Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation.

Mesenchymal stromal cells (MSCs) have the potential to suppress pathological activation of immune cells and have therefore been considered for the treatment of Graft-versus-Host-Disease. The clinical application of MSCs requires a process validation to ensure consistent quality. A flow cytometry-based mixed lymphocyte reaction (MLR) was developed to analyse the inhibitory effect of MSCs on T cell proliferation. Monoclonal antibodies were used to stimulate T cell expansion and determine the effect of MSCs after four days of co-culture based on proliferation tracking with the violet proliferation dye VPD450. Following the guidelines of the International Council for Harmonisation (ICH) Q2 (R1), the performance of n = 30 peripheral blood mononuclear cell (PBMC) donor pairs was assessed. The specific inhibition of T cells by viable MSCs was determined and precision values of <10% variation for repeatability and <15% for intermediate precision were found. Compared to a non-compendial reference method, a linear correlation of r = 0.9021 was shown. Serial dilution experiments demonstrated a linear range for PBMC:MSC ratios from 1:1 to 1:0.01. The assay was unaffected by PBMC inter-donor variability. In conclusion, the presented MLR can be used as part of quality control tests for the validation of MSCs as a clinical product.

Rules of thumb to obtain, isolate, and preserve porcine peripheral blood mononuclear cells.

One of the most used biospecimens in immunology are peripheral blood mononuclear cells (PBMC). PBMC are particularly useful when evaluating immunity through responses of circulating B- and T-cells, during an infection, or after a vaccination. While several reviews and research papers have been published aiming to point out critical steps when sampling, isolating, and cryopreserving human PBMC -or even analyzing any parameter before sampling that could impair the immune assays' outcomes-, there are almost no publications in swine research dealing with these topics. As it has been demonstrated, several factors, such as stress, circadian rhythmicity, or the anticoagulant used have serious negative impact, not only on the separation performance of PBMC, but also on the ulterior immune assays. The present review aims to discuss studies carried out in humans that could shed some light for swine research. When possible, publications in pigs are also discussed. The main goal of the review is to encourage swine researchers to standardize protocols to obtain, manage and preserve porcine PBMC, as well as to minimize, or at least to consider, the bias that some parameters might induce in their studies before, during and after isolating PBMC.

[Expression Level of SOCS3 in Acute Lymphoblastic Leukemia Cells Affects the Cytotoxicity of NK Cells].

OBJECTIVE: To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells. METHODS: The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells. RESULTS: Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells. CONCLUSION: SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.

CSF-1-induced DC-SIGN+ macrophages are present in the ovarian endometriosis.

BACKGROUND: Researchers have found that macrophages are the predominant cells in the peritoneal fluid (PF) of endometriosis patients. CSF-1 has been found to accumulate in the lesions and PF of endometriosis patients, and CSF-1 induces THP-1-derived macrophages to polarize toward a CD169+ DC-SIGN+ phenotype. Does the cytokine CSF-1 induce monocytes to differentiate into macrophages with a DC-SIGN+ phenotype in endometriosis? METHODS: The level of CSF-1 in the endometrium of control subjects, and the eutopic, and ectopic endometrium of endometriosis patients was evaluated by real-time polymerase chain reaction (qRT-PCR) and was determined by enzyme-linked immunosorbent assay (ELISA) in the PF of control and endometriosis patients. CSF-1 expression was examined with a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. DC-SIGN+ macrophages were detected by immunohistochemical staining of tissues and flow cytometric analysis of the PF of control subjects (N = 25) and endometriosis (N = 35) patients. The phenotypes and biological activities of CSF-1 -induced macrophages were compared in an in vitro coculture system with peripheral blood lymphocytes from control subjects. RESULTS: In this study, we found that the proportion of DC-SIGN+ CD169+ macrophages was higher in the abdominal immune microenvironment of endometriosis patients. CSF-1 was primarily secreted from ectopic lesions and peritoneum in mice with endometriosis. In addition, CSF-1 induced the polarization of macrophages toward a DC-SIGN+ CD169+ phenotype; this effect was abolished by the addition of an anti-CSF-1R antibody. CSF-1 induced the generation of DC-SIGN+ macrophages, leading to a depressed status of peripheral blood lymphocytes, including a high percentage of Treg cells and a low percentage of CD8+ T cells. Similarly, blockade with the anti-CSF-1R antibody abrogated this biological effect. CONCLUSIONS: This is the first study on the role of DC-SIGN+ macrophages in the immune microenvironment of endometriosis. Further study of the mechanism and biological activities of CSF-1-induced DC-SIGN+ macrophages will enhance our understanding of the physiology of endometriosis.

