Naive arthritogenic SKG T cells have a defect in anergy and a repertoire pruned by superantigen

How autoreactive CD4 T cells develop to cause rheumatoid arthritis remains unknown. We used a reporter for antigen-receptor signaling in the SKG autoimmune arthritis model to profile a T cell subpopulation enriched for arthritogenic naive CD4 T cells before arthritis onset by bulk and single cell RNA and T cell antigen-receptor (TCR) sequencing. Our analyses reveal that despite their impaired proximal TCR signaling, a subset of SKG naive CD4 T cells that have recently encountered endogenous antigen upregulate gene programs associated with positive regulation of T cell activation and cytokine signaling at higher levels than wild type cells in the pre-disease state. These arthritogenic cells also induce genes associated with negative regulation of T cell activation but do so less efficiently than wild type cells. Furthermore, their TCR sequences exhibit a previously unrecognized biased peripheral TCR Vβ repertoire likely driven by endogenous viral superantigens. These particular Vβs, known to recognize endogenous mouse mammary tumor virus (MMTV) superantigen, are further expanded in arthritic joints. Our results demonstrate that autoreactive naive CD4 T cells which recognize endogenous viral superantigens are poised to cause disease by their altered transcriptome.

Vitamin D3 Treatment Alters Thyroid Functional Morphology in Orchidectomized Rat Model of Osteoporosis

Vitamin D plays an essential role in prevention and treatment of osteoporosis. Thyroid hormones, in addition to vitamin D, significantly contribute to regulation of bone remodeling cycle and health. There is currently no data about a possible connection between vitamin D treatment and the thyroid in the context of osteoporosis. Middle-aged Wistar rats were divided into: sham operated (SO), orchidectomized (Orx), and cholecalciferol-treated orchidectomized (Orx + Vit. D3; 5 µg/kg b.m./day during three weeks) groups (n = 6/group). Concentration of 25(OH)D in serum of the Orx + Vit. D3 group increased 4 and 3.2 times (p < 0.0001) respectively, compared to Orx and SO group. T4, TSH, and calcitonin in serum remained unaltered. Vit. D3 treatment induced changes in thyroid functional morphology that indicate increased utilization of stored colloid and release of thyroid hormones in comparison with hormone synthesis, to maintain hormonal balance. Increased expression of nuclear VDR (p < 0.05) points to direct, TSH independent action of Vit. D on thyrocytes. Strong CYP24A1 immunostaining in C cells suggests its prominent expression in response to Vit. D in this cell subpopulation in orchidectomized rat model of osteoporosis. The indirect effect of Vit. D on bone, through fine regulation of thyroid function, is small.

Safety and Long-term Improvement of Mesenchymal Stromal Cell Infusion in Critically COVID-19 Patients: A Randomized Clinical Trial.

Background: COVID-19 is a multisystem disease that presents acute and persistent symptoms, the PostAcute Sequelae (PASC). Long-term symptoms may be due to consequences from organ or tissue injury caused by SARS-CoV-2, associated clotting or inflammatory processes during acute COVID-19. Various strategies are being chosen for by clinicians to prevent severe cases of COVID-19; however, a single treatment would not be efficient in treating such a complex disease. Mesenchymal stromal cells (MSCs) are known for their immunomodulatory properties and regeneration ability; therefore, they are a promising tool for treating disorders involving immune dysregulation and extensive tissue damage, as is the case with COVID-19. This study aimed to assess the safety and explore the long-term efficacy of three intravenous doses of UC-MSCs (umbilical cord-MSCs) as an adjunctive therapy in the recovery and postacute sequelae reduction caused by COVID-19. To our knowledge, this is the first report that presents the longest follow-up after MSC treatment in COVID-19 patients. Methods: This was a phase I/II, prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. Seventeen patients diagnosed with COVID-19 who require intensive care surveillance and invasive mechanical ventilation – critically ill patients – were included. The patient infusion was three doses of 5x105 cells/kg UC-MSCs, with a dosing interval of 48 hours (n=11) or placebo (n=6). These evaluations consisted of a clinical assessment, viral load, laboratory testing, including blood count, serologic, biochemical, cell subpopulation, cytokines and CT scan. Results: The results revealed that in the UC-MSC group, there was a reduction in the levels of ferritin, IL-6 and MCP1-CCL2 on the fourteen day. In the second month, a decrease in the levels of reactive C-protein, D-dimer, and neutrophils and an increase in the numbers of TCD3, TCD4 and NK lymphocytes were observed. A decrease in lung extension was observed at the fourth month. The improvement in all these parameters was maintained until the end of patient follow-up. Conclusions: UC-MSCs infusion is safe and can play an important role as an adjunctive therapy, both in the early stages, preventing severe complications and in the chronic phase with postacute sequelae reduction in critically ill COVID-19 patients. Trial registration: Brazilian Registry of Clinical Trials (ReBEC), UTN code - U1111-1254-9819. Registered 31 October 2020 - Retrospectively registered, https://ensaiosclinicos.gov.br/rg/RBR-3fz9yr

