Cost-Effective Whole Transcriptome Sequencing Landscape and Diagnostic Potential Biomarkers in Active Tuberculosis.

Tuberculosis (TB) is a prevalent and severe infectious disease that poses a significant threat to human health. However, it is frequently disregarded as there are not enough quick and accurate ways to diagnose tuberculosis. Here, we develop a strategy for tuberculosis detection to address the challenges, including an experimental strategy, namely, Double Adapter Directional Capture sequencing (DADCSeq), an easily operated and low-cost whole transcriptome sequencing method, and a computational method to identify hub differentially expressed genes as well as the diagnosis of TB based on whole transcriptome data using DADCSeq on peripheral blood mononuclear cells (PBMCs) from active TB and latent TB or healthy control. Applying our approach to create a robust and stable TB multi-mRNA risk probability model (TBMMRP) that can accurately distinguish active and latent TB patients, including active TB and healthy controls in clinical cohorts, this diagnostic biomarker was successfully validated by several independent cross-platform cohorts with favorable performance in differentiating active TB from latent TB or active TB from healthy controls and further demonstrated superior or similar diagnostic accuracy compared to previous diagnostic markers. Overall, we develop a low-cost and effective strategy for tuberculosis diagnosis; as the clinical cohort increases, we can expand to different disease kinds and learn new features through our disease diagnosis strategy.

Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.

Decoding the transcriptional heterogeneity, differentiation lineage, clinical significance in tissue-resident memory CD8 T cell of the small intestine by single-cell analysis.

Resident memory T (Trm) cells which are specifically located in non-lymphoid tissues showed distinct phenotypes and functions compared to circulating memory T cells and were vital for the initiation of robust immune response within tissues. However, the heterogeneity in the transcriptional features, development pathways, and cancer response of Trm cells in the small intestine was not demonstrated. Here, we integrated scRNA-seq and scTCR-seq data pan-tissue T cells to explore the heterogeneity of Trm cells and their development pathways. Trm were enriched in tissue-specific immune response and those in the DUO specially interacted with B cells via TNF and MHC-I signatures. T cell lineage analyses demonstrated that Trm might be derived from the T_CD4/CD8 subset within the same organ or migrated from spleen and mesenteric lymph nodes. We compared the immune repertoire of Trm among organs and implied that clonotypes in both DUO and ILE were less expanded and hydrophilic TRB CDR3s were enriched in the DUO. We further demonstrated that Trm in the intestine infiltrated the colorectal cancer and several effector molecules were highly expressed. Finally, the TCGA dataset of colorectal cancer implied that the infiltration of Trm from the DUO and the ILE was beneficial for overall survival and the response to immune checkpoint blockade.

Cell volume controlled by LRRC8A-formed volume-regulated anion channels fine-tunes T cell activation and function.

Biosynthesis drives the cell volume increase during T cell activation. However, the contribution of cell volume regulation in TCR signaling during T lymphoblast formation and its underlying mechanisms remain unclear. Here we show that cell volume regulation is required for optimal T cell activation. Inhibition of VRACs (volume-regulated anion channels) and deletion of leucine-rich repeat-containing protein 8A (LRRC8A) channel components impair T cell activation and function, particularly under weak TCR stimulation. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles, indicating the prominent effects of cell volume regulation for T cell functions. Moreover, cell volume regulation via LRRC8A controls T cell-mediated antiviral immunity and shapes the TCR repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via regulated volume decrease (RVD) during T cell blast formation to keep the TCR signaling molecules at an adequate density. Together, our results show a further layer of T cell activation regulation that LRRC8A functions as a cell volume controlling "valve" to facilitate T cell activation.

Aspirin induces immunogenic cell death and enhances cancer immunotherapy in colorectal cancer.

The use of aspirin is associated with reduced incidence of colorectal cancer (CRC). However, the detailed mechanism remains unclear. In this study, we reported that colon cancer cells treated with aspirin showed the hallmarks of immunogenic cell death (ICD), including surface expression of calreticulin (CRT) and heat shock protein 70 (HSP70). Mechanistically, aspirin induced endoplasmic reticulum (ER) stress in colon cancer cells. In addition, aspirin decreased the expression of the glucose transporters, GLUT3, and reduced the key enzyme of glycolysis, including HK2, PFKM, PKM2 and LDHA. The changes of tumor glycolysis after aspirin treatment were associated with c-MYC downregulation. Moreover, aspirin potentiated the antitumor efficacy of anti-PD-1 antibody and anti-CTLA-4 antibody in CT26 tumors. However, this antitumor activity of aspirin in combination with anti-PD-1 antibody was abolished by the depletion of CD8+ T cells. Vaccination with tumor antigens is one of the strategies for activating T-cell response against tumors. Here, we demonstrated that aspirin-treated tumor cells in combination with tumor antigens (AH1 peptide) or protective substituted peptide (A5 peptide) could be served as a potent vaccine to eradicate tumors. Overall, our data indicated that aspirin can be used as an inducer of ICD for CRC therapy.