Oligodendrocyte progenitor cell-specific delivery of lipid nanoparticles loaded with Olig2 synthetically modified messenger RNA for ischemic stroke therapy.

The transcription factor Olig2 is highly expressed throughout oligodendroglial development and is needed for the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes and remyelination. Although Olig2 overexpression in OPCs is a possible therapeutic target for enhancing myelin repair in ischemic stroke, achieving Olig2 overexpression in vivo remains a formidable technological challenge. To address this challenge, we employed lipid nanoparticle (LNP)-mediated delivery of Olig2 synthetically modified messenger RNA (mRNA) as a viable method for in vivo Olih2 protein overexpression. Specifically, we developed CD140a-targeted LNPs loaded with Olig2 mRNA (C-Olig2) to achieve targeted Olig2 protein expression within PDGFRα+ OPCs, with the goal of promoting remyelination for ischemic stroke therapy. We show that C-Olig2 promotes the differentiation of PDGFRα+ OPCs derived from mouse neural stem cells into mature oligodendrocytes in vitro, suggesting that mRNA-mediated Olig2 overexpression is a rational approach to promote oligodendrocyte differentiation and remyelination. Furthermore, when C-Olig2 was administered to a murine model of ischemic stroke, it led to improvements in blood‒brain barrier (BBB) integrity, enhanced remyelination, and rescued learning and cognitive deficits. Our comprehensive analysis, which included bulk RNA sequencing (RNA-seq) and single-nucleus RNA-seq (snRNA-seq), revealed upregulated biological processes related to learning and memory in the brains of mice treated with C-Olig2 compared to those receiving empty LNPs (Mock). Collectively, our findings highlight the therapeutic potential of multifunctional nanomedicine targeting mRNA expression for ischemic stroke and suggest that this approach holds promise for addressing various brain diseases. STATEMENT OF SIGNIFICANCE: While Olig2 overexpression in OPCs represents a promising therapeutic avenue for enhancing remyelination in ischemic stroke, in vivo strategies for achieving Olig2 expression pose considerable technological challenges. The delivery of mRNA via lipid nanoparticles is considered aa viable approach for in vivo protein expression. In this study, we engineered CD140a-targeted LNPs loaded with Olig2 mRNA (C-Olig2) with the aim of achieving specific Olig2 overexpression in mouse OPCs. Our findings demonstrate that C-Olig2 promotes the differentiation of OPCs into oligodendrocytes in vitro, providing evidence that mRNA-mediated Olig2 overexpression is a rational strategy to foster remyelination. Furthermore, the intravenous administration of C-Olig2 into a murine model of ischemic stroke not only improved blood-brain barrier integrity but also enhanced remyelination and mitigated learning and cognitive deficits. These results underscore the promising therapeutic potential of multifunctional nanomedicine targeting mRNA expression in the context of ischemic stroke.

Dual-Targeting Nanovesicles Carrying CSF1/CD47 Identified from Single-Cell Transcriptomics of Innate Immune Cells in Heart Transplant for Alleviating Acute Rejection.

Although immunosuppressive drugs for targeting T cells are the standard of care in acute transplantation rejection, the role of innate immune cells should not be ignored. Here, single-cell RNA sequencing (scRNA-seq) and flow cytometry are performed to reveal the dynamic changes of innate immune cells within the acute rejection time and find a significantly-increased presence of Ly6G- Ly6C+ inflammatory macrophages and decreased presence of neutrophils among all types of immune cells. Next, to further explore potential targets regulating Ly6G- Ly6C+ inflammatory macrophages, scRNA-seq is used to analyze the reciprocal signaling of both neutrophils and macrophages, along with the surface genes of macrophages. It is found that activating colony-stimulating factor 1/ colony-stimulating factor 1 receptor (CSF1/CSF1R) andcluster of differentiation 47/signal regulatory protein α (CD47/SIRPα) signaling may serve as a strategy to relieve Ly6G- Ly6C+ inflammatory macrophage-mediated early graft rejection. To investigate this hypothesis, CSF1/CD47 dual-targeting nanovesicles (NVs) derived from IFN-γ-stimulated induced pluripotent stem cell-derived mesenchymal stem cells ( iPSC-MSCs )are designed and constructed. It is confirmed that CSF1/CD47 NVs synergistically induce the differentiation of Ly6G- Ly6C- M2 inhibitory macrophages by the CSF1/CSF1R pathway, and inhibit the phagocytosis of inflammatory macrophages and inflammatory response by the CD47/SIRPα pathway, ultimately relieving immune rejection. This study highlights the power of dual-targeting CSF1/CD47 NVs as an immunosuppressant against early innate immune responses with the potential for broad clinical applications.