Altered Frequencies and Functions of Innate Lymphoid Cells in Melanoma Patients Are Modulated by Immune Checkpoints Inhibitors.

Monoclonal antibodies targeting immune checkpoints improved clinical outcome of patients with malignant melanoma. However, the mechanisms are not fully elucidated. Since immune check-point receptors are also expressed by helper innate lymphoid cells (ILCs), we investigated the capability of immune checkpoints inhibitors to modulate ILCs in metastatic melanoma patients as well as melanoma cells effects on ILC functions. Here, we demonstrated that, compared to healthy donors, patients showed a higher frequency of total peripheral ILCs, lower percentages of CD117+ ILC2s and CD117+ ILCs as well as higher frequencies of CD117- ILCs. Functionally, melanoma patients also displayed an impaired TNFα secretion by CD117- ILCs and CD117+ ILCs. Nivolumab therapy reduced the frequency of total peripheral ILCs but increased the percentage of CD117- ILC2s and enhanced the capability of ILC2s and CD117+ ILCs to secrete IL-13 and TNFα, respectively. Before Nivolumab therapy, high CCL2 serum levels were associated with longer Overall Survival and Progression Free Survival. After two months of treatment, CD117- ILC2s frequency as well as serum concentrations of IL-6, CXCL8 and VEGF negatively correlated with both the parameters. Moreover, melanoma cells boosted TNFα production in all ILC subsets and increased the number of IL-13 producing ILC2s in vitro. Our work shows for the first time that PD-1 blockade is able to affect ILCs proportions and functions in melanoma patients and that a specific subpopulation is associated with the therapy response.

Inhibition of microRNA-448 suppresses CD4+ T cell inflammatory activation via up-regulating suppressor of cytokine signaling 5 in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. MicroRNA-448 (miR-448) has a pro-inflammatory effect in various inflammation-related diseases and is up-regulated in serum of patients with SLE. However, the role of miR-448 in SLE development remains elusive. In our study, we found high expression of miR-448 in peripheral blood mononuclear cells (PBMCs) of SLE patients, and miR-448 level was positively associated with disease severity. Besides, miR-448 level was up-regulated during the growth of MRL/lpr mice. To investigate the function of miR-448 in SLE, we subjected 8-week MRL/lpr mice to injection of lentivirus (LV)-mediated anti-miR-448. Inhibition of miR-448 reduced serum IgG and anti-dsDNA IgG contents, 24 h urine protein and blood urea nitrogen (BUN) levels, increased complement C3 concentration, and ameliorated splenomegaly and lymphadenectasis in MRL/lpr mice. MiR-448 inhibition alleviated renal inflammatory infiltration and glycogen deposition. Moreover, miR-448 inhibition promoted Treg cell activation and inhibited Th17 cell proportion in naïve CD4+ T cells from spleens, along with elevated interleukin (IL)-10 and reduced IL-17A levels. In vitro, miR-448 inhibition diminished CD4+ T cell polarization toward Th17 cells under Th17-polarizing conditions. Further, luciferase reporter assay revealed that miR-448 binds to the 3'UTR of suppressor of cytokine signaling 5 (SOCS5). SOCS5 expression was down-regulated in the spleens of MRL/lpr mice and induced Th17 cells. SOCS5 deficiency partially reversed the role of miR-448 in Th17 differentiation and IL-17A expression in SLE. Taken together, inhibition of miR-448 impedes Th17 cell activation and tissue damages via targeting SOCS5 in SLE.

Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Chloroquine Suppresses Effector B-Cell Functions and Has Differential Impact on Regulatory B-Cell Subsets.

Objectives: Chloroquine (CQ) is approved for treatment of B-cell mediated diseases such as rheumatoid arthritis and systemic lupus erythematosus. However, the exact mode of action in these diseases has not been studied and it remains unclear which effect CQ has on B-cells. Thus, it was the aim of this study to investigate to which extent CQ affects functionality of effector and regulatory B-cell. Methods: For this purpose, B-cells were isolated from peripheral blood of healthy controls and renal transplant patients. B-cells were stimulated in presence or absence of CQ and Interleukin-10 (IL-10) and Granzyme B (GrB) secretion were assessed. In addition, effector functions such as plasma cell formation, and Immunoglobulin G (IgG) secretion were studied. Results: CQ suppressed Toll-Like-Receptor (TLR)-9 induced B-cell proliferation in a dose-dependent manner. IL-10pos regulatory B-cells were suppressed by CQ already at low concentrations whereas anti-IgG/IgM-induced GrB secreting regulatory B-cells were less susceptible. Plasma blast formation and IgG secretion was potently suppressed by CQ. Moreover, purified B-cells from renal transplant patients were also susceptible to CQ-induced suppression of effector B-cell functions as observed by diminished IgG secretion. Conclusion: In conclusion, CQ had a suppressive effect on IL-10 regulatory B-cells whereas GrB secreting regulatory B-cells were less affected. Effector functions of B-cells such as plasma blast formation and IgG secretion were also inhibited by CQ. Effector B-cells derived from renal transplant patients already under immunosuppression could be suppressed by CQ. These findings may partly explain the clinical efficacy of CQ in B-cell mediated autoimmune diseases. The application of CQ in other disease contexts where suppression of effector B-cells could offer a benefit, such as renal transplantation, may hypothetically be advantageous.

Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single-cell sequencing.

BACKGROUND: Immune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited. METHODS: In this study, we compared the single-cell transcriptomes of immune cells from 12 species. Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. RESULTS: Our data revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular crosstalks and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells. CONCLUSIONS: This study is the first to provide a comprehensive analysis of the cross-species single-cell transcriptome atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders.

An atlas of dynamic peripheral blood mononuclear cell landscapes in human perioperative anaesthesia/surgery.

BACKGROUND: The number of patients receiving anaesthesia is increasing, but the impact of general anaesthesia on the patient's immune system remains unclear. The aim of the present study is to investigate dynamics of systemic immune cell responses to anaesthesia during perioperative period at a single-cell solution. METHODS: The peripheral blood mononuclear cells (PBMCs) and clinical phenomes were harvested and recorded 1 day before anaesthesia and operation, just after anaesthesia (0 h), and 24 and 48 h after anaesthesia. Single-cell sequencing of PBMCs was performed with 10× genomics. Subsequently, data analysis was performed with R packages: Seurat, clusterProfiler and CellPhoneDB. RESULTS: We found that the cluster of CD56+ NK cells changed at 0 h and the cluster of monocytes increased at 24 and 48 h after anaesthesia. The characteristic genes of CD56+ NK cells were mainly enriched in the Jak-STAT signalling pathway and in cell adhesion molecules (24 h) and carbon metabolism (48 h). The communication between CD14+ monocytes and other cells decreased substantially 0 and 48 h after operation. The number of plasma cells enriched in protein export in men was substantially higher than that in women, although the total number in patients decreased 24 h after operation. CD14+ monocytes dominated that cell-cell communications appeared in females, while CD8+ NKT cells dominated that cell-cell communications appeared in male. The number of plasma cells increased substantially in patients with major surgical trauma, with enrichments of pentose phosphate pathway. The communications between plasma cells with other cells varied between surgical severities and anaesthetic forms. The intravenous anaesthesia caused major alterations of cell types, including CD14+ monocytes, plasmas cells and MAIT cells, as compared with inhalation anaesthesia. CONCLUSION: We initially reported the roles of perioperative anaesthesia/surgery in temporal phenomes of circulating immune cells at a single-cell solution. Thus, the protection against immune cell changes would benefit the recovery from anaesthesia/surgery.