Dissecting the Single-Cell Transcriptome Network in Superficial/Deep Tumor Tissues of Diffuse Gastric Cancer

Background and purpose: Gastric cancer is a type of highly heterogeneous malignant tumor and the prognosis of gastric cancer is hard to be improved due to limited knowledge on the molecular mechanism of heterogeneity. Single-cell sequencing technology is recently widely used for the investigation of both inter-tumoral heterogeneity and intra-tumoral heterogeneity. The present study aims to explore the potential oncogene by analyzing the single-cell data in the GSE167297 dataset.Methods: The GSE167297 dataset was downloaded from the GEO database, followed by quality control to remove data with lower quality. The division on cell subtypes was determined by the characteristic marker expressed in each cell subpopulation. Wilcoxon rank-sum test was used to screen out differentially expressed genes. Survival analysis was performed to evaluate the prognostic value of G-protein subunit g 11 (GNG11) gene which was significantly overexpressed in deep tumor tissues of diffuse gastric cancer.Results: In both normal tissues and tumor tissues, subtypes of immune cells and stromal cells were identified, with a higher proportion of infiltrated macrophages observed in deep tumor tissues. EPCAM was found significantly highly expressed in a cell subpopulation from gastric tumor tissues. 515 differentially expressed genes (| log2FC | > 2 and FDR < 1e-5) were screened out between normal tissues and tumor tissues. 86 differentially expressed genes (| log2FC | > 1 and FDR < 0.01) were screened out between superficial and deep tumor tissues, in which GNG11 was most highly expressed in deep tumor tissues (mean expression value: 0.1247, FC value: 52.2109). Disease-specific survival analysis on GNG11 results showed that the HR [95%CI] in the constructed univariate Cox proportional risk model was 4.419 [1.399-13.96] and the P-value in the log-rank test was 0.0056.Conclusion: Differentially expression profiles were provided both extratumorally and intratumorally, indicating a higher infiltration of macrophages in deep tumor tissues. Additionally,GNG11 was screened out to be a significant risk factor in STAD patients.

Regulatory T Cells as Predictors of Clinical Course in Hospitalised COVID-19 Patients

BackgroundThe host immune response has a prominent role in the progression and outcome of SARS-CoV-2 infection. Lymphopenia has been described as an important feature of SARS-CoV-2 infection and has been associated with severe disease manifestation. Lymphocyte dysregulation and hyper-inflammation have been shown to be associated with a more severe clinical course; however, a T cell subpopulation whose dysfunction correlate with disease progression has yet to be identify.MethodsWe performed an immuno-phenotypic analysis of T cell sub-populations in peripheral blood from patients affected by different severity of COVID-19 (n=60) and undergoing a different clinical evolution. Clinical severity was established based on a modified WHO score considering both ventilation support and respiratory capacity (PaO2/FiO2 ratio). The ability of circulating cells at baseline to predict the probability of clinical aggravation was explored through multivariate regression analyses.ResultsThe immuno-phenotypic analysis performed by multi-colour flow cytometry confirmed that patients suffering from severe COVID-19 harboured significantly reduced circulating T cell subsets, especially for CD4+ T, Th1, and regulatory T cells. Peripheral T cells also correlated with parameters associated with disease severity, i.e., PaO2/FiO2 ratio and inflammation markers. CD4+ T cell subsets showed an important significant association with clinical evolution, with patients presenting markedly decreased regulatory T cells at baseline having a significantly higher risk of aggravation. Importantly, the combination of gender and regulatory T cells allowed distinguishing between improved and worsened patients with an area under the ROC curve (AUC) of 82%.ConclusionsThe present study demonstrates the association between CD4+ T cell dysregulation and COVID-19 severity and progression. Our results support the importance of analysing baseline regulatory T cell levels, since they were revealed able to predict the clinical worsening during hospitalization. Regulatory T cells assessment soon after hospital admission could thus allow a better clinical stratification and patient management.

