IL-17 Expression In CD4 Research Articles

The Effects of GM‐CSF and IL‐5 as Molecular Adjuvants on Immune Responses and Contraception Induced by mZP3 DNA Vaccination

Various approaches have been developed to improve the antibody response of zona pellucida glycoprotein-3 (ZP3) vaccination. In this study, we investigated whether GM-CSF and IL-5 can be used as cytokine adjuvants to increase the humoral immune response generated by mouse ZP3 (mZP3) DNA vaccine. Mice in experimental group were injected by GM-CSF 4days before the co-immunization of IL-5 and mZP3 DNA vaccine. The contraception and the correlation with humoral and cellular immune responses were analyzed after immunization and mating. The effect of cytokine adjuvant on the maturation of DCs was evaluated. Co-immunization of GM-CSF and IL-5 with mZP3 DNA vaccine induced the highest level of serum IgG and IL-4 expression in CD4(+) T cells. Importantly, this strategy reduced mice fertility without disrupting normal ovarian morphology. GM-CSF enhanced the maturation of DCs evidenced by up-regulating the expression of MHC-II and CD86. GM-CSF and IL-5 co-administration enhanced humoral immune responses to mZP3, and this may be a potential strategy for development of immunocontraceptive vaccine.

TGF-β Is Selectively Expressed on Lymphoma B Cells and Regulates the Differentiation of Intratumoral T Cells in B-Cell Non-Hodgkin Lymphoma (NHL)

Abstract 1586Transformation growth factor (TGF-β) is a highly pleiotropic cytokine critical to a variety of cellular events such as cell differentiation and apoptosis. TGF-β is synthesized as a prepro-TGF-β precursor and secreted after being processed in Golgi apparatus as a latent form that non-covalently combines both TGF-β and latency-associated protein (LAP). Our previous work in B-cell NHL has shown that the intratumoral T cell composition results in the establishment of a profoundly inhibitory tumor microenvironment. However, the underlying mechanism is only partially understood. In this study, using patient specimens and lymphoma cell lines, we evaluated the role of TGF-β in the tumor microenvironment and determined the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in B-cell NHL. First, we determined expression of TGF-β and found that a latent form of TGF-β was specifically expressed on the surface of CD19+ B cells, but not on other types of cells from B-cell lymphoma biopsy specimens. By screening cell lines, we found that latent TGF-β was also expressed on the surface of lymphoma cell lines, confirming the finding. Second, we tested whether surface expression by lymphoma cells led to the secretion of TGF-β in culture medium. Using an ELISA assay, we detected variable levels of latent TGF-β in the culture medium of primary malignant B cells (median 100 pg/ml per million cells, range: undetectable −229 pg/ml, n=7). Similarly, lymphoma cell lines secreted variable amounts of TGF-β from undetectable to 200 pg/ml per million cells. Next, we determined the effect of TGF-b on intratumoral T cell proliferation and differentiation. As expected, exogenous addition of TGF-β inhibited the proliferation of T cells. Notably, the proliferation of intratumoral T cells was significantly reduced when co-cultured with lymphoma cells bearing an active form of TGF-β compared to that with lymphoma cells without TGF-β. Using flow cytometry, we showed that the addition of exogenous TGF-β enhanced Foxp3 expression in activated CD4+, CD4+CD45RA+ or CD4+CD45RO+ intratumoral T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed the expression of IFN-γ in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, up-regulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell differentiation and the host immune response. Disclosures:No relevant conflicts of interest to declare.

β-arrestin2 stimulates interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model.