Nanocages engineered from Bacillus Calmette-Guerin facilitate protective Vγ2Vδ2 T cell immunity against Mycobacterium tuberculosis infection.

Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a top killer among infectious diseases. While Bacillus Calmette-Guerin (BCG) is the sole TB vaccine, the clumped-clustered features of BCG in intradermal immunization appear to limit both the BCG protection efficacy and the BCG vaccination safety. We hypothesize that engineering of clumped-clustered BCG into nanoscale particles would improve safety and also facilitate the antigen-presenting-cell (APC)'s uptake and the following processing/presentation for better anti-TB protective immunity. Here, we engineered BCG protoplasts into nanoscale membraned BCG particles, termed as "BCG-Nanocage" to enhance the anti-TB vaccination efficiency and safety. BCG-Nanocage could readily be ingested/taken by APC macrophages selectively; BCG-Nanocage-ingested macrophages exhibited better viability and developed similar antimicrobial responses with BCG-infected macrophages. BCG-Nanocage, like live BCG bacilli, exhibited the robust capability to activate and expand innate-like T effector cell populations of Vγ2+ T, CD4+ T and CD8+ T cells of rhesus macaques in the ex vivo PBMC culture. BCG-Nanocage immunization of rhesus macaques elicited similar or stronger memory-like immune responses of Vγ2Vδ2 T cells, as well as Vγ2Vδ2 T and CD4+/CD8+ T effectors compared to live BCG vaccination. BCG-Nanocage- immunized macaques developed rapidly-sustained pulmonary responses of Vγ2Vδ2 T cells upon Mtb challenge. Furthermore, BCG- and BCG-Nanocage- immunized macaques, but not saline controls, exhibited undetectable Mtb infection loads or TB lesions in the Mtb-challenged lung lobe and hilar lymph node at endpoint after challenge. Thus, the current study well justifies a large pre-clinical investigation to assess BCG-Nanocage for safe and efficacious anti-TB vaccination, which is expected to further develop novel vaccines or adjuvants.

Platinum(IV) complexes as inhibitors of CD47-SIRPα axis for chemoimmunotherapy of cancer.

Phagocytosis of cancer cells by antigen presenting cells (APCs) is critical to activate the host's immune responses. However, the targeting ability of APCs to cancer cells is limited by the upregulation of transmembrane protein CD47 on the cancer cell surface. Blocking CD47 can affect the macrophage-mediated phagocytosis. Two platinum-based immunomodulators MUP and DMUP were synthesized to enhance the phagocytic activity of macrophages by blocking the CD47-SIRPα axis. These PtIV complexes not only showed high antiproliferative activity against a panel of human cancer cell lines, but also cooperated with human peripheral blood mononuclear cells (PBMCs) to suppress cancer cells. They acted as immune checkpoint inhibitors to modulate the immune responses of both cancer and immune cells. In particular, DMUP decreased the expression of CD47 in tumor tissues and promoted the polarization of macrophages from M2 to M1 phenotype in a mouse model of non-small cell lung cancer, thereby enhancing the anticancer effect. By interfering with DNA synthesis and stimulating immune system, DMUP takes the advantage of chemotherapy and immunotherapy to inhibit cancer cells. The dual efficacy of DMUP makes it a potential chemoimmunotherapeutic agent in cancer therapy.

Discovery of quinazoline derivatives as novel small-molecule inhibitors targeting the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction.