Single-cell and spatial transcriptome analyses revealed cell heterogeneity and immune environment alternations in metastatic axillary lymph nodes in breast cancer.

BACKGROUND: Tumor heterogeneity plays essential roles in developing cancer therapies, including therapies for breast cancer (BC). In addition, it is also very important to understand the relationships between tumor microenvironments and the systematic immune environment. METHODS: Here, we performed single-cell, VDJ sequencing and spatial transcriptome analyses on tumor and adjacent normal tissue as well as axillar lymph nodes (LNs) and peripheral blood mononuclear cells (PBMCs) from 8 BC patients. RESULTS: We found that myeloid cells exhibited environment-dependent plasticity, where a group of macrophages with both M1 and M2 signatures possessed high tumor specificity spatially and was associated with worse patient survival. Cytotoxic T cells in tumor sites evolved in a separate path from those in the circulatory system. T cell receptor (TCR) repertoires in metastatic LNs showed significant higher consistency with TCRs in tumor than those in nonmetastatic LNs and PBMCs, suggesting the existence of common neo-antigens across metastatic LNs and primary tumor cites. In addition, the immune environment in metastatic LNs had transformed into a tumor-like status, where pro-inflammatory macrophages and exhausted T cells were upregulated, accompanied by a decrease in B cells and neutrophils. Finally, cell interactions showed that cancer-associated fibroblasts (CAFs) contributed most to shaping the immune-suppressive microenvironment, while CD8+ cells were the most signal-responsive cells. CONCLUSIONS: This study revealed the cell structures of both micro- and macroenvironments, revealed how different cells diverged in related contexts as well as their prognostic capacities, and displayed a landscape of cell interactions with spatial information.

Dynamic changes in intrinsic subtype, immunity status, and risk score before and after neoadjuvant chemo- and HER2-targeted therapy without pCR in HER2-positive breast cancers: A cross-sectional analysis.

Very few studies have been done in HER2 positive patients without complete pathological response (pCR) after combined neoadjuvant chemo- and HER2-target therapy to investigate changes in intrinsic subtype, risk of recurrence (ROR) score, and immunity status before and after treatment. Patients with nonmetastatic HER2-positive breast cancer failed to achieve pCR after neoadjuvant chemotherapy plus trastuzumab were included in current study. We examined the distribution of PAM50 subtypes, ROR score and immunity score in 25 paired baseline and surgical samples. The Miller-Payne grading system was used to evaluate the efficacy of the neoadjuvant therapy. It was observed that the distribution of intrinsic subtype, ROR category and immunity subgroup varied according to hormone receptor (HR) status. HER2-enriched and basal-like subtypes, median-high ROR categories and immunity-weak subgroup were dominant in baseline tumors. Compared to baseline samples, conversion of intrinsic subtype, ROR categories and immunity subgroups were found in 15 (60.0%), 13(52.0%), and 11(44.0%) surgical samples, respectively. The PAM50 subtype, ROR category, and immunity subgroup were concordant between baseline and surgical samples where nonluminal subtypes, median-high ROR categories and i-weak subgroup were still common. In conclusion, the HER2-positive breast cancer is highly heterogeneous with a distribution of 72-gene expression varying according to HR co-expression. The dynamics of the 72-gene expression pre- and posttreatment may become novel biomarker for guiding adjuvant therapy and hence warrant further investigation.

Single-cell transcriptome analysis revealed the heterogeneity and microenvironment of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the human gastrointestinal tract. In this study, we performed single-cell RNA sequencing (RNA-seq) on intra- and peri-tumor tissues from GIST patients with the aim of discovering the heterogeneity of tumor cells in GIST and their interactions with other cells. We found four predominating cell types in GIST tumor tissue, including T cells, macrophages, tumor cells, and NK cells. Tumor cells could be clustered into two groups: one was highly proliferating and associated with high risk of metastasis, the other seemed "resting" and associated with low risk. Their clinical relevance and prognostic values were confirmed by RNA-seq of 65 GIST samples. T cells were the largest cell type in our single-cell data. Two groups of CD8+ effector memory (EM) cells were in the highest clonal expansion and performed the highest cytotoxicity but were also the most exhausted among all T cells. A group of macrophages were found polarized to possess both M1 and M2 signatures, and increased along with tumor progression. Cell-to-cell interaction analysis revealed that adipose endothelial cells had high interactions with tumor cells to facilitate their progression. Macrophages were at the center of the tumor microenvironment, recruiting immune cells to the tumor site and having most interactions with both tumor and nontumor cells. In conclusion, we obtained an overview of the GIST microenvironment and revealed the heterogeneity of each cell type and their relevance to risk classifications, which provided a novel theoretical basis for learning and curing GISTs.