Combinatorial immunotherapy induces tumor-infiltrating CD8+ T cells with distinct functional, migratory, and stem-like properties

BackgroundProgrammed death (ligand) 1 (PD-(L)1) blockade and OX40/4-1BB costimulation have been separately evaluated in the clinic to elicit potent antitumor T cell responses. The precise mechanisms underlying single agent activity are incompletely understood. It also remains unclear if combining individual therapies leads to synergism, elicits novel immune mechanisms, or invokes additive effects.MethodsWe performed high-dimensional flow cytometry and single-cell RNA sequencing-based immunoprofiling of murine tumor-infiltrating lymphocytes (TILs) isolated from hosts bearing B16 or MC38 syngeneic tumors. This baseline infiltrate was compared to TILs after treatment with either anti-PD-(L)1, anti-OX40, or anti-4-1BB as single agents or as double and triple combinatorial therapies. Fingolimod treatment and CXCR3 blockade were used to evaluate the contribution of intratumoral versus peripheral CD8+ T cells to therapeutic efficacy.ResultsWe identified CD8+ T cell subtypes with distinct functional and migratory signatures highly predictive of tumor rejection upon treatment with single agent versus combination therapies. Rather than reinvigorating terminally exhausted CD8+ T cells, OX40/4-1BB agonism expanded a stem-like PD-1loKLRG-1+Ki-67+CD8+ T cell subpopulation, which PD-(L)1 blockade alone did not. However, PD-(L)1 blockade synergized with OX40/4-1BB costimulation by dramatically enhancing stem-like TIL presence via a CXCR3-dependent mechanism.ConclusionsOur findings provide new mechanistic insights into the interplay between components of combinatorial immunotherapy, where agonism of select costimulatory pathways seeds a pool of stem-like CD8+ T cells more responsive to immune checkpoint blockade (ICB).

Colorectal Cancer Stem Cells: An Overview of Evolving Methods and Concepts

Colorectal cancer (CRC) represents one of the most deadly cancers worldwide. Colorectal cancer stem cells (cCSCs) are the driving units of CRC initiation and development. After the concept of cCSC was first formulated in 2007, a huge bulk of research has contributed to expanding its definition, from a cell subpopulation defined by a fixed phenotype in a plastic entity modulated by complex interactions with the tumor microenvironment, in which cell position and niche-driven signals hold a prominent role. The wide development of cellular and molecular technologies recent years has been a main driver of advancements in cCSCs research. Here, we will give an overview of the parallel role of technological progress and of theoretical evolution in shaping the concept of cCSCs.

Expression of L-selectin (CD62L) and CD44 on Bovine Lymphocytes and WC1.1+ γδ T Cells under Stimulation with Staphylococcus aureus

The present study explored the expression of CD62L and CD44 by bovine peripheral blood mononuclear cells (PBMCs) and WC1.1+ &gamma;&delta; T cells under Staphylococcus aureus cell culture stimu-lation. In this study, peripheral blood cells were isolated from ten dairy cows and cocultured with S. aureus. Afterward, the &gamma;&delta; T cell subpopulation and the expression of CD44, CD62L and prolif-erative (Ki67+) cells were evaluated by flow cytometry. Our results showed that the percentages of proliferative PBMCs and WC1.1+ &gamma;&delta; T cells were higher when stimulated with S. aureus. The percentage of CD44+ cells increased in S. aureus-stimulated cultured PMBCs and WC1.1+ &gamma;&delta; T cells, as did the CD44 geometric mean fluorescence intensity (GMFI). The rate of CD62L cells did not differ among groups for either PBMCs or WC1.1+ &gamma;&delta; T cells. A higher GMFI of CD62L in prolif-erative PBMCs than nonproliferative PBMCs upon stimulation with S. aureus was detected, whereas no impact on the GMFI of CD62L was observed in WC1.1+ cells. In summary, our study identified that S. aureus was associated with high expression of CD44 in overall PBMCs and WC1.1+ &gamma;&delta; T cells, and they could generate memory WC1.1+ &gamma;&delta; T cells, preferably central memory cells.

scSemiAE: A Deep Model with Semi-Supervised Learning for Single-Cell Transcriptomics

Background: With the development of modern sequencing technology, hundreds of thousands of single-cell RNA-sequencing(scRNA-seq) profiles allow to explore the heterogeneity in the cell level, but it faces the challenges of high dimensions and high sparsity. Dimensionality reduction is essential for downstream analysis, such as clustering to identify cell subpopulations. Usually, dimensionality reduction follows unsupervised approach. Results: In this paper, we introduce a semi-supervised dimensionality reduction method named scSemiAE, which is based on an autoencoder model. It transfers the information contained in available datasets with cell subpopulation labels to guide the search of better low-dimensional representations, which can ease further analysis. Conclusions: Experiments on five public datasets show that, scSemiAE outperforms both unsupervised and semi-supervised baselines whether the transferred information embodied in the number of labeled cells and labeled cell subpopulations is much or less.