Development of small molecule PD-1/PD-L1 inhibitors as a novel immunotherapy strategy exhibits great promise. Herein, a novel series of quinazoline derivatives were designed, synthesized and their inhibitory activity against the PD-1/PD-L1 interaction was evaluated through a homogenous time-resolved fluorescence (HTRF) assay. Among them, the compound 39 exhibited the most potent inhibitory activity with an IC50 value of 1.57 nM. Furthermore, the cellular level assays revealed that 39 could inhibit the PD-1/PD-L1 interaction and restore T-cell function, and showed low toxicity on the PBMCs. In addition, the structure-activity relationships (SARs) of the novel quinazoline derivatives were explored and the binding mode of 39 with dimeric PD-L1 was analyzed by molecular docking. This work demonstrates that incorporation of pyrimidine group between the 2 and 3-positions of the biphenyl structure is an effective strategy for designing novel and more potent small molecule PD-1/PD-L1 inhibitors, and 39 can be regarded as a promising lead compound for further investigation.

Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma.

Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

An active learning approach for clustering single-cell RNA-seq data.

Single-cell RNA sequencing (scRNA-seq) data has been widely used to profile cellular heterogeneities with a high-resolution picture. Clustering analysis is a crucial step of scRNA-seq data analysis because it provides a chance to identify and uncover undiscovered cell types. Most methods for clustering scRNA-seq data use an unsupervised learning strategy. Since the clustering step is separated from the cell annotation and labeling step, it is not uncommon for a totally exotic clustering with poor biological interpretability to be generated-a result generally undesired by biologists. To solve this problem, we proposed an active learning (AL) framework for clustering scRNA-seq data. The AL model employed a learning algorithm that can actively query biologists for labels, and this manual labeling is expected to be applied to only a subset of cells. To develop an optimal active learning approach, we explored several key parameters of the AL model in the experiments with four real scRNA-seq datasets. We demonstrate that the proposed AL model outperformed state-of-the-art unsupervised clustering methods with less than 1000 labeled cells. Therefore, we conclude that AL model is a promising tool for clustering scRNA-seq data that allows us to achieve a superior performance effectively and efficiently.

Expression Level of SOCS3 in Acute Lymphoblastic Leukemia Cells Affects the Cytotoxicity of NK Cells / 中国实验血液学杂志

OBJECTIVE@#To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells.@*METHODS@#The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells.@*RESULTS@#Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells.@*CONCLUSION@#SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Peripheral Mononuclear Cells in Patients With Ankylosing Spondylitis.

Objective: Genetic studies on ankylosing spondylitis (AS) have identified more than 100 pathogenic genes. Building a bridge between these genes and biologically targeted therapies is the current research hotspot. Methods: We integrated single-cell assaying transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to explore the key genes and related mechanisms associated with AS pathogenesis. Results: We identified 18 cell types in peripheral mononuclear cells from patients with AS and normal controls and summarized the cell-type-specific abnormal genes by scRNA-seq. Interestingly, we found that the pathogenic gene NFKB involved in AS progression originated from CD8+ T cells. Moreover, we observed an abnormal tumor TNF pathway mediated by abnormal expression of TNF, NFKB, FOS, JUN, and JUNB, and scATAC-seq results confirmed the abnormal accessible binding sites of transcriptional factors FOS, JUN, and JUNB. The final magnetic bead sorting and quantitative real-time PCR(RT-qPCR) confirmed that NFKB, FOS, JUN, and JUNB in CD8+ T cells differed in the AS group. Conclusions: Our results revealed a possible mechanism by which NFKB abnormally regulates FOS, JUN, and JUNB and drives AS progression, providing a novel perspective from a single cell point of view in AS.