Allergic asthma is a complex and chronic inflammatory airway disease. Interleukin-17 is a pro-inflammatory cytokine which plays critical role in the pathogenesis of allergic asthma. It has been reported that β-arrestin2 regulated the development of allergic asthma at a proximal step in the inflammatory cascade. In this study, the influence of β-arrestin2 on Interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model was investigated. Splenic CD4+ T lymphocytes from wild-type mice and those from a murine asthma model were purified. CD4+ T lymphocytes from a murine asthma model were transfected with siRNAs targeting the β-arrestin2 or were pretreated with the ERK1/2 inhibitor, PD98059. After stimulation, the protein expression of β-arrestin2、phosphorylated-ERK1/2 and IL-17 were detection by Western blot; the mRNA expression of IL-17 were detected by real-time PCR; the accumulation of IL-17 in supernatants were detected by ELISA. We found that β-arrestin2、phosphorylated-ERK1/2 and IL-17 expression in CD4+ T lymphocytes from a murine asthma model were increased compared with those from wild-type mice (p < 0.01). Treatment of CD4+ T lymphocytes with siRNAs targeting the β-arrestin2 down-regulated phosphorylated- ERK 1/2 and IL-17 expression (p < 0.01). PD98059 decreased IL-17 production and expression in CD4+ T lymphocytes in a murine asthma model (p < 0.05). We conclude that β-arrestin2 stimulated IL-17 production and expression of CD4+ T lymphocytes in a murine asthma model. The effect was partly mediated by ERK 1/2 activation. Targeting β-arrestin2 biological activity could be a valid therapeutic approach for the treatment of allergic asthma.

β-arrestin2 stimulates interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model.

Allergic asthma is a complex and chronic inflammatory airway disease. Interleukin-17 is a pro-inflammatory cytokine which plays critical role in the pathogenesis of allergic asthma. It has been reported that β-arrestin2 regulated the development of allergic asthma at a proximal step in the inflammatory cascade. In this study, the influence of β-arrestin2 on Interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model was investigated. Splenic CD4+ T lymphocytes from wild-type mice and those from a murine asthma model were purified. CD4+ T lymphocytes from a murine asthma model were transfected with siRNAs targeting the β-arrestin2 or were pretreated with the ERK1/2 inhibitor, PD98059. After stimulation, the protein expression of β-arrestin2、phosphorylated-ERK1/2 and IL-17 were detection by Western blot; the mRNA expression of IL-17 were detected by real-time PCR; the accumulation of IL-17 in supernatants were detected by ELISA. We found that β-arrestin2、phosphorylated-ERK1/2 and IL-17 expression in CD4+ T lymphocytes from a murine asthma model were increased compared with those from wild-type mice (p < 0.01). Treatment of CD4+ T lymphocytes with siRNAs targeting the β-arrestin2 down-regulated phosphorylated- ERK 1/2 and IL-17 expression (p < 0.01). PD98059 decreased IL-17 production and expression in CD4+ T lymphocytes in a murine asthma model (p < 0.05). We conclude that β-arrestin2 stimulated IL-17 production and expression of CD4+ T lymphocytes in a murine asthma model. The effect was partly mediated by ERK 1/2 activation. Targeting β-arrestin2 biological activity could be a valid therapeutic approach for the treatment of allergic asthma.

Induction of regulatory T cells by green tea polyphenol EGCG

Regulatory T cells (Treg) are critical in maintaining immune tolerance and suppressing autoimmunity. The transcription factor Foxp3 serves as a master switch that controls the development and function of Treg. Foxp3 expression is epigenetically regulated by DNA methylation, and DNA methyltransferase (DNMT) inhibitors can induce Foxp3 expression in naive CD4+ T cells. We showed that EGCG, a major green tea polyphenol, could act as a dietary DNMT inhibitor, and induced Foxp3 and IL-10 expression in CD4+ Jurkat T cells at physiologically relevant concentrations in vitro. We further showed that mice treated with EGCG in vivo had significantly increased Treg frequencies and numbers in spleen and lymph nodes and had inhibited T cell response. Induction of Foxp3 expression correlated with a concomitant reduction in DNMT expression and a decrease in global DNA methylation. Our data suggested that EGCG can induce Foxp3 expression and increase Treg frequency via a novel epigenetic mechanism. While the DNMT inhibitory effects of EGCG was not as potent as pharmacologic agents such as 5-aza-2′-deoxycytidine, the ability of dietary agents to target similar mechanisms offers opportunities for potentially sustained and longer-term exposures with lower toxicity. Our work provides the foundation for future studies to further examine and evaluate dietary strategies to modulate immune function.

Mesenchymal Stromal Cells Derived From Bone Marrow of Chronic Lymphocytic Leukemia Patients Promote Th17 Cell Differentiation

AbstractAbstract 2414 Background:Mesenchymal stromal cells (MSC) derived from normal subjects are known to have immunosuppressive capacity by virtue of inhibiting T- and B-cell activation. A novel subset of T cells, Th17, plays an important role in inflammation and autoimmunity. A recent report demonstrated that normal MSC ameliorates experimental autoimmune encephalomyelitis by inhibiting CD4+ Th17 cells in a chemokine ligand 2-dependent manner (J Immunol. 2009, 182: 5994). It remains unknown if MSC derived from leukemic or cancer patients play a role in Th17 cell differentiation. In particular this would be of interest to study in B-Chronic Lymphocytic Leukemia (CLL) where immunosuppression is evident even in early stage disease. Methods:MSC derived from bone marrow of CLL patients or normal subjects were expanded in vitro as previously described by us (Br J Haematol. 2009, 147:471). CD4+ cells positively selected from normal peripheral blood mononuclear cells were co-cultured with either CLL MSC or normal MSC at a ratio of 50:1 for 3 days with stimulation via CD3/CD28 beads, as well as interleukin-1β (IL-1β; 50 ng/ml). Then phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (500 ng/ml) were introduced into the co-culture for 5 hrs in the presence of brefeldin A. Subsequently, cells were stained with CD4-phycoerythrin (PE) and IL-17-Alexa647 using intracellular flow to analyze the percent expression of IL-17 in CD4 + cells. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) were tested using a commercial multiplex cytokine array (Invitrogen, CA). This array measures the level of 30 different cytokines. Results:Positively selected CD4+ cells from peripheral blood of normal donors contain minimal percentages of Th17 cells (range: 0.48–0.71%). IL-1β stimulation induced increased IL-17 expression (range: 1.05–1.12%). Co-culture of CLL MSC with CD4+ cells induced significantly increased IL-17 expression in the CD4+ T cells (range: 1.16–1.32%). The promoting effect of CLL MSC on these Th17 cells appeared to be mediated by direct contact since the CM of CLL MSC was not able to induce increased IL-17 expression (mean = 0.66%) to a similar level as direct co-culture. When IL-1b was used to stimulate Th17 cell differentiation from CD4+ cells, CLL MSC were able to further promote the level of Th17 cell differentiation (range: 2.01–2.63%), indicating synergistic function for CLL MSC with IL-1β. This latter finding again appeared to be more pronounced for CLL MSC as normal MSC with IL-1β was not able to promote Th17 cell differentiation to a similar degree. To further investigate the mechanism of CLL MSC on Th17 cell differentiation, we assessed the cytokine production for resting CLL MSC and normal MSC compared to cytokine production of CLL and normal MSC stimulated with IL-17. The data from multiplex cytokine arrays revealed that the cytokine profiles were not different between resting CLL and normal MSC; however, when MSC were stimulated with IL-17, there were significant differences between CLL and normal MSC in terms of IL-6 and MCP-1 production (IL-6, CLL vs. normal, 957.9 ± 98 vs. 554.2 ± 92.3 pg/ml, p = 0.01; MCP-1, CLL vs. normal, 787.7 ± 166.9 vs. 330.2 ± 116.5 pg/ml, p = 0.04, n = 7). Since both IL-6 and MCP-1 have been demonstrated to play important roles in Th17 differentiation, we are conducting further studies to dissect the mechanism of CLL MSC in the promotion of Th17 cell differentiation. Conclusions:These results indicate that MSC derived from CLL patients promotes Th17 cell differentiation in vitro, which is in contrast to the previous published suppressive role of normal MSC on Th17 cell differentiation. Recent findings have indeed demonstrated that CLL patients do have high percentage of Th17 cells (Cancer Res. 2009. 69: 5922) when compared to other lymphoproliferative diseases. Given this data we believe that CLL MSC are intrinsically different from normal MSC in terms of immune regulation and cytokine production. This may occur as a result of the bi-directional activation that we found to be present between MSC and CLL leukemic cells (Br. J Haematol. 2009. 147:471). In total, our findings demonstrated that the dynamic interactions between the CLL leukemic cells and MSC appear to influence the Th 17 cell levels. This is of biological and clinical interest in that Th17 cells have the potential to regulate the immune environment to favor tumor proliferation and progression. Disclosures:No relevant conflicts of interest to declare.

Targeted Drug Delivery to Lymphocytes: A Route to Site-Specific Immunomodulation?

Lymphocytes are central to the progression of autoimmune disease, transplant rejection, leukemia, lymphoma and lymphocyte-resident viral diseases such as HIV/AIDs. Strategies to target drug treatments to lymphocytes, therefore, represent an opportunity to enhance therapeutic outcomes in disease states where many current treatment regimes are incompletely effective and promote significant toxicities. Here we demonstrate that highly lipophilic drug candidates that preferentially access the intestinal lymphatics after oral administration show significantly enhanced access to lymphocytes leading to improved immunomodulatory activity. When coadministered with such drugs, lipids enhance lymphocyte targeting via a three tiered action: promotion of drug absorption from the gastrointestinal tract, enhancement of lymphatic drug transport and stimulation of lymphocyte recruitment into the lymphatics. This strategy has been exemplified using a highly lipophilic immunosuppressant (JWH015) where coadministration with selected lipids led to significant increases in lymphatic transport, lymphocyte targeting and IL-4 and IL-10 expression in CD4+ and CD8+ lymphocytes after ex vivo mitogen stimulation. In contrast, administration of a 2.5-fold higher dose of JWH015 in a formulation that did not stimulate lymph transport had no effect on antiinflammatory cytokine levels, in spite of equivalent drug exposure in the blood. The current data suggest that complementary drug design and delivery strategies that combine highly lipophilic, lymphotropic drug candidates with lymph-directing formulations provide enhanced selectivity, potency and therapeutic potential for drug candidates with lymphocyte associated targets.

IL-17 is a potent synergistic factor with GM-CSF in mice in stimulating myelopoiesis, dendritic cell expansion, proliferation, and functional enhancement

Interleukin (IL)-17, which now defines the Th(17) immune response, is a critical cytokine expressed and required for stress granulopoiesis during microbial invasion. Dendritic cells(DC) can instigate this response by inducing IL-17 expression in CD4(+) T cells. Besides IL-17, microbial invasion also stimulates production of the DC growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The objective was the in vitro and in vivo investigation of IL-17 on DC proliferation and function in mice. Murine IL-17 (mIL-7) or murine GM-CSF (mGM-CSF), or both, was expressed in C57BL6 mice using adenoviral technology to assess hematopoietic and DC changes. The E-22 tymoma tumor cell line using a previously described vaccinia virus ovalbumin/LacZ murine tumor model was employed to study effects on tumor rejection. The combination of mIL-17 and mGM-CSF increased peripheral neutrophila by 28-fold and splenic colonies by 11- and 14-fold over each individual factor in mice, respectively. The effect of mIL-17 by itself on murine DCs in vitro and in vivo was minimal; however, the combination greatly enhanced the stimulating effects of mGM-CSF, increasing the total numbers of CD14b/c(+) spleen DC by fourfold, as well as their function measured by enhanced endocytosis. Mixed lymphocyte reactions using mIL-17/mGM-CSF cultured DCs stimulator cells enhanced lymphocyte responses by twofold over mGM-CSF alone. Vaccination against LacZ in the C57BL6 E22 syngenic thymoma tumor model effectively delayed tumor growth in animals pretreated with the mIL-17/mGM-CSF combination prior to vaccination. mIL-17 effectively synergizes with mGM-CSF in stimulating granulopoiesis and DC expansion, as well as in functional enhancement of DCs.

Malignant B cells skew the balance of regulatory T cells and TH17 cells in B-cell non-Hodgkin's lymphoma.

Using biopsy specimens from patients with B-cell non-Hodgkin's lymphoma, we observed a significantly low frequency of T(H)17 cells, including several samples with no detectable amount of interleukin (IL)-17-producing cells present in the tumor microenvironment. We found that, in the absence of lymphoma B cells, treatment with IL-1beta/IL-6 or lipopolysaccharide (LPS) enhanced IL-17 expression in CD4(+) T cells and this enhancement was attenuated when CD4(+) T cells were cocultured with lymphoma B cells. Blockade of CD27-CD70 or CD28-CD80/86 interactions by anti-CD70 or anti-CD80/86 antibodies restored LPS-mediated induction of IL-17 expression in CD4(+) T cells cocultured with lymphoma B cells. Because a subset of lymphoma B cells express IL-2 and given that IL-2 signaling is critically important in the development of regulatory T (T(reg)) cells, we tested the role of IL-2 signaling in T(H)17 cell development. We found that treatment with anti-IL-2 antibody to interrupt IL-2 signaling significantly inhibited Foxp3 expression in CD4(+) T cells. In contrast, interruption of IL-2 signaling up-regulated IL-17 expression in CD4(+) T cells and restored lymphoma-mediated down-regulation of IL-17-producing cells. Furthermore, the reversal of T(reg) cell activity by LPS or CpG-A resulted in an enhancement of IL-17-producing cells. Taken together, our study indicated that lymphoma B cells play an important role in skewing the balance between T(reg) and T(H)17 cells resulting in the establishment of a profoundly inhibitory tumor microenvironment.

Differential Regulation of Foxp3 and IL-17 Expression in CD4 T Helper Cells by IRAK-1

Host immune responses are finely regulated by the opposing effects of Th17 and T regulatory (Treg) cells. Treg cells help to dampen inflammatory processes and Th17 cells facilitate various aspects of immune activation. The differentiation of Th cells depends on a unique combination of stimulants and subsequent activation of diverse transcription factors. In particular, cooperative activation of NFAT and Smad3 leads to the induction of Treg cells, and cooperation among STAT3 and Smad3 switches to the induction of Th17 cells. We have previously shown that the IL-1 receptor associated kinase 1 (IRAK-1) selectively activates STAT3 and inactivates NFAT. Physiological studies have shown that IRAK-1(-/-) mice are protected from developing various inflammatory diseases, including experimental autoimmune encephalomyelitis and atherosclerosis with unknown mechanism. In this study, we demonstrate that IRAK-1 plays a critical modulatory role in the differentiation of Th17 and Treg cells. Following stimulation with TCR agonists and TGFbeta, IRAK-1(-/-) CD4 Th cells display elevated nuclear NFATc2 levels and increased interaction of NFATc2 and Smad3, resulting in increased expression of Foxp3, a key marker for Treg cells. IRAK-1(-/-) mice have constitutively higher populations of Treg cells. In contrast, when stimulated with TCR agonists together with IL-6 and TGF-beta, IRAK-1(-/-) CD4 Th cells exhibit attenuated STAT3 Ser727 phosphorylation and reduced expression of IL-17 and RORgamma t compared with wild-type cells. Correspondingly, IRAK-1 deletion results in decreased IL-17 expression and dampened inflammatory responses in acute and chronic inflammatory mice models. Our data provides mechanistic explanation for the anti-inflammatory phenotypes of IRAK-1(-/-) mice.

Ikaros regulates IL-10 expression in CD4+ T cells (38.27)

Abstract IL-10 is a regulatory cytokine critical for controlling inflammatory responses. However, little is known about the molecular mechanisms controlling Il10 gene expression, although GATA-3 is known to be involved in Il10 expression by Th2 cells. Here we show that Ikaros, a zinc finger DNA-binding protein, plays an important role in the regulation of IL-10 in T cells. Upon initial stimulation by T cell receptor, cells deficient in Ikaros (Ikaros null) express significantly lower levels of IL-10 compared to wild type T cells. In addition, under Th2 skewing conditions, which induce high levels of IL-10 production by wild type cells, Ikaros null T cells are unable to properly differentiate and produce only low levels of IL-10. The use of a retroviral vector system to express a dominant negative isoform of Ikaros in wild type Th2 cells represses IL-10 production but does not significantly alter the levels of transcription factors Gata3 and Tbet. Further, expression of Ikaros in Ikaros null T cells restores IL-10 expression. We found that Ikaros binds to conserved regulatory regions of the Il10 gene locus in Th2 cells, supporting a direct role for Ikaros in Il10 expression. In addition, we have evidence to suggest a coordinate role for Ikaros and Gata3 in the regulation of Il10. A direct interaction of these proteins is being examined. Thus, we suggest of novel role of Ikaros in regulating Il10 gene expression.

Monocytes and T lymphocytes contribute to a predominance of interleukin 6 and interleukin 10 in systemic lupus erythematosus

To investigate the contribution of T lymphocytes and monocytes to cytokine production in systemic lupus erythematosus (SLE). Forty-five SLE patients and 19 healthy volunteers were included. Serum levels of tumor necrosis factor alpha (TNFalpha), interferon gamma (IFN gamma), interleukin (IL)-6, and IL10 were quantified by ELISA. The cytokine production capacities of peripheral blood mononuclear cells were assessed by culturing in vitro with PMA+Ionomycin or LPS. The intracellular cytokine expression was measured by flow cytometry in T lymphocytes and monocytes, respectively. The influence of the disease activity (measured as the SLE-disease activity index; SLEDAI) and the treatment the patients were receiving was evaluated. Serum IL10, IL6, and TNFalpha levels were increased in patients (P <or= 0.01), and a higher spontaneous (without stimuli) intracellular expression of IL10 in CD4+ and CD8+ T lymphocytes (P < 0.05) and of IL6 in monocytes (P = 0.01) were found. After stimulation, patients presented a higher percentage of CD4+ and CD8+ T lymphocytes producing IL4 and IL10 (P <or= 0.01), and of monocytes producing IL6 (P = 0.04) and IL10 (P = 0.008). The SLEDAI score was positively correlated with the percentage of CD4+IL10+ and CD8+IL10+ T lymphocytes (P < 0.01), and inversely correlated with CD8+TNFalpha+ (P= 0.02), CD4+IFN gamma+ (P = 0.04) and CD8+ IFN gamma+ (P = 0.002) T lymphocytes. Patients receiving high dose prednisone produced higher IL10, but they also were the patients with a more active disease. Monocytes and T lymphocytes (CD4+ and CD8+) contribute to an overproduction of IL6 and IL10 in SLE; this correlates with the disease activity but is independent of the treatment the patients are receiving.

Malignant B Cell-Derived TGF-β Controls the Generation of TH1, TH17 and Treg Cells in the Tumor Microenvironment of B-Cell Non-Hodgkin Lymphoma (NHL).

Background: Our previous work has shown that malignant B cells induce the development of intratumoral Treg cells that inhibit the host anti-tumor response. In contrast to an increase in Treg cells, we found that the number of effector T helper cells (TH1, TH2 and TH17) was low in B-cell NHL tumors, suggesting an imbalance between Treg and TH cells in the tumor microenvironment. Understanding the mechanism(s) of this imbalance is important to the development of treatments to enhance host immunity and in previous work we have shown that signaling through CD70, CD80 and CD86 plays a role. Since soluble factors, particularly TGF-β, have an important role in directing T-cell differentiation, we evaluated in this study the role of TGF-β in the lymphoma microenvironment.Goal: To determine the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in human B-cell NHL.Results: Human B-cell NHL specimens were obtained from consenting patients and were used for all experiments. Using an ELISA assay, we found that malignant B cells variably secrete TGF-β - median 100 pg/ml per million cells (range: undetectable −229 pg/ml, n=7). Using flow cytometry, we showed that addition of exogenous TGF-β enhanced the expression of Foxp3+ in activated CD4+ or CD4+CD45RA+ or CD4+CD45RO+ nodal T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed expression of IFN-g in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, upregulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results indicate that TGF-β plays an important role in the regulation of intratumoral TH17 cell generation. In additional experiments, TGF-β was found to exert a suppressive effect on the proliferation of both CD4+ and CD8+ intratumoral T cells. However, treatment with TGF-β enhanced IL-2 production by intratumoral CD4+ T cells detected by intracellular staining of flow cytometry. Interruption of IL-2 signaling by anti-IL-2 Ab abolished the upregulation of TGF-β-mediated Foxp3 expression and enhanced the production of IL-17 in CD4+ T cells. Furthermore, treatment with anti-IL-2 Ab reversed the inhibition of NHL B cell-mediated TH17 cell generation.Conclusion: These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL through IL-2. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell generation and the host immune response.

Increased Interleukin-17 Production Both in Helper T Cell Subset Th17 And CD4-Negative T Cells in Human Immunodeficiency Virus Infection

Interleukin (IL)-17 is produced mainly by activated CD4(+) T cells, currently known as Th17. Human immunodeficiency virus (HIV) pathogenesis leads to CD4(+) T cell depletion. This is the first report of IL-17 in HIV infection. We assessed IL-17 expression in the CD4(+) T cells (Th17) of 40 asymptomatic HIV-infected treatment-naive patients compared with 40 HIV-seronegative volunteers. Peripheral blood mononuclear cells (PBMCs), with/without phorbol myristate acetate (PMA)/ionomycin stimulation, were stained with CD3, CD4, IL-17, and interferon (IFN)-gamma antibodies and analyzed by four-color flow cytometry. Both groups had comparable baseline data, except for age (mean+/-SD): 36 +/- 9 versus 30 +/- 9 yr (p= 0.001), CD4(+) T cell counts (median): 218 versus 623 cells/microL (p < 0.0001), CD8(+) T cell counts (median): 875.5 versus 382.5 cells/microL ((p) < 0.0001), and CD4(+)/CD8(+) cell ratios (median): 0.225 versus 1.45 (p< 0.0001). Without stimulation, the percentages of IL-17(+) CD3(+) CD4() and IL-17(+) CD3(+) CD4() cells among HIV-seropositive and -seronegative volunteers (median) were as follows: 0.68 versus 0.12% (p< 0.0001) and 0.92 versus 0.09% (p< 0.0001), respectively. With PMA/ionomycin stimulation, the percent IL-17 expression in CD4(+) cells (median) was 1.45 versus 0.65 (p< 0.0001) and in CD4() T cells it was 1.0 versus 0.12 (p< 0.0001). In conclusion, HIV infection is associated with a significant increase in IL-17 production in both CD4(+) and CD4() T cells in peripheral blood. IL-17 expression was further inducible by PMA/ionomycin stimulation in vitro only in CD4(+) T cells. The roles of IL-17 and Th17 in HIV viral replication and immunopathogenesis are under further investigation.

Induction of IL-4 Expression in CD4+ T Cells by Thymic Stromal Lymphopoietin

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic inflammation in both humans and mice. Although the underlying mechanism is not known, TSLP-stimulated dendritic cells have been shown to prime human CD4(+) T cells into Th2 cytokine-producing cells. However, its direct effect on CD4(+) T cells has not been extensively investigated. In this study, we show that TSLP can drive Th2 differentiation in the absence of exogenous IL-4 and APCs. IL-4 blockade inhibited TSLP-mediated Th2 differentiation, demonstrating that IL-4 is involved in this process. Further analysis has shown that TSLP-induced Th2 differentiation is dependent on Stat6 and independent of IL-2 and that TSLP treatment leads to immediate, direct Il-4 gene transcription. Taken together, these data demonstrate that TSLP is directly involved in Th2-mediated responses via the induction of IL-4 production.

Innate and Acquired Immune System in Patients Developing Interferon-α-Related Autoimmune Thyroiditis: A Prospective Study

In this prospective study, we investigated whether the development of interferon (IFN)-alpha-related autoimmune thyroiditis (IFN-AT) was correlated with the sequential changes of cytokine pattern induced by IFNalpha in the peripheral lymphocytes. We enrolled 18 hepatitis C virus (HCV)-positive patients who developed IFN-AT, eight patients with euthyroidism [IFN-AT(Eu)] and 10 with thyroid dysfunction [IFN-AT(Dy)]. Twenty HCV-positive patients without IFN-AT acted as control group (Co-HCV+). Intracellular expression of IFNgamma and IL-4 was evaluated by multicolor flow-cytometry analysis in peripheral lymphocytes in vitro stimulated by phorbol-12-myristate-13-acetate (PMA) (25 ng/ml) and ionomycin (1 mug/ml) in presence of monensin (5 microm). At the appearance of