SARS-CoV-2: minireview and review of first case in Foshan, China.

The first case of #COVID19 in Foshan provides a reference for the treatment of severe #SARSCoV2 pneumonia https://bit.ly/3eD81qj.

The variations of TRBV genes usages in the peripheral blood of a healthy population are associated with their evolution and single nucleotide polymorphisms.

T cell receptors (TCRs) are a class of T cell surface molecules that recognize the antigen-derived peptides presented by the major histocompatibility complex (MHC) and are able to trigger a series of immune responses. TCRs are important members of the adaptive immune system that arose in the jawed fish 500 million years ago. T cell receptor beta variable (TRBV) genes have been widely used to characterize TCR repertoires. Studying the evolution of TRBV may help us to better understand the adaptive immune system. To investigate TRBV evolution and its impacts on the usages of TRBV genes in human populations, we compared the TRBV genes and their homologous sequences among humans, mouse, rhesus and chimpanzee, analyzed the single-nucleotide polymorphisms (SNPs) located at TRBV loci, and sequenced TCR repertoires in the peripheral blood of 97 healthy donors. We found that functional TRBVs are more evolutionarily conserved but possess more SNPs in human populations than do nonfunctional (pseudo) TRBVs. Based on the conservation levels in the four species, we classified the functional TRBVs into 2 groups: old (conserved between mouse and humans) and new (conserved only in primates). The new TRBVs evolve faster and possess more SNPs than the old TRBVs. The variations in TRBV genes frequencies in the peripheral blood of healthy donors are negatively correlated with SNP density. These observations suggest that TRBV usages may be influenced by TCR-MHC co-evolution.

TCR Repertoire as a Novel Indicator for Immune Monitoring and Prognosis Assessment of Patients With Cervical Cancer.

There is increasing evidence that deep sequencing-based T cell repertoire can sever as a biomarker of immune response in cancer patients; however, the characteristics of T cell repertoire including diversity and similarity, as well as its prognostic significance in patients with cervical cancer (CC) remain unknown. In this study, we applied a high throughput T cell receptor (TCR) sequencing method to characterize the T cell repertoires of peripheral blood samples from 25 CC patients, 30 cervical intraepithelial neoplasia (CIN) patients and 20 healthy women for understanding the immune alterations during the cervix carcinogenesis. In addition, we also explored the signatures of TCR repertoires in the cervical tumor tissues and paired sentinel lymph nodes from 16 CC patients and their potential value in predicting the prognosis of patients. Our results revealed that the diversity of circulating TCR repertoire gradually decreased during the cervix carcinogenesis and progression, but the circulating TCR repertoires in CC patients were more similar to CIN patients than healthy women. Interestingly, several clonotypes uniquely detected in CC patients tended to share similar CDR3 motifs, which differed from those observed in CIN patients. In addition, the TCR repertoire diversity in sentinel lymphatic nodes from CC patients was higher than in tumor tissues. More importantly, less clonotypes in TCR repertoire of sentinel lymphatic node was associated with the poor prognosis of the patients. Overall, our findings suggested that TCR repertoire might be a potential indicator of immune monitoring and a biomarker for predicting the prognosis of CC patients. Although functional studies of T cell populations are clearly required, this study have expanded our understanding of T cell immunity during the development of CC and provided an experimental basis for further studies on its pathogenesis and immunotherapy.

Circulating CD8+ T-cell repertoires reveal the biological characteristics of tumors and clinical responses to chemotherapy in breast cancer patients.

PURPOSE: CD8+ T cells are primarily cytotoxic cells that provide immunological protection against malignant cells. Considerable evidence suggests that the T-cell repertoire is closely associated with the host immune response and the development of cancer. In this study, we explored the characteristics of the circulating CD8+ T-cell repertoire and their potential value in predicting the clinical response of breast cancer patients to chemotherapy. EXPERIMENTAL DESIGN: We applied a high-throughput TCR ß-chain sequencing method to characterize the CD8+ T-cell repertoire of the peripheral blood from 26 breast cancer patients. In addition, changes in the circulating CD8+ T-cell repertoire during chemotherapy were analyzed. RESULTS: We found that the HEC ratios of the CD8+ T-cell repertoires from HER2+ breast cancer patients were significantly higher than those of HER2- patients, suggesting that the HER2 protein is released into circulation where it is targeted by CD8+ T cells. Several Vß and CDR3 motifs preferentially used in HER2+ patients were identified. Besides, we found that the circulating CD8+ T-cell repertoires evolved during chemotherapy and correlated with patient clinical responses to chemotherapy. Increased CD8+ T-cell repertoire heterogeneity during chemotherapy was associated with a better clinical response. CONCLUSIONS: Although functional studies of clonally expanded CD8+ T-cell populations are clearly required, our results suggest that the circulating CD8+ T-cell repertoire reflects the characteristics of the tumor-associated biomolecules released into the blood and correlates with the clinical responses of the patients to chemotherapy which might assist in making treatment decisions.

TCR repertoire profiling of tumors, adjacent normal tissues, and peripheral blood predicts survival in nasopharyngeal carcinoma.

The T-cell immune responses in nasopharyngeal carcinoma patients have been extensively investigated recently for designing adoptive immunotherapy or immune checkpoint blockade therapy. However, the distribution characteristics of T cells associated with NPC pathogenesis are largely unknown. We performed deep sequencing for TCR repertoire profiling on matched tumor/adjacent normal tissue from 15 NPC patients and peripheral blood from 39 NPC patients, 39 patients with other nasopharyngeal diseases, and 33 healthy controls. We found that a lower diversity of TCR repertoire in tumors than paired tissues or a low similarity between the paired tissues was associated with a poor prognosis in NPC. A more diverse TCR repertoire was identified in the peripheral blood of NPC patients relative to the controls; this was related to a significant decrease in the proportion of high-frequency TCR clones in NPC. Higher diversity in peripheral blood of NPC patients was associated with a worse prognosis. Due to the peculiarity of the Vß gene usage patterns in the peripheral blood of NPC patients, 15 Vß genes were selected to distinguish NPC patients from controls by the least absolute shrinkage and selection operator analysis. We identified 11 clonotypes shared by tumors and peripheral blood samples from different NPC patients, defined as "NPC-associated" that might have value in adoptive immunotherapy. In conclusion, we here report the systematic and overall characteristics of the TCR repertoire in tumors, adjacent normal tissues, and peripheral blood of NPC patients. The data obtained may be relevant to future clinical studies in the setting of immunotherapy for NPC patients.

Characterization of peripheral blood TCR repertoire in patients with ankylosing spondylitis by high-throughput sequencing.

Ankylosing spondylitis (AS) is a chronic and progressive autoimmune disease affecting the invasion of the spine, sacroiliac joints and peripheral joints. T cells play a vital role in the underlying pathogenesis of AS, which mediated autoimmune and inflammatory responses via specific recognition of autoantigen peptides presented by susceptibility HLA. Antigen-specific T cells triggered by HLA/antigen complexes will undergo a massive expansion that forming an uneven T cell repertoire. To enhance our understanding of T-cell-mediated autoimmune in AS, we applied TCR ß chains high-throughput sequencing to AS patients for in-depth TCR repertoire analysis. A significantly lower TCR repertoire diversity was observed in peripheral blood of AS patients relative to controls. And severe patients in our AS cohort have a more restricted TCR repertoire than mild patients, suggesting that the TCR repertoire diversity might be associated with the clinical severity of disease. No V, J and VJ pairs with significant biased usage were identified, which indicated that the usage frequency deviation of certain V/J/V-J genes in AS patients is little. This is a pilot study with potentially interesting observation on reduced diversity of T cells repertoire in peripheral blood of AS patients and further studies are needed.

Evolutionary comparisons of miRNA regulation system in six model organisms.

miRNAs are a class of endogenous small non-coding regulatory RNAs, that can mediate the transcriptional gene silencing as well as gene expression activation. miRNAs, which are found in a wide range of species, participate in cell differentiation, proliferation, development, apoptosis, tumorigenesis, metabolism, immune system, and signaling pathways. Here, we focused on the relationship between evolution and the miRNA system, with an emphasis on both miRNAs and their target genes. Six species from the evolutionary ladder were selected as a focus of this study. Public data were retrieved and combined to compare miRNAs abundance, miRNA families, molecular functions of target genes, biological processes of target genes, protein families of target gene products, transcription factors regulated by the miRNAs, signaling pathways and tissues across the six species. We found that the expansion rate of miRNAs was significantly higher compared to other genes in human evolution. Newborn miRNA families, which were quantitatively larger than dead miRNA families, seem to be closely related to the species complexity and tissue specificity. Additionally, miRNAs in higher order species were more likely to target genes related to signaling and the immune system, while miRNAs from lower order species preferred to target genes related to the embryonic development process, reproduction and growth. Meanwhile, miRNA systems displayed diversity in regulating transcription factors, signaling pathways and tissues. Our research suggested that the miRNA system might promote evolution, especially in higher species.