scholarly journals miR-20b Inhibits T Cell Proliferation and Activation via NFAT Signaling Pathway in Thymoma-Associated Myasthenia Gravis

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Yanzhong Xin ◽  
Hongfei Cai ◽  
Tianyu Lu ◽  
Yan Zhang ◽  
Yue Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cell ◽  
Myasthenia Gravis ◽  
Cultured Cells ◽  
T Cell Proliferation ◽  
Marker Genes ◽  
Specific Marker ◽  
Expression Levels ◽  
Qrt Pcr

Purpose. We examined the role of miR-20b in development of thymoma-associated myasthenia gravis, especially in T cell proliferation and activation.Materials and Methods. Using qRT-PCR, we assessed expression levels of miR-20b and its target genes in cultured cells and patient samples and examined the proliferation of cultured cells, using MTT cell proliferation assays and flow cytometry based cell cycle analysis. Activation of T cells was determined by both flow cytometry and qRT-PCR of activation-specific marker genes.Results. Expression of miR-20b was downregulated in samples of thymoma tissues and serum from patients with thymoma-associated myasthenia gravis. In addition, T cell proliferation and activation were inhibited by ectopic overexpression of miR-20b, which led to increased T cell proliferation and activation. NFAT5 and CAMTA1 were identified as targets of miR-20b. Expression levels of NFAT5 and CAMTA1 were inhibited by miR-20b expression in cultured cells, and the expression levels of miR-20b and NFAT5/CAMTA1 were inversely correlated in patients with thymoma-associated myasthenia gravis.Conclusion. miR-20b acts as a tumor suppressor in the development of thymoma and thymoma-associated myasthenia gravis. The tumor suppressive function of miR-20b in thymoma could be due to its inhibition of NFAT signaling by repression of NFAT5 and CAMTA1 expression.

scholarly journals Gag-Specific CD8 T-Cell Proliferation Is Associated With Higher Peripheral Blood Levels of Transforming Growth Factor-β and Gut-Homing T Cells in Youths Perinatally Infected With Human Immunodeficiency Virus-1: The ANRS-EP38-IMMIP Study

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Josiane Warszawski ◽  
Véronique Avettand-Fenoel ◽  
Christine Rouzioux ◽  
Daniel Scott-Algara ◽  
Thomas Montange ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Immunodeficiency Virus ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Transforming Growth Factor ◽  
Cd8 T Cell ◽  
T Cell Proliferation ◽  
Immunodeficiency Virus ◽  
Tgf Β1

Abstract Background Gag-specific T lymphocytes play a key role in the control of human immunodeficiency virus (HIV) replication. Their restoration will be important for future reservoir targeting strategies. In this study, we aimed to identify immune correlates of Gag-specific CD8 T-cell proliferation in youths with perinatally acquired HIV-1 infection. Methods The ANRS-EP38-IMMIP study included youths of 15 to 24 years of age. Fifty-three were taking combination anti-retroviral therapy and aviremic at the time of the study and had undergone valid 5-6-carboxyfluorescein diacetate succimidyl ester-based flow cytometry T-cell proliferation assays. Plasma analytes were quantified by enzyme-linked immunosorbent assay or multiplex assays. Peripheral blood cells were phenotyped by flow cytometry. Logistic regression was used to study the association between Gag-specific T-cell proliferation and immune markers. Results Patients with Gag-specific CD8 T-cell proliferation had higher levels of plasma transforming growth factor (TGF)-β1, a lower proportion of naive cells among regulatory T cells (Tregs), and higher percentages of CD4 and CD8 T cells expressing the α4β7 integrin or CD161 molecule than those without a Gag-specific response. These associations were significant based on analyses including potential confounders. Conclusions Preserved Gag-specific CD8 T-cell proliferation was associated with higher TGF-β1 levels and increased percentages of T cells with a gut-homing phenotype at least 15 years after HIV infection during the perinatal period.

scholarly journals High Beta-Chemokine Expression Levels in Lymphoid Tissues of Simian/Human Immunodeficiency Virus 89.6-Vaccinated Rhesus Macaques Are Associated with Uncontrolled Replication of Simian Immunodeficiency Virus Challenge Inoculum

2004 ◽  
Vol 78 (12) ◽  
pp. 6399-6408 ◽  
Author(s):  
Lisa LaFranco-Scheuch ◽  
Kristina Abel ◽  
Norbert Makori ◽  
Kristina Rothaeusler ◽  
Christopher J. Miller
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
T Cell Proliferation ◽  
Expression Levels ◽  
Immunodeficiency Virus

ABSTRACT Viral suppression by noncytolytic CD8+ T cells, in addition to that by classic antiviral CD8+ cytotoxic T lymphocytes, has been described for human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. However, the role of soluble effector molecules, especially beta-chemokines, in antiviral immunity is still controversial. In an attenuated vaccine model, approximately 60% of animals immunized with simian/human immunodeficiency virus (SHIV) 89.6 and then challenged intravaginally with SIVmac239 controlled viral replication (viral RNA level in plasma, <104 copies/ml) and were considered protected (K. Abel, L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller, J. Virol. 77:3099-3118, 2003). To determine the in vivo importance of beta-chemokine secretion and CD8+-T-cell proliferation in the control of viral replication in this vaccine model, we examined the relationship between viral RNA levels in the axillary and genital lymph nodes of vaccinated, protected (n = 20) and vaccinated, unprotected (n = 11) monkeys by measuring beta-chemokine mRNA levels and protein expression, the frequency of CD8+ T cells expressing beta-chemokines, and the extent of CD8+-T-cell proliferation. Tissues from uninfected (n = 3) and unvaccinated, SIVmac239-infected (n = 9) monkeys served as controls. Axillary and genital lymph nodes from unvaccinated and vaccinated, unprotected monkeys had significantly higher beta-chemokine mRNA expression levels and increased numbers of beta-chemokine-positive cells than did vaccinated, protected animals. Furthermore, the lymph nodes of vaccinated, unprotected monkeys had significantly higher numbers of beta-chemokine+ CD8+ T cells than did vaccinated, protected monkeys. Lymph nodes from vaccinated, unprotected animals also had significantly more CD8+-T-cell proliferation and marked lymph node hyperplasia than the lymph nodes of vaccinated, protected monkeys. Thus, higher levels of virus replication were associated with increased beta-chemokine secretion and there is no evidence that beta-chemokines contributed to the SHIV89.6-mediated control of viral replication after intravaginal challenge with SIVmac239.

Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2577-2577
Author(s):  
Richard W. Joseph ◽  
Tae Kon Kim ◽  
Lisa St. John ◽  
Jahan Khalili ◽  
Uday R Popat ◽  
...  
Keyword(s):  
Vitamin D ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
Activation Markers

Abstract Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p&lt;0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p&lt;0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease.

Blocking VIP Signaling Abrogates Upregulation Of PD1 and Increases Proliferation Of Allo-Reactive T-Cells In a Mixed Lymphocyte Reaction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1041-1041
Author(s):  
Emily R Summerbell ◽  
Cynthia R. Giver ◽  
Sravanti Rangaraju ◽  
Katarzyna Anna Darlak ◽  
Edmund K. Waller
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Immunity ◽  
T Cell Proliferation ◽  
Cell Immunity

Abstract Introduction Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1 immunity and inhibits antiviral immunity. Decreased Th1 immunity is problematic for allogeneic bone marrow transplant (allo-BMT) patients requiring T-cell immunity against blood cancers (Graft-versus-Tumor) and against secondary infections such as CMV. VIPhyb, a modified VIP peptide, is a VIP receptor antagonist that decreases VIP signaling. VIP-knockout mice and mice treated with VIPhyb after allo-BMT are known to have better antiviral immunity and survival after CMV infection without increasing GvHD (Li et al. PLoS One. 2013 May 27;8(5):e63381) (Li et al. Blood. 2013 Mar 21;121(12):2347-51.), thus making VIPhyb of interest for pharmacological use in humans to improve the efficacy of allo-BMT The effects of VIPhyb on T-cell immunity are not yet fully profiled. This study aimed to analyze the effects of VIPhyb on CD4+ and CD8+ T-cell proliferation and activation in order to better understand the mechanistic implications of VIP inhibition on T-cell adaptive immunity. This study also aimed to show that mixed lymphocyte reactions (MLRs), an in vitro allo-BMT model, could be used to provide rapid and reliable results that are consistent with in vivo data. It was hypothesized that VIPhyb would increase T-cell immunity as profiled by: increased T-cell proliferation, CD69 and PD1 co-upregulation in early T-cell activation, and PD1 downregulation in T-cells after initial activation. Methods Splenocytes from two histoincompatible mice were cultured together at 37°C in a 1:1 ratio in a one-way MLR. BALB/c splenocytes (stimulators) were irradiated at 20Gy, and Pepboy splenocytes (responders) were labeled with CFSE to trace proliferation. VIPhyb was added daily to the cell cultures in doses of 0.1μM, 0.3μM, 1μM, or 3μM. Treatment groups were compared to a PBS control. Proliferation, CD69, and PD1 were assessed by flow cytometry on the BD FACSAria. All results are shown as mean ± SEM (n=3). One-way ANOVA tests with Dunnett post-tests were calculated using Prism software. *p < 0.05; **p < 0.01; ***p < 0.001 Results VIPhyb increased CD4+ and CD8+ T-cell proliferation: 3, 5, and 7 days after initiating a one-way MLR, CFSE expression of Pepboy responder T-cells was assessed using flow cytometry (Figure 1). As the VIPhyb dose increased, the percentage of initial splenocytes that underwent proliferation increased in both CD4+ and CD8+ T-cells. VIPhyb increased early T-cell CD69 expression and abrogated later PD1 upregulation in CD8+ T-cells: 3, 5, and 7 days after initiating a one-way MLR, expression levels of CD69 and PD1 on Pepboy responder T-cells were assessed by flow cytometry. Significant upregulation of CD69 on CD4+ and CD8+ T-cells on day 3 occurred with increasing VIPhyb doses (Figures 2A and 2B). PD1 was co-upregulated with CD69 during early activation, and VIPhyb significantly decreased PD1 expression on CD8+ T-cells on days 5 and 7 (Figures 2C and 2D). Conclusions VIPhyb increased T-cell proliferation; CD8+ T-cells were affected more significantly. VIPhyb increased early co-upregulation of CD69 and PD1 in all T-cells and significantly decreased later CD8+ T-cell PD1 expression, indicating that VIPhyb increases T-cell activation. We hypothesize that the decreased PD1 expression will be critical for understanding the pathways involved in VIP inhibition. Importantly, since it has been shown in vivo that VIPhyb does not increase GvHD, then it can be assumed that the VIPhyb-induced T-cell proliferation and activation will increase GvL and adaptive immunity without increasing alloreactivity. Notably, these results are consistent with published in vivo data, which demonstrates that the MLR can be used as a faster method of analyzing pharmacological compounds than in vivo experiments. Given these results, VIPhyb is still of interest as a potential therapy for allo-BMT patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CXCL9 secreted by tumor-associated dendritic cells up-regulates PD-L1 expression in bladder cancer cells by activating the CXCR3 signaling

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Weigang Xiu ◽  
Jingjing Luo
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Bladder Cancer ◽  
Dendritic Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Human Peripheral Blood ◽  
T Cell Proliferation ◽  
T24 Cells ◽  
Cell Immunity

Abstract Background Tumor-associated dendritic cells (TADCs) can interact with tumor cells to suppress anti-tumor T cell immunity. However, there is no information on whether and how TADCs can modulate programmed death-ligand 1 (PD-L1) expression by cancer cells. Methods Human peripheral blood monocytes were induced for DCs and immature DCs were cultured alone, or co-cultured with bladder cancer T24 or control SV-HUC-1 cells, followed by stimulating with LPS for DC activation. The activation status of DCs was characterized by flow cytometry and allogenic T cell proliferation. The levels of chemokines in the supernatants of co-cultured DCs were measured by CBA-based flow cytometry. The impacts of CXCL9 on PD-L1, STAT3 and Akt expression and STAT3 and Akt phosphorylation in T24 cells were determined by flow cytometry and Western blot. Results Compared with the control DCs, TADCs exhibited immature phenotype and had significantly lower capacity to stimulate allogenic T cell proliferation, particularly in the presence of recombinant CXCL9. TADCs produced significantly higher levels of CXCL9, which enhanced PD-L1 expression in T24 cells. Pre-treatment with AMG487 abrogated the CXCL9-increased PD-L1 expression in T24 cells. Treatment with CXCL9 significantly enhanced STAT3 and Akt activation in T24 cells. Conclusions TADCs produced high levels of CXCL9 that increased PD-L1 expression in bladder cancer T24 cells by activating the CXCR3-related signaling. Our findings may shed new lights in understanding the regulatory roles of TADCs in inhibiting antitumor T cell responses and promoting tumor growth.

Pharmacological Inhibition Of Semi-Direct Alloantigen Presentation and The Generation Of Cross-Dressed Antigen Presenting Cells: A Novel approach To Limit Graft Versus Host Disease Following Allogeneic Hematopoetic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 294-294
Author(s):  
Sravanti Rangaraju ◽  
Junghwa Choi ◽  
Cynthia R. Giver ◽  
Edmund K. Waller
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Proliferation ◽  
Mhc Ii ◽  
Direct Pathway ◽  
Antigen Presenting

Abstract Background Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant (allo-HSCT) is caused by CD4+ and CD8+ donor T cells directed against mismatched recipient antigens, presented in the context of donor MHC-II (indirect pathway) and recipient MHC-I (direct pathway). Recently, the presence of 'cross-dressed' CD11c+ antigen presenting cells (APCs) expressing both donor and recipient type MHC-I molecules has been demonstrated in animal organ and HSCT transplant models supporting 'semi-direct' pathway of allo-activation (Wang et al, Blood. 2011).These APCs can efficiently present allo-antigens to both CD4+ and CD8+ T cells and activate immune responses that could lead to allograft rejection or GVHD. Exchange of membrane fragments and associated proteins between cells, termed trogocytosis, generates cross-dressed APCs.We sought to test whether cross-dressed APCs facilitate antigen presentation to donor T cells and initiate GVHD following allo-HSCT. Further, we tested an array of drugs as inhibitors of trogocytosis, to interrupt the semi-direct pathway of allo-antigen presentation. Methods In vivo experiments used a B6(H2Kb) ˆ B10.BR(H2Kk) murine transplant model. Spleens of transplanted mice were analyzed on days 10, 15, 20 post-transplant for presence of cross dressed CD11c+cells, and their expression of CD80, CD86 and MHC-II by flow cytometry. Cross dressed donor CD11c+ FACS sorted cells from recipient spleens were co-cultured with CFSE labeled donor type T-cells for 6 days, and T-cell proliferation was measured as dilution of CFSE by flow cytometry. In vitro experiments used primary MLR consisting of CFSE labeled B6 bone marrow cells co-cultured with PKH26 (membrane dye) labeled B10.BR splenocytes. B6 antigen presenting cells were analyzed by flow cytometry for the presence of CFSE+PKH26+ double positive cells generated by trogocytosis. Pharmacological inhibitors of cytoskeleton function were added to the primary MLR and their effect on trogocytosis as well as T cell proliferation was assessed. Results Cross-dressed donor CD11c+ APCs were generated in vivo following allo-HSCT (Figure 1). Recipient spleens showed that 50%, 28.6% (p=0.01) and 12% (p=0.02) of donor type CD11c+ cells were cross dressed on days 10, 15 and 20 respectively post transplant (n=5). These cross dressed APCs expressed higher levels of co-stimulatory molecules CD80 (p<0.001) and CD86 (p<0.001), and MHC-II compared to non-cross-dressed donor CD11c+ cells (Figure 2). Sorted cross dressed CD11c+ cells from recipient mice were able to induce in vitro proliferation of co-geneic CD8 T-cells, while their non-crossdressed counterparts did not. We demonstrated that cross-dressed CD11c+ cells were generated in vitro, by exchange of plasma membrane fragments and could be inhibited in vitro by low doses of paclitaxel and VIP antagonist (Figure 3), while preserving cell viability. Further more, bone marrow treated with 0.05uM of paclitaxel, caused significantly decreased T cell proliferation in primary MLR compared to non drug treated bone marrow. Discussion The high frequencies of cross-dressed donor CD11c+ APCs following allo-HSCT suggests that semi-direct allo-antigen presentation may play a key role in the initiation of GVHD, while the decreasing trend could reflect replacement of host cells by donor hematopoetic cells. Reducing the generation of cross-dressed APCs by pharmacological inhibition of trogocytosis is a novel approach to reduce GVHD post allo-HSCT, targeting the semi-direct pathway of allo-antigen presentaion. Our data shows that very low doses of paclitaxel, a microtubule inhibitor and VIPHyb, an antagonist of Vasoactive Intestinal Peptide signaling, can reduce semi-direct presentaion of allo-antigen to Tcells and reduce alloreactivity without direct cytotoxic effect. Disclosures: No relevant conflicts of interest to declare.

scholarly journals T-Cell Defect in Diffuse Large B-Cell Lymphomas Involves Expansion of Myeloid Derived Suppressor Cells Expressing IL-10, PD-L1, and S100A12

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1478-1478 ◽  
Author(s):  
Imane Azzaoui ◽  
Fabrice Uhel ◽  
Delphine Rossille ◽  
Céline Pangault ◽  
Joelle Dulong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cell ◽  
B Cell ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Arginase 1 ◽  
Culture Supernatants ◽  
Large B Cell

Abstract Introduction: In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cells include monocytic and granulocytic myeloid derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory or infectious diseases as well as in numerous tumors including multiple myeloma, chronic lymphocytic leukemia and DLBCL. However, their mechanisms of action in DLBCL remain unclear. The aim of the study was to investigate the presence and mechanisms of suppression of MDSC subsets in DLBCL. Methods: Whole blood from 56 DLBCL at diagnosis and 45 healthy donors (HD) were analyzed by flow cytometry. Quantitative Real Time PCR were analyzed on Taqman@ array microfluidic cards. Cytokine levels in plasma and culture supernatants were measured by Luminex and Elisa. T-cell proliferation after monocyte depletion or treatment with inhibitors, was analyzed by CFSE assay. Results: By flow cytometry, we identified an expansion of G-MDSC (Linneg HLA-DRneg CD33pos CD11bpos) and M-MDSC (CD14pos HLA-DRlow) in DLBCL compared to HD (p<0.001). Interestingly, only M-MDSC number was correlated with the International Prognostic Index (IPI), the event-free survival (EFS), and the number of circulating Treg. Furthermore, T-cell proliferation was restored after monocyte depletion. To identify the mechanism of suppression involved, we evaluated the gene expression of key enzymes (ARG1, IDO1, NOS2, HO-1 and PTGS2) or immunomodulatory molecules (IL10, TGF β1, CD274/PD-L1, S100A8, S100A9, S100A12) involved in MDSC biology. ARG1, IDO1, IL10, CD274/PD-L1 and S100A12 were significantly up-regulated in DLBCL (p<0.05). At the protein level arginase 1, IL-10 and S100A12 were increased in DLBCL plasma, whereas PD-L1 expression on monocyte was increased in DLBCL compared to HD (p=0.03). Arginase 1 and IDO activities where increased in DLBCL patients. In DLBCL, IL-10 and S100A12 were detected in culture supernatants from stimulated PBMC and these molecules were decreased after CD14pos depletion (p<0.05). Finally, we defined 2 groups of DLBCL depending on M-MDSC content and analyzed the percentage of proliferating T cells after CD3/CD28 stimulation in the presence of various inhibitors or blocking antibodies (L-1MT, nor-NOHA, anti-IL-10, anti-PD-1 and anti-S100A12). In the group of patients with circulating MDSC, neutralizing IL-10, PDL-1 and S100A12 resulted in an increase of CD4 and/or CD8 T-cell proliferation. Conclusion: In summary, we identified MDSC subsets expanded in DLBCL as well as new mechanisms of immunosuppression in DLBCL. Myeloid-dependent T-cell suppression is attributed to a release of IL-10 and S100A12 and an increase of PD-L1 expression. Disclosures Cartron: Roche: Consultancy, Honoraria; GSK: Honoraria; Celgene: Honoraria; Sanofi: Honoraria; Gilead: Honoraria.

scholarly journals Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

2021 ◽  
Author(s):  
Jamie L McCall ◽  
Melinda E Varney ◽  
Sebastian A. Dziadowicz ◽  
Casey Hall ◽  
Kathryn E Blethen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Protein Levels ◽  
Cd Exposure

Objective: Prenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation. Methods: RNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Results: We identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Conclusion: Prenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

scholarly journals Prenatal Cadmium Exposure Alters Proliferation in Mouse CD4+ T Cells via LncRNA Snhg7

2022 ◽  
Vol 12 ◽  
Author(s):  
Jamie L. McCall ◽  
Melinda E. Varney ◽  
Emily Rice ◽  
Sebastian A. Dziadowicz ◽  
Casey Hall ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Protein Levels ◽  
Cd Exposure

ObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA host gene 7 (lncSnhg7) in T cell proliferation.MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Finally, lncSnhg7 was knocked down in splenic CD4+ T cells with lentivirus to assess its effect on proliferation.ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Knockdown on lncSnhg7 inhibits proliferation of CD4+ T cells.ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression and knockdown of lncSnhg7 inhibited proliferation suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

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