A pilot study of high-intensity interval training in older adults with treatment naïve chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the USA, affecting predominantly older adults. CLL is characterized by low physical fitness, reduced immunity, and increased risk of secondary malignancies and infections. One approach to improving CLL patients' physical fitness and immune functions may be participation in a structured exercise program. The aims of this pilot study were to examine physical and immunological changes, and feasibility of a 12-week high-intensity interval training (HIIT) combined with muscle endurance-based resistance training on older adults with treatment naïve CLL. We enrolled eighteen participants with CLL aged 64.9 ± 9.1 years and assigned them to groups depending on distance lived from our fitness center. Ten participants (4 M/6F) completed HIIT and six participants (4 M/2F) completed a non-exercising control group (Controls). HIIT consisted of three 30-min treadmill sessions/week plus two concurrent 30-min strength training sessions/week. Physical and immunological outcomes included aerobic capacity, muscle strength and endurance, and natural killer (NK) cell recognition and killing of tumor cells. We confirmed feasibility if > 70% of HIIT participants completed > 75% of prescribed sessions and prescribed minutes, and if > 80% of high-intensity intervals were at a heart rate corresponding to at least 80% of peak aerobic capacity (VO2peak). Results are presented as Hedge's G effect sizes (g), with 0.2, 0.5 and 0.8 representing small, medium and large effects, respectively. Following HIIT, leg strength (g = 2.52), chest strength (g = 1.15) and seated row strength (g = 3.07) were 35.4%, 56.1% and 39.5% higher than Controls, respectively, while aerobic capacity was 3.8% lower (g = 0.49) than Controls. Similarly, following HIIT, in vitro NK-cell cytolytic activity against the K562 cell line (g = 1.43), OSU-CLL cell line (g = 0.95), and autologous B-cells (g = 1.30) were 20.3%, 3.0% and 14.6% higher than Controls, respectively. Feasibility was achieved, with HIIT completing 5.0 ± 0.2 sessions/week and 99 ± 3.6% of the prescribed minutes/week at heart rates corresponding to 89 ± 2.8% of VO2peak. We demonstrate that 12-weeks of supervised HIIT combined with muscle endurance-based resistance training is feasible, and that high adherence and compliance are associated with large effects on muscle strength and immune function in older adults with treatment naïve CLL.Trial registration: NCT04950452.

T-Lymphocyte proliferative activity in early pregnancy and outside pregnancy state.

T-lymphocytes are present in the endometrium before pregnancy and their number varies depending on menstrual cycle stage. Despite T-lymphocyte population heterogeneity, there is no clear vision of general mechanisms of decidua T-lymphocyte pool formation. One of the assumed variants is T-lymphocyte proliferation in situ. The study objective is to evaluate variations of peripheral blood T-lymphocyte proliferative activity in the presence of trophoblast cells. The peripheral blood was sampled from healthy nonpregnant women in the proliferative (n = 29) and secretory (n = 32) menstrual cycle phases and also from women on 6-7 weeks stage of physiological pregnancy (n = 30). Jeg-3 (ATCC) line cells were applied as trophoblast cells within in vitro model system. T-lymphocyte proliferation was determined by estimating the Ki-67 expression and T-lymphocyte relative number. It was established that trophoblast cells perform inhibiting effect on Ki-67 by T-lymphocytes in all groups of examined women both in course of PBMC cultivation and in case of preliminarily isolated T-lymphocytes. During cultivation in the presence of IL-2 and trophoblasts, PBMC T-lymphocytes in pregnant women are more resistant to trophoblast cells inhibition than in nonpregnant women. In case of isolated T-lymphocytes, decreased T-lymphocyte proliferation during pregnancy was observed as compared to the proliferative cycle phase hence pointing to necessity of T-lymphocyte contact with microenvironment cells for self-support.

Selective Biological Effects of Selenium-Enriched Polysaccharide (Se-Le-30) Isolated from Lentinula edodes Mycelium on Human Immune Cells.

A common edible mushroom Lentinula edodes, is an important source of numerous biologically active substances, including polysaccharides, with immunomodulatory and antitumor properties. In the present work, the biological activity of the crude, homogenous (Se)-enriched fraction (named Se-Le-30), which has been isolated from L. edodes mycelium by a modified Chihara method towards human peripheral blood mononuclear cells (PBMCs) and peripheral granulocytes, was investigated. The Se-Le-30 fraction, an analog of lentinan, significantly inhibited the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated, and down-regulated the production of tumor necrosis factor (TNF)-α by CD3+ T cells. Moreover, it was found that Se-Le-30 significantly reduced the cytotoxic activity of human natural killer (NK) cells. The results suggested the selective immunosuppressive activity of this fraction, which is non-typical for mushroom derived polysaccharides.