CD28-stimulated CD4 Research Articles

Persons who inject drugs (PWID) retain functional NK cells, dendritic cell stimulation, and adaptive immune recall responses despite prolonged opioid use.

Previous literature suggests that acute opioid use results in the functional impairment of the immune response, thereby decreasing resistance to viral infection. Here, we assessed if innate and adaptive immune responses are compromised ex vivo in persons who inject drugs (PWID) and whether long-term injection drug use may impact host susceptibility to in vitro HIV infection. We measured the frequency, activation state, and functional profile of NK cells, dendritic cells, and CD4+ and CD8+ T cells in low-risk PWID who do not share needles, high-risk needle-sharing PWID, and control donors who did not inject drugs. We also assessed plasma levels of inflammatory markers and CD4+ T cell susceptibility to HIV infection. We observed a significant increase in the amount of sCD14 (P=0.0023, n=16) and sCD163 (P=0.0001, n=16) in the plasma of PWID compared to controls. Evidence of constitutive activation was noted in PWID as compared to controls with increased CD69 expression in CD56dim NK cells (P=0.0103, n=26) and increased CD38 and HLA-DR expression in CD4+ T cells (P=0.0355, n=23). However, no innate or adaptive functional differences were detected between PWID and controls, including: NK cell direct or antibody-dependent cellular cytotoxicity poly-functional response, TLR-stimulated dendritic cell/NK crosstalk, CD8+ T cell response to Staphylococcal enterotoxin B or CMV/EBV/FLU peptides, or constitutive or anti-CD3/CD28-stimulated CD4+ T cell infectivity with CCR5-tropic or CXCR4-tropic HIV-1 isolates. Our data indicate that PWID who utilize opioids over as prolonged time frame can retain a functional ex vivo immune response without a measurable increase in CD4+ T cell infectivity suggesting that leukocytes from PWID are not intrinsically more susceptibility to infection with HIV than non-PWID controls.

Monocytic myeloid-derived suppressor cells generated from rhesus macaque bone marrow enrich for regulatory T cells

Putative monocytic myeloid-derived suppressor cells (mMDSC; lineage−HLA-DR−/lo) were generated in 7-day cultures from normal rhesus macaque bone marrow (BM) cells in GM-CSF and IL-6. Three subsets were identified based on their differential expression of CD14, CD33, CD34 and CD11b. Following flow sorting, assessment of the capacity of these subsets to suppress anti-CD3/CD28-stimulated CD4 and CD8 T cell proliferation revealed that the most potent population was CD14hiCD33−/loCD34loCD11bhi. These BM-derived mMDSC markedly increased the incidence of CD4+CD25+CD127−Foxp3+ regulatory T cells in responder T cell populations. They offer potential value in testing the therapeutic efficacy of immunoregulatory mMDSC for the promotion of tolerance in nonhuman primate transplant models.

Transcriptome analysis of low-dose ionizing radiation-impacted genes in CD4+ T-cells undergoing activation and regulation of their expression of select cytokines

Immune cells are known as the most sensitive tissue for ionizing radiation. Numerous reports relating with the effect of low-dose ionizing radiation (LDIR) on immune activities showed that LDIR can induce immune-potentiation via modulating the activity of B-, T-, and NK cells, or macrophages, whereas high-dose radiation induces genome-wide apoptotic/necrotic tissue injury and immune suppression. Generally, CD4+ T-cells play pivotal roles in immune systems via cytokines and cell-surface molecules to activate other types of immune cells to eliminate the pathogen. In spite of the significance of CD4+ T-cells in the immune system, mechanism of how LDIR regulates CD4+ T-cell gene expression is poorly investigated. Thus, RNA-Seq and Gene-Set Enrichment Analysis (GSEA) analysis were done with low-dose irradiated (γ-radiation, 50 mGy, 204 mGy/h)/anti-CD3/CD28-stimulated CD4+ T-cells to explore the LDIR-specific regulation of CD4+ T-cell gene expression. The results indicated that the genes related to mRNA translation processes, mitochondrial function, cell cycle regulation, and cytokine induction were upregulated in irradiated cells. Moreover, this study showed that the expression of T-helper cell Type 1 (TH1) or type 2 (TH2) cytokine genes, such as those for interferon (IFN)-γ, interleukin (IL)-4, and IL-5 were increased by at least 1.4-fold in acute (204 mGy/h) or chronic (10 mGy/h) low-dose (10 or 50 mGy) irradiated/anti-CD3/CD28 stimulated CD4+ T-cells, whereas the T-regulatory (Treg) cell cytokine gene, transforming growth factor (TGF)-β was decreased. Overall, these findings demonstrated that LDIR could cause an upregulation of selected immune product genes and, in turn, might modulate the activity of CD4+ T-cells undergoing activation via an impact on cytokine gene regulation.

Anti-inflammatory Effects of Carbon Monoxide-Releasing Molecule on Trinitrobenzene Sulfonic Acid-Induced Colitis in Mice

Recent findings indicate that carbon monoxide (CO) in non-toxic doses exerts a beneficial anti-inflammatory action in various experimental models. However, the precise anti-inflammatory mechanism of CO in the intestine remains unclear. Here, we assessed the effects of a novel water-soluble CO-releasing molecule, CORM-3, on trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. To induce colitis, C57BL/6 male mice received an enema of TNBS. CORM-3 or its inactive compound, iCORM-3, were administered intraperitoneally, once immediately before, and twice daily after receiving an enema of TNBS. Three days after TNBS administration, the distal colon was removed, assessed for colonic damage and histological scores, polymorphonuclear leukocyte recruitment (tissue-associated myeloperoxidase, MPO activity), and TNF-α, IFN-γ and IL-17A expression (mRNA and protein levels in the colon mucosa). CD4(+) T cells isolated from murine spleens were stimulated with anti-CD3/CD28, in the presence or absence of CORM-3/iCORM-3. The cell supernatants were assessed for TNF-α and IFN-γ expression, 24h following stimulation. Colonic damage and histological scores were significantly increased in TNBS-induced mice compared to sham-operated mice. Tissue-associated MPO activity and expression of TNF-α, IFN-γ, and IL-17A in the colonic mucosa were higher in TNBS-induced colitis mice. The above changes were attenuated in CORM-3-treated mice. Further, CORM-3 was effective in reducing TNF-α and IFN-γ production in anti-CD3/CD28-stimulated CD4(+) T cells. These findings indicate that CO released from CORM-3 ameliorates inflammatory responses in the colon of TNBS-challenged mice at least in part through a mechanism that involves the suppression of inflammatory cell recruitment/activation.

Activated T cells modulate immunosuppression by embryonic-and bone marrow-derived mesenchymal stromal cells through a feedback mechanism

Human embryonic stem cell (hESC)-derived mesenchymal stromal cells (MSC) (hESC-MSC) are an alternative source of MSC to bone marrow (BM)-derived MSC (BM-MSC), which are being investigated in clinical trials for their immunomodulatory potential. hESC-MSC have the advantage of being consistent because each batch can be generated from hESC under defined conditions. In contrast, BM-MSC have a limited proliferative capacity. The ability to suppress the proliferation of anti-CD3/CD28-stimulated CD4 (+) T cells by hESC-MSC was compared with adult BM-MSC and neonatal foreskin fibroblast (Fb). hESC-MSC suppress the proliferation of CD4 (+) T cells in both contact and transwell systems, although inhibition is less in the transwell system. hESC-MSC are approximately 2-fold less potent (67 cells/100 T cells) than BM-MSC and Fb (37 and 34 cells/100 T cells, respectively) at suppressing T-cell proliferation by 50% in a transwell [inhibitory concentration(IC)(50)]. The anti-proliferative effect is not contact-dependent but requires the presence of factors such as interferon (IFN)-γ produced by activated T cells. IFN-γ induces the expression of indoleamine-2,3-dioxygenase (IDO) in hESC-MSC, BM-MSC and Fb, contributing to their immunosuppressive property. The feedback loop between MSC or Fb and activated T cells may limit the immunosuppressive effects of MSC and Fb to sites containing ongoing immunologic or inflammatory responses where activated T cells induce the up-regulation of IDO and immunomodulatory properties of MSC and Fb. These data demonstrate that hESC-MSC may be evaluated further as an allogeneic cell source for therapeutic applications requiring immunosuppression.

Interleukin-35 Suppresses Allergen-specific Th2 Response in Patients with Grass pollen induced Seasonal Allergic Rhinitis

Regulatory T cells maintain peripheral tolerance to allergens in non-atopic individuals. IL-35 is a novel cytokine consisting of EBI3 and p35 subunit of IL-12. Recent studies have revealed IL-35 in regulating immunologic responses through induction of inducible IL-35-producing Treg cells. We hypothesized that IL-35 suppresses T effector (Teff) functions. We further hypothesized that IL-35 suppresses grass pollen-specific Th2 responses following in-vitro stimulation. CD4+CD25- T effector cells were purified from PBMCs (8 non-atopic and 7 grass pollen allergics) by magnetic separation. Teff cells were stimulated with anti-CD3/CD28 +/- rhIL-35:Fc for 6 days. The effect of rhIL-35:fc on grass pollen-specific Teff cell proliferative responses (n=6) was assessed. Gene expression was assessed by RT-PCR. A dose-dependent suppression of anti-CD3/CD28-stimulated CD4+CD25- Teff cell proliferation was demonstrated. This suppression was optimal at 10ng/mL of rhIL-35:Fc (n=15; p<0.005) and was associated with increases in relative gene expression of EBI3 (p=0.03), IL-12p35 (p=0.02), IL-12p28 (p=0.02), IFN-γ (p=0.02), FoxP3 (p=0.03) but not IL-10. A downward trend in IL-4 mRNA was demonstrated (p=0.06). An increase in CD4+CD25hiFoxp3+ (p=0.04) and CD4+CD25+TGF-β+ (p=0.009) T cells was demonstrated in the presence of IL-35; however, PBMC proliferative responses were not affected. Timothy grass pollen-driven CD4+CD25- Teff cell proliferative responses (n=6; p=0.03) and Th2 cytokine production (IL-4, p=0.03; IL-9, p=0.03 and IL-13, p=0.02) were suppressed by rhIL-35. IL-35 supresses antigen-specific T cell responses and is associated with the induction of CD4+CD25hiFoxp35+ and inducible IL-35+CD4+CD25+ (EBI3+IL-12p35+) Tregs. The mechanisms by which iIL35 Treg cells immunomodulate Th2 responses remains to be fully determined.

Platelets regulate CD4+ T-cell differentiation via multiple chemokines in humans

Atherosclerosis is an inflammatory and thrombotic disease. Both platelets and lymphocytes play important roles in atherogenesis. However, information on their interaction is limited. We therefore studied how platelets regulate CD4+ T cell activation and differentiation. Human CD4+ T cells and autologous platelets were co-cultured. Platelets concentration-dependently enhanced anti-CD3/CD28-induced IFNγ production by CD4+ T cells, but attenuated their proliferation. Abrogation of heterotypic cell-cell contact partially reversed the enhancement, and supernatant from activated platelets partially mimicked the enhancement, suggesting that platelets exert their effects via both soluble mediators and direct cell-cell contact. Platelets enhanced the production of IL-10 and cytokines characteristic for type 1 T helper (TH1) (IFNγ/TNFα) and TH17 (IL-17) cells, but influenced TH2 cytokines (IL-4/IL-5) little. The cytokine responses were accompanied by enhanced TH1/TH17/TReg differentiation. Using neutralising antibodies and recombinant PF4, RANTES, and TGFβ, we found that platelet-derived PF4 and RANTES enhanced both pro- and anti-inflammatory cytokine production, whilst recombinant TGFβ enhanced IL-10 but not TNFα production. In conclusion, platelets enhance the differentiation and cytokine production of anti-CD3/CD28-stimulated CD4+ T cells via both multiple chemokines and direct cell-cell contact. Our study provides new insights into the cross-talk between thrombosis and adaptive immunity, and indicates that platelets can enhance T-effector cell development.

Anti-inflammatory effects of Artemisia princeps in antigen-stimulated T cells and regulatory T cells

Abstract Objectives The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4+CD25- T cells and antigen-expanded regulatory T cells. Methods CD4+CD25- T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [3H]thymidine and, after harvesting the cells, [3H] thymidine incorporation was measured. For analysis of interleukin-2 and interferon-γ secreted from CD4+CD25- T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4+CD25- T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography–mass spectrometry. Key findings A. princeps (30 μg/ml) effectively suppressed proliferation of CD4+CD25- T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-γ in anti-CD3/CD28-stimulated CD4+CD25- T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. Conclusions The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4+CD25- T cells and increasing activity of expanded regulatory T cells.

Anti-inflammatory effects of &lt;I&gt;Artemisia princeps&lt;/I&gt; in antigen-stimulated T cells and regulatory T cells

The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells and antigen-expanded regulatory T cells. CD4(+)CD25(-) T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [(3)H]thymidine and, after harvesting the cells, [(3)H]thymidine incorporation was measured. For analysis of interleukin-2 and interferon-gamma secreted from CD4(+)CD25(-) T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4(+)CD25(-) T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography-mass spectrometry. A. princeps (30 microg/ml) effectively suppressed proliferation of CD4(+)CD25(-) T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-gamma in anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4(+)CD25(-) T cells and increasing activity of expanded regulatory T cells.

P38 MAPK plays a role in IL-4 synthesis in jacalin plus CD28-stimulated CD4+ T cells—II

We have previously shown that jacalin, a CD4+ T cell lectin, induces phosphorylation of intracellular events, moderate levels of interleukin (IL)-2 secretion. We have also shown that in the presence of CD28 costimulation, jacalin induces IL-4 secretion. In the present study, we showed that stimulation of normal CD4+ T cells with jacalin plus CD28 cross-linking (CD28XL) resulted in phosphorylation of signal transducer and activator of transcription (STAT)-6 and expression of Bcl-2 and Bcl-xL, which were inhibited significantly when cells were cultured in the presence of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. We further generated jacalin-induced CD4+ T cell blasts, examined the effects of CD28XL, and observed enhanced up-regulation of p38 and activation of STAT-6, Bcl-2, and Bcl-xL. Engagement of CD28 alone induced a marked degree of phosphorylation of p38 MAPK and IL-4 secretion in memory T cells (jacalin blasts), whereas in naïve T cells, jacalin plus CD28XL was required to induce these molecules. Incubation of cells with p38 inhibitor prior to CD28XL resulted in down-modulation of all these molecules. Further treatment with IL-4 has not reversed this trend. Our studies imply that p38 MAPK may play an important role in induction of these molecules and a putative role in protecting cells from undergoing apoptosis.

Dietary n-3 polyunsaturated fatty acids modulate purified murine T-cell subset activation.

Studies in humans and murine disease models have clearly shown dietary fish oil to possess anti-inflammatory properties, apparently mediated by the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To determine the mechanisms by which dietary EPA and DHA modulate mouse T-cell activation, female C57BL/6 mice were fed diets containing either 2% safflower oil (SAF), 2% fish oil (FO), or a 2% purified EPA/DHA ethyl ester mixture for 14 days. Splenic CD4 T cells ( approximately 90% purity) or CD8 T cells ( approximately 85% purity) were incubated with agonists which act at the plasma membrane receptor level [anti(alpha)-CD3/anti(alpha)-CD28], the intracellular level (PMA/Ionomycin), or at both the receptor and intracellular levels (alphaCD3/PMA). CD4 T cells stimulated with alphaCD3/alphaCD28 or PMA/Ionomycin proliferated and produced principally IL-2 (i.e. a Th1 phenotype), whereas the proliferation of CD4 T cells stimulated with alphaCD3/PMA was apparently driven principally by IL-4 (i.e. a Th2 phenotype). The IL-4 driven proliferation of putative Th2 CD4 cells was enhanced by dietary n-3 fatty acids (P = 0.02). Conversely, IL-2 production by alphaCD3/alpha CD28-stimulated CD4 T cells was reduced in FO-fed animals (P < 0.0001). The alphaCD3/alphaCD28-stimulated CD8 cells cultured from FO-fed animals exhibited a significant decrease (P < 0.05) in proliferation. There were no dietary effects seen in alphaCD3/PMA-stimulated CD8 cells, which produced both IL-2 and IL-4, or in PMA/Ionomycin-stimulated CD8 cells, which produced principally IL-2. These data suggest that dietary n-3 fatty acids down-regulated IL-2 driven CD4 and CD8 activation, while up-regulating the activation of the Th2 CD4 T-cell subset. Thus, the anti-inflammatory effects of n-3 fatty acids may result in both the direct suppression of IL-2-induced Th1 cell activation and the indirect suppression of Th1 cells by the enhanced cross-regulatory function of Th2 cells.