor cells (CTCs). A total of 224 and 134 patients diagnosed with NSCLC were respectively gathered in 2018 and 2019 in this study. There were totally 1197 radiomics features that were extracted and quantified from the images produced by computed tomography. Then we selected the radiomics features with predictive value by least absolute shrinkage and selection operator and combined them into radiomics signature. Logistic regression models were built using radiomics signature as the only predictor, which were then converted to nomograms for individualized predictions. Finally, the performance of the nomograms was assessed on both cohorts. Additionally, immunohistochemical correlation analysis was also performed. As for discrimination, the area under the curve of pathological diagnosis nomogram and progression diagnosis nomogram in NSCLC were both higher than 90% in the training cohort and higher than 80% in the validation cohort. The performance of the CTC-diagnosis nomogram was somehow unexpected where the area under the curve were range from 60% to 70% in both cohorts. As for calibration, nonsignificant statistics (P > .05) yielded by Hosmer-Lemeshow tests suggested no departure between model prediction and perfect fit. Additionally, decision curve analyses demonstrated the clinically usefulness of the nomograms. We developed radiomics-based nomograms for pathological, progression and CTC diagnosis prediction in NSCLC respectively. Nomograms for pathological and progression diagnosis were demonstrated well-performed to facilitate the individualized preoperative prediction, while the nomogram for CTC-diagnosis prediction needed improvement.

A Unique Gene Signature Predicting Recurrence Free Survival in Stage IA Lung Adenocarcinoma.

Objective: Resected stage IA lung adenocarcinoma (LUAD) has a reported 5-year recurrence free survival (RFS) of 63-81%. A unique gene signature stratifying patients with early stage LUAD as high or low-risk of recurrence would be valuable. Methods: GEO datasets combining European and North American LUAD patients (n=684) were filtered for stage IA (n=105) to develop a robust signature for recurrence (RFSscore). Univariate Cox proportional hazard regression model was used to assess associations of gene expression with RFS and OS. Leveraging a bootstrap approach of these identified upregulated genes allowed construction of a model which was evaluated by Area Under the Received Operating Characteristics. The optimal signature has RFSscore calculated via a linear combination of expression of selected genes weighted by the corresponding Cox regression derived coefficients. Log-rank analysis calculated RFS and OS. Results were validated using the LUAD TCGA transcriptomic NGS based dataset. Results: Rigorous bioinformatic analysis identified a signature of 4 genes: KNSTRN, PAFAH1B3, MIF, CHEK1. Kaplan-Meier analysis of stage IA LUAD with this signature resulted in 5-year RFS for low-risk of 90% compared to 53% for high-risk (HR 6.55, 95%CI 2.65-16.18, p-value <0.001), confirming the robustness of the gene signature with its clinical significance. Validation of the signature using TCGA dataset resulted in an AUC of 0.797 and 5-year RFS for low and high-risk stage IA patients being 91% and 67%, respectively (HR 3.44, 95%CI 1.16-10.23, p-value=0.044). Conclusions: This 4 gene signature stratifies European and North American patients with pathologically confirmed stage IA LUAD into low and high-risk groups for OS and more importantly RFS.

Racial Disparities in MiT Family Translocation Renal Cell Carcinoma.

Racial disparities have been documented in the biology and outcome of certain renal cell carcinomas (RCCs) among Black patients. However, little is known about racial differences in MiT family translocation RCC (TRCC). To investigate this issue, we performed a case-control study using data from The Cancer Genome Atlas (TCGA) and the Chinese OrigiMed2020 cohort. A total of 676 patients with RCC (14 Asian, 113 Black, and 525 White) were identified in TCGA, and TRCC was defined as RCC with TFE3/TFEB translocation or TFEB amplification, leading to 21 patients with TRCC (2 Asian, 8 Black, 10 White, and 1 unknown). Asian (2 of 14 [14.3%] vs 10 of 525 [1.9%]; P = .036) and Black (8 of 113 [7.1%] vs 1.9%; P = .007) patients with RCC showed significantly higher prevalence of TRCC compared with White patients with RCC. The overall mortality rate of TRCC was slightly higher in Asian and Black patients compared with White patients (HR: 6.05, P = .069). OrigiMed2020 Chinese patients with RCC had a significantly higher proportion of TRCC with TFE3 fusions than TCGA White patients with RCC (13 of 250 [5.2%] vs 7 of 525 [1.3%]; P = .003). Black patients with TRCC were more likely to exhibit the proliferative subtype than White patients (6 of 8 [75%] vs 2 of 9 [22.2%]; P = .057) for those who had RNA-seq profiles. We present evidence of higher prevalence of TRCC in Asian and Black patients with RCC compared with White patients and show that these tumors in Asian and Black patients have distinct transcriptional signatures and are associated with poor outcomes.

Persistent T cell proliferation and MDSCs expansion precede incomplete CD4+ T cell recovery in people with acute HIV-1 infection with early ART.

HIV-1 infection causes T cell dysfunction that cannot be fully restored by anti-retroviral therapy (ART). Myeloid-derived suppressor cells (MDSCs) expand and suppress T cell function during viral infection. In this study, we evaluated the dynamics of phenotypes and function of T cells and MDSCs and the effects of their interaction on CD4+ T cell reconstitution in people with acute HIV-1 infection (PWAH) with early ART. Flow cytometry was used to detect the phenotypic dynamics and function of T cells and MDSCs at pre-ART, 4, 24, 48, and 96 weeks of ART. We observed that T cells were hyper-activated and hyper-proliferative in PWAH at pre-ART. Early ART normalized T cell activation but not their proliferation. T cell proliferation, enriched in PD-1+ T cells, was persisted and negatively associated with CD4+ T-cell counts after ART. Moreover, M-MDSCs frequency was increased and positively correlated with T cell proliferation after 96 weeks of ART. M-MDSCs persisted and inhibited T cell proliferation ex vivo, which could be partially reversed by PD-L1 blockade. Further, we found higher frequencies of proliferative CD4+ T cells and M-MDSCs in PWAH with lower CD4+ T cell numbers (<500 cells/µL) compared to PWAH with higher CD4+ T cell numbers (>600 cells/µL) after 96 weeks of ART. Our findings indicate that persistent T cell proliferation, MDSCs expansion, and their interaction may affect CD4+ T-cell recovery in PWAH with early ART.

Tong Sai granule improves AECOPD via regulation of MAPK-SIRT1-NF-κB pathway and cellular senescence alleviation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tong Sai granule (TSG) a traditional Chinese medicine, are used to treat acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Cellular senescence is considered the mechanism underlying AECOPD progression. AIM OF THE STUDY: This study aimed to investigate the therapeutic mechanisms of TSG in an AECOPD rat model (established using cigarette smoke exposure and bacterial infection) and focused on the inhibition of cellular senescence in vivo and in vitro. MATERIALS AND METHODS: Histological changes and levels of inflammatory cytokines, matrix metalloproteinases (MMPs), p53, and p21 were determined. A cellular senescence model was established by challenging airway epithelial cells with cigarette smoke extract (CSE) and lipopolysaccharide (LPS). Quantitative PCR, western blotting, and immunofluorescence were used to measure mRNA and protein levels. Additionally, UPLC-Q-Extractive-Orbitrap MS analysis, network analysis, and transcriptomics were used to analyze the potential compounds and molecular mechanisms of TSG. RESULTS: The results showed that oral administration of TSG significantly reduced the severity of AECOPD in rats by ameliorating lung function decline and pathological injuries and increasing the levels of C-reactive protein and serum amyloid A, two well-known proinflammatory mediators of the acute phase response. Oral TSG administration also decreased the expression levels of proinflammatory cytokines (e.g., IL-6, IL-1ß, and TNF-α), MMPs (e.g., MMP-2 and MMP-9), critical regulators of senescence such as p21 and p53, and the apoptotic marker γH2AX, all of which are factors in cellular senescence in lung tissue. TSG4 was isolated from TSGs using macroporous resin and found to significantly suppress cellular senescence in CSE/LPS-induced bronchial epithelial cells. Furthermore, 26 of 56 compounds identified in TSG4 were used to predict 882 potential targets. Additionally, 317 differentially expressed genes (DEGs) were detected in CSE/LPS-treated bronchial epithelial cells. Network analysis of the 882 targets and 317 DEGs revealed that TSG4 regulated multiple pathways, among which the mitogen-activated protein kinase-sirtuin 1-nuclear factor kappa B (MAPK-SIRT1-NF-κB) pathway is important in terms of antisenescent mechanisms. Moreover, in CSE/LPS-induced bronchial epithelial cells, p-p38, p-ERK1/2, p-JNK, and p-p65 levels were increased and SIRT1 levels were decreased after TSG4 treatment. Additionally, oral TSG administration decreased p-p38 and p-p65 levels and increased SIRT1 levels in the lung tissues of AECOPD model rats. CONCLUSION: Collectively, these results indicate that TSGs ameliorate AECOPD by regulating the MAPK-SIRT1-NF-κB signaling pathway and subsequently suppressing cellular senescence.

Transforming Growth Factor ß Signaling Promotes HIV-1 Infection in Activated and Resting Memory CD4+ T Cells.

Understanding the facilitator of HIV-1 infection and subsequent latency establishment may aid the discovery of potential therapeutic targets. Here, we report the elevation of plasma transforming growth factor ß (TGF-ß) during acute HIV-1 infection among men who have sex with men (MSM). Using a serum-free in vitro system, we further delineated the role of TGF-ß signaling in mediating HIV-1 infection of activated and resting memory CD4+ T cells. TGF-ß could upregulate both the frequency and expression of the HIV-1 coreceptor CCR5, thereby augmenting CCR5-tropic viral infection of resting and activated memory CD4+ T cells via Smad3 activation. The production of live HIV-1JR-FL upon infection and reactivation was increased in TGF-ß-treated resting memory CD4+ T cells without increasing CD4 expression or inducing T cell activation. The expression of CCR7, a central memory T cell marker that serves as a chemokine receptor to facilitate T cell trafficking into lymphoid organs, was also elevated on TGF-ß-treated resting and activated memory CD4+ T cells. Moreover, the expression of CXCR3, a chemokine receptor recently reported to facilitate CCR5-tropic HIV-1 infection, was increased on resting and activated memory CD4+ T cells upon TGF-ß treatment. These findings were coherent with the observation that ex vivo CCR5 and CXCR3 expression on total resting and resting memory CD4+ T cells in combination antiretroviral therapy (cART)-naive and cART-treated patients were higher than in healthy individuals. Overall, the study demonstrated that TGF-ß upregulation induced by acute HIV-1 infection might promote latency reservoir establishment by increasing infected resting memory CD4+ T cells and lymphoid organ homing of infected central memory CD4+ T cells. Therefore, TGF-ß blockade may serve as a potential supplementary regimen for HIV-1 functional cure by reducing viral latency. IMPORTANCE Incomplete eradication of HIV-1 latency reservoirs remains the major hurdle in achieving a complete HIV/AIDS cure. Dissecting the facilitator of latency reservoir establishment may aid the discovery of druggable targets for HIV-1 cure. This study showed that the T cell immunomodulatory cytokine TGF-ß was upregulated during the acute phase of infection. Using an in vitro serum-free system, we specifically delineated that TGF-ß promoted HIV-1 infection of both resting and activated memory CD4+ T cells via the induction of host CCR5 coreceptor. Moreover, TGF-ß-upregulated CCR7 or CXCR3 might promote HIV-1 latent infection by facilitating lymphoid homing or IP-10-mediated viral entry and DNA integration, respectively. Infected resting and central memory CD4+ T cells are important latency reservoirs. Increased infection of these cells mediated by TGF-ß will promote latency reservoir establishment during early infection. This study, therefore, highlighted the potential use of TGF-ß blockade as a supplementary regimen with cART in acute patients to reduce viral latency.

Dysregulation of memory B cells and circulating T follicular helper cells is a predictor of poor immune recovery in HIV-infected patients on antiretroviral therapy.

T follicular helper (Tfh) cells and their interactions with B cells within the germinal center play extensive roles in human immunodeficiency virus (HIV) pathology. However, their association with immune reconstitution during antiretroviral therapy (ART) is still unclear. The aim of this study was to determine the impact of Tfh and memory B cell function on T helper cell recovery in patients with acute or chronic HIV infection. A total of 100 HIV-infected individuals were enrolled in our study, classified into acute and chronic HIV infection groups (60 and 40, respectively), and subsequently classified into immunological responder (IR) and immunological nonresponder (INR) subgroups according to immune recovery outcomes after 96 weeks of ART. Liquid chromatography-mass spectrometry was used to quantify the temporal regulation patterns of B and CD4+ T-cell profiles among patients, and flow cytometry was used to investigate certain subsets of B and T cells. Here we showed that the prevalence of Tfh cells in the T helper cell population correlated negatively with CD4+ T-cell recovery. The proportion of CXCR3-  Tfh cells in patients with acute or chronic infection was associated with CD4+ T-cell count recovery, and the proportion of CD21+ memory B cells at baseline was significantly higher in those with improved immune recovery outcomes. Universal proteomic dysregulation of B and CD4+ T cells at baseline was detected in patients with acute infected and poor CD4+ T-cell recovery. Proteomics analysis revealed distinct temporal regulation profiles of both T helper cells and B cells between IRs and INRs among patients with acute infection. Our results suggest that the functions of memory B cells in INRs are dysregulated at the early stage of ART, possibly through disruption of Tfh cell function. The frequency and function of Tfh cells and their subsets are potential predictors of poor immune recovery.

TIMEAS, a promising method for the stratification of testicular germ cell tumor patients with distinct immune microenvironment, clinical outcome and sensitivity to frontline therapies.

PURPOSE: With the heterogeneous genetic background, prognosis prediction and therapeutic targets for testicular germ cell tumors (TGCTs) are still unclear. We defined the tumor immune microenvironment activation status (TIMEAS). METHODS: We collected a total of 314 TGCT patients from four cohorts, including a 48-case microarray. A nonnegative matrix factorization algorithm was applied to identify the "immune factor", derived the top 150 weighted genes to divide patients into immune and non-immune classes, and further separated the immune class into activated and exhausted subgroups by nearest template prediction. Tumor mutant burden, gene mutation, and copy number alteration were compared with our recently developed package "MOVICS". A random forest algorithm was performed to establish a prediction model with fewer genes. Immunohistochemistry staining was performed to identify TIMEAS in the microarray. RESULTS: We constructed the TIMEAS in the TCGA-TGCT cohort and further validated it in the GSE3218 and GSE99420 cohorts. The immune class contained the activated status of T-lymphocytes, B-lymphocytes, and macrophages, while Treg cells and the WNT/TGFß signature were more activated in the immune-suppressed subgroup. Patients in the immune-exhausted subgroup had the worst prognosis, and 22.9% of patients in the immune-activated subgroup had KRAS mutations, which might stimulate the response of the immune system and lead to a favorable prognosis. The immune-exhausted group benefited more from chemotherapy, while the immune-activated subgroup responded well to anti-PD-1/PD-L1 therapy. FSCN1 was validated as the target of the immune-exhausted microenvironment by immunohistochemistry. CONCLUSION: TIMEAS classification can separate TGCT patients; patients in the immune-activated subgroup could benefit more from anti-PD-L1 immunotherapy, and those in the immune-exhausted subgroup are more suitable for chemotherapy.

Online solid-phase extraction based on size-controllable spherical covalent organic framework for efficient determination of polybrominated diphenyl ethers in foods.

An analytical method of microspheric brominated covalent organic framework (Br-COF)-online solid-phase extraction integrated with high-performance liquid chromatography (online SPE-HPLC) was proposed for efficiently enriching six polybrominated diphenyl ethers (PBDEs) in foods. The Br-COF microspheres were facilely prepared with uniformity and dispersion by a size-controllable synthesis at the room temperature. Attributed to multiple interactions of the halogen bonding, Van der Waals forces, hydrophobic interaction along with size-matching effect, Br-COF performed satisfactory extraction capacity for PBDEs compared with commercial adsorbents. Five primary influencing factors were optimized, including loading solvent, loading flow rate, elution solvent, elution flow rate and elution volume. Under the optimal parameters, the implement displayed excellent linear ranges (0.5-500 ng mL-1) and low detection limits (0.01-0.05 ng mL-1). The relative recoveries in six spiked food samples ranged from 87.8 to 119.7 % with relative standard deviations below 10 %. This research estabished a promising platform for quantitatively determining trace PBDEs in complex foods.

Combined Ultrasound-Guided Thoracic Paravertebral Nerve Block with Subcostal Transversus Abdominis Plane Block for Analgesia After Total Minimally Invasive Mckeown Esophagectomy: A Randomized, Controlled, and Prospective Study.

INTRODUCTION: Thoracic paravertebral block (TPVB) and subcostal transverse abdominis plane block (TAP) have been considered to provide an effective analgesic effect for laparoscopic and thoracoscopic surgery, respectively. The purpose of this randomized, controlled, and prospective study was to evaluate the analgesic effect of TPVB combined with TAP in patients undergoing total minimally invasive Mckeown esophagectomy. METHODS: Between February 2020 and December 2021, a total of 168 esophageal cancer patients undergoing McKeown esophagectomy at the Cancer Center of Sun Yat-Sen University, China, were randomly assigned to receive patient-controlled epidural analgesia alone (group PCEA, n = 56), patient-controlled intravenous analgesia alone (group PCIA, n = 56), and TPVB combined with TAP and patient-controlled intravenous analgesia (group PVB, n = 56). The primary outcome was a visual analogue scale (VAS) pain score on movement 48 h postoperatively. Secondary endpoints were pain scores at other points, intervention-related side effects, surgical complications, and length of intensive care unit and hospital stay. For the VAS pain score, the Kruskal-Wallis method was conducted for comparison of 3 treatment groups and further pairwise comparison with Bonferroni correction. RESULTS: On movement, the VAS in the PVB group was higher than that in the PCEA group at 48 h, 72 h, 96 h, and 120 h postoperatively (p < 0.05) except in the postoperative anesthesia care unit (PACU) and 24 h postoperatively. The VAS in the PCIA group was higher than the PCEA and PVB groups in the first 4 days after surgery. The pulmonary complication rate in the PCIA group was significantly higher than the rate in the PCEA [95% Confidence Interval 0.214 (0.354, 0.067), p = 0.024]. CONCLUSIONS: Combined TPVB and TAP was more effective than intravenous opioid analgesia alone, while PCEA was more effective than TPVB combined with TAP and intravenous opioid analgesia for patients after McKeown esophagectomy. TRIAL REGISTRATION: Chinese Clinical Trial Registry; ChiCTR2000029588.

Clustering analysis revealed the autophagy classification and potential autophagy regulators' sensitivity of pancreatic cancer based on multi-omics data.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is unresponsive to conventional therapeutic modalities due to its high heterogeneity, expounding the necessity, and priority of searching for effective biomarkers and drugs. Autophagy, as an evolutionarily conserved biological process, is upregulated in PDAC and its regulation is linked to a poor prognosis. Increased autophagy sequestered MHC-I on PDAC cells and weaken the antigen presentation and antitumor immune response, indicating the potential therapeutic strategies of autophagy inhibitors. METHODS: By performing 10 state-of-the-art multi-omics clustering algorithms, we constructed a robust PDAC classification model to reveal the autophagy-related genes among different subgroups. OUTCOMES: After building a more comprehensive regulating network for potential autophagy regulators exploration, we concluded the top 20 autophagy-related hub genes (GAPDH, MAPK3, RHEB, SQSTM1, EIF2S1, RAB5A, CTSD, MAP1LC3B, RAB7A, RAB11A, FADD, CFKN2A, HSP90AB1, VEGFA, RELA, DDIT3, HSPA5, BCL2L1, BAG3, and ERBB2), six miRNAs, five transcription factors, and five immune infiltrated cells as biomarkers. The drug sensitivity database was screened based on the biomarkers to predict possible drug-targeting signal pathways, hoping to yield novel insights, and promote the progress of the anticancer therapeutic strategy. CONCLUSION: We succefully constructed an autophagy-related mRNA/miRNA/TF/Immune cells network based on a 10 state-of art algorithm multi-omics analysis, and screened the drug sensitivity dataset for detecting potential signal pathway which might be possible autophagy modulators' targets.

Changing roles of CD3 + CD8 low T cells in combating HIV-1 infection.

BACKGROUND: Cluster of differentiation 8 (CD8 T) cells play critical roles in eradicating human immunodeficiency virus (HIV)-1 infection, but little is known about the effects of T cells expressing CD8 at low levels (CD8 low ) or high levels (CD8 high ) on HIV-1 replication inhibition after HIV-1 invasion into individual. METHODS: Nineteen patients who had been acutely infected with HIV-1 (AHI) and 20 patients with chronic infection (CHI) for ≥2 years were enrolled in this study to investigate the dynamics of the quantity, activation, and immune responses of CD3 + CD8 low T cells and their counterpart CD3 + CD8 high T cells at different stages of HIV-1 infection. RESULTS: Compared with healthy donors, CD3 + CD8 low T cells expanded in HIV-1-infected individuals at different stages of infection. As HIV-1 infection progressed, CD3 + CD8 low T cells gradually decreased. Simultaneously, CD3 + CD8 high T cells was significantly reduced in the first month of AHI and then increased gradually as HIV-1 infection progressed. The classical activation of CD3 + CD8 low T cells was highest in the first month of AHI and then reduced as HIV-1 infection progressed and entered the chronic stage. Meanwhile, activated CD38 - HLA-DR + CD8 low T cells did not increase in the first month of AHI, and the number of these cells was inversely associated with viral load ( r = -0.664, P = 0.004) but positively associated with the CD4 T-cell count ( r = 0.586, P = 0.014). Increased programmed cell death protein 1 (PD-1) abundance on CD3 + CD8 low T cells was observed from the 1st month of AHI but did not continue to be enhanced, while a significant T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) abundance increase was observed in the 12th month of infection. Furthermore, increased PD-1 and TIGIT abundance on CD3 + CD8 low T cells was associated with a low CD4 T-cell count (PD-1: r = -0.456, P = 0.043; TIGIT: r = -0.488, P = 0.029) in CHI. Nonetheless, the nonincrease in PD-1 expression on classically activated CD3 + CD8 low T cells was inversely associated with HIV-1 viremia in the first month of AHI ( r = -0.578, P = 0.015). Notably, in the first month of AHI, few CD3 + CD8 low T cells, but comparable amounts of CD3 + CD8 high T cells, responded to Gag peptides. Then, weaker HIV-1-specific T-cell responses were induced in CD3 + CD8 low T cells than CD3 + CD8 high T cells at the 3rd and 12th months of AHI and in CHI. CONCLUSIONS: Our findings suggest that CD3 + CD8 low T cells play an anti-HIV role in the first month of infection due to their abundance but induce a weak HIV-1-specific immune response. Subsequently, CD3 + CD8 low T-cell number decreased gradually as infection persisted, and their anti-HIV functions were inferior to those of CD3 + CD8 high T cells.

Single-cell Transcriptomics Reveals Early Molecular and Immune Alterations Underlying the Serrated Neoplasia Pathway Toward Colorectal Cancer.

BACKGROUND & AIMS: Approximately one-third of colorectal cancers develop from serrated lesions (SLs), including hyperplastic polyp (HP), sessile serrated lesion (SSL), traditional serrated adenoma (TSA), and SSL with dysplasia (SSLD) through the serrated neoplasia pathway, which progresses faster than the conventional adenoma-carcinoma pathway. We sought to depict the currently unclarified molecular and immune alterations by the single-cell landscape in SLs. METHODS: We performed single-cell RNA sequencing of 16 SLs (including 4 proximal HPs, 5 SSLs, 2 SSLDs, and 5 TSAs) vs 3 normal colonic tissues. RESULTS: A total of 60,568 high-quality cells were obtained. Two distinct epithelial clusters with redox imbalance in SLs were observed, along with upregulation of tumor-promoting SerpinB6 that regulated ROS level. Epithelial clusters of SSL and TSA showed distinct molecular features: SSL-specific epithelium manifested overexpressed proliferative markers with Notch pathway activation, whereas TSA-specific epithelium showed Paneth cell metaplasia with aberrant lysozyme expression. As for immune contexture, enhanced cytotoxic activity of CD8+ T cells was observed in SLs; it was mainly attributable to increased proportion of CD103+CD8+ tissue-resident memory T cells, which might be regulated by retinoic acid metabolism. Microenvironment of SLs was generally immune-activated, whereas some immunosuppressive cells (regulatory T cells, anti-inflammatory macrophages, MDK+IgA+ plasma cells, MMP11-secreting PDGFRA+ fibroblasts) also emerged at early stage and further accumulated in SSLD. CONCLUSION: Epithelial, immune, and stromal components in the serrated pathway undergo fundamental alterations. Future molecular subtypes of SLs and potential immune therapy might be developed.

An Enhancer Demethylator Phenotype Converged to Immune Dysfunction and Resistance to Immune Checkpoint Inhibitors in Clear-Cell Renal Cell Carcinomas.

PURPOSE: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of patients with clear-cell renal cell carcinomas (ccRCC). Although analyses of transcriptome, genetic alterations, and the tumor microenvironment (TME) have shed light into mechanisms of response and resistance to these agents, the role of epigenetic alterations in this process remains fully unknown. EXPERIMENTAL DESIGN: We investigated the methylome of six ccRCC cohorts as well as one cell line dataset. Of note, we took advantage of the BIONIKK trial aiming to tailor treatments according to Paris Descartes 4-gene expression subgroups, and performed Illumina EPIC profiling for 46 samples related to patients treated with ipilimumab plus nivolumab, and 17 samples related to patients treated with sunitinib. RESULTS: A group of tumors associated with enhancer demethylation was discovered, namely TED. TED was associated with tumors with sarcomatoid differentiation and poor clinical outcome. TED harbored TET1 promoter demethylation, activated the gene expression signature of epithelial-mesenchymal transition and IL6/JAK/STAT3 pathways, and displayed a TME characterized by both immune activation and suppressive populations, fibroblast infiltration, and endothelial depletion. In addition, TED was a predictive factor of resistance to the combination of first-line ipilimumab-nivolumab in the BIONIKK clinical trial. Finally, TED was associated with activation of specific regulons, which we also found to be predictive of resistance to immunotherapy in an independent cohort. CONCLUSIONS: We report on the discovery of a novel epigenetic phenotype associated with resistance to ICIs that may pave the way to better personalizing patients' treatments. See related commentary by Zhou and Kim, p. 1170.