Glioma-derived ANXA1 suppresses the immune response to TLR3 ligands by promoting an anti-inflammatory tumor microenvironment.

A highly immunosuppressive tumor microenvironment (TME) and the presence of the blood‒brain barrier are the two major obstacles to eliciting an effective immune response in patients with high-grade glioma (HGG). Here, we tried to enhance the local innate immune response in relapsed HGG by intracranially injecting poly(I:C) to establish a robust antitumor immune response in this registered clinical trial (NCT03392545). During the follow-up, 12/27 (44.4%) patients who achieved tumor control concomitant with survival benefit were regarded as responders in our study. We found that the T-cell receptor (TCR) repertoire in the TME was reshaped after poly(I:C) treatment. Based on the RNA-seq analysis of tumor samples, the expression of annexin A1 (ANXA1) was significantly upregulated in the tumor cells of nonresponders, which was further validated at the protein level. In vitro and in vivo experiments showed that ANXA1 could induce the production of M2-like macrophages and microglia via its surface receptor formyl peptide receptor 1 (FPR1) to establish a Treg cell-driven immunosuppressive TME and suppress the antitumor immune response facilitated by poly(I:C). The ANXA1/FPR1 signaling axis can inhibit the innate immune response of glioma patients by promoting an anti-inflammatory and Treg-driven TME. Moreover, ANXA1 could serve as a reliable predictor of response to poly(I:C), with a notable predictive accuracy rate of 92.3%. In light of these notable findings, this study unveils a new perspective of immunotherapy for gliomas.

Case report: A rare multidrug-resistant Escherichia coli causes fatal neonatal meningoencephalitis.

Neonatal meningitis is rare but devastating disease. Multidrug-resistant (MDR, multi-drug resistant) bacteria are a major global health risk. We report an Escherichia coli meningitis isolate with multiple resistance patterns and unusual serotype (O75) that caused sudden neonatal death. The isolate was resistant to antibiotics other than cefoperazone/sulbactam and imipenem, challenging the combination of antibiotics commonly used in the empirical treatment of neonatal sepsis. Despite aggressive symptomatic and supportive treatment of the infant based on laboratory tests and clinical practice, the infant eventually died. This is the first case of meningoencephalitis due to serotype O75 reported in China. The presence of highly pathogenic multidrug-resistant microorganisms isolated in neonates underscores the need to implement rapid resistance diagnostic methods and should prompt consideration of alternatives to empiric treatment of neonatal bacterial meningitis.

Efficacy of pp65-specific TCR-T cell therapy in treating cytomegalovirus infection after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65+ HLA+ tumor cells, TCR-T cells induced dramatic tumor regression and exhibited long-term persistence. In a phase I clinical trial (NCT04153279), CMV TCR-T cells were applied to treat patients with CMV reactivation after HSCT. Except one patient who withdrew at early treatment stage, all other six patients were well-tolerated and achieved complete response (CR), no more than grade 2 cytokine release syndrome (CRS) and other adverse events were observed. CMV TCR-T cells persisted up to 3 months. Among them, two patients have survived for more than 1 year. This study demonstrates the great potential in the treatment and prevention of CMV infection following HSCT or other organ transplantation.

Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors.

Chimeric antigen receptor T (CAR-T) cell therapies have demonstrated high response rate and durable disease control for the treatment of B cell malignancies. However, in the case of solid tumors, CAR-T cells have shown limited efficacy, which is partially attributed to intrinsic defects in CAR signaling. Here, we construct a double-chain chimeric receptor, termed as synthetic T cell receptor (TCR) and antigen receptor (STAR), which incorporates antigen-recognition domain of antibody and constant regions of TCR that engage endogenous CD3 signaling machinery. Under antigen-free conditions, STAR does not trigger tonic signaling, which has been reported to cause exhaustion of traditional CAR-T cells. Upon antigen stimulation, STAR mediates strong and sensitive TCR-like signaling, and STAR-T cells exhibit less susceptibility to dysfunction and better proliferation than traditional 28zCAR-T cells. In addition, STAR-T cells show higher antigen sensitivity than CAR-T cells, which holds potential to reduce the risk of antigen loss-induced tumor relapse in clinical use. In multiple solid tumor models, STAR-T cells prominently outperformed BBzCAR-T cells and generated better or equipotent antitumor effects to 28zCAR-T cells without causing notable toxicity. With these favorable features endowed by native TCR-like signaling, STAR-T cells may provide clinical benefit in treating refractory solid tumors.

CXCR2-modified CAR-T cells have enhanced trafficking ability that improves treatment of hepatocellular carcinoma.

Unlike hematological malignancies, solid tumors have proved to be less susceptible to chimeric antigen receptor (CAR)-T cell therapy, which is partially caused by reduced accumulation of therapeutic T cells in tumor site. Since efficient trafficking is the precondition and pivotal step for infused CAR-T cells to exhibit their anti-tumor function, strategies are highly needed to improve the trafficking ability of CAR-T cells for solid tumor treatment. Here, based on natural lymphocyte chemotaxis theory and characteristics of solid tumor microenvironments, we explored the possibility of enhancing CAR-T cell trafficking by using chemokine receptors. Our study found that compared with other chemokines, several CXCR2 ligands showed relatively high expression level in human hepatocellular carcinoma tumor tissues and cell lines. However, both human peripheral T cells and hepatocellular carcinoma tumor infiltrating T cells lacked expression of CXCR2. CXCR2-expressing CAR-T cells exhibited identical cytotoxicity but displayed significantly increased migration ability in vitro. In a xenograft tumor model, we found that expressing CXCR2 in CAR-T cells could significantly accelerate in vivo trafficking and tumor-specific accumulation, and improve anti-tumor effect of these cells.

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

BACKGROUND: Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. RESULTS: We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. CONCLUSIONS: Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

Mapping the Mouse Cell Atlas by Microwell-Seq.

Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

A molecular roadmap for induced multi-lineage trans-differentiation of fibroblasts by chemical combinations.

Recent advances have demonstrated the power of small molecules in promoting cellular reprogramming. Yet, the full potential of such chemicals in cell fate manipulation and the underlying mechanisms require further characterization. Through functional screening assays, we find that mouse embryonic fibroblast cells can be induced to trans-differentiate into a wide range of somatic lineages simultaneously by treatment with a combination of four chemicals. Genomic analysis of the process indicates activation of multi-lineage modules and relaxation of epigenetic silencing programs. In addition, we identify Sox2 as an important regulator within the induced network. Single cell analysis uncovers a novel priming state that enables transition from fibroblast cells to diverse somatic lineages. Finally, we demonstrate that modification of the culture system enables directional trans-differentiation towards myocytic, glial or adipocytic lineages. Our study describes a cell fate control system that may be harnessed for regenerative medicine.

[Optimization of vacuum belt drying process of Gardeniae Fructus in Reduning injection by Box-Behnken design-response surface methodology].

To optimize the belt drying process conditions optimization of Gardeniae Fructus extract from Reduning injection by Box-Behnken design-response surface methodology, on the basis of single factor experiment, a three-factor and three-level Box-Behnken experimental design was employed to optimize the drying technology of Gardeniae Fructus extract from Reduning injection. With drying temperature, drying time, feeding speed as independent variables and the content of geniposide as dependent variable, the experimental data were fitted to a second order polynomial equation, establishing the mathematical relationship between the content of geniposide and respective variables. With the experimental data analyzed by Design-Expert 8. 0. 6, the optimal drying parameter was as follows: the drying temperature was 98.5 degrees C , the drying time was 89 min, the feeding speed was 99.8 r x min(-1). Three verification experiments were taked under this technology and the measured average content of geniposide was 564. 108 mg x g(-1), which was close to the model prediction: 563. 307 mg x g(-1). According to the verification test, the Gardeniae Fructus belt drying process is steady and feasible. So single factor experiments combined with response surface method (RSM) could be used to optimize the drying technology of Reduning injection Gardenia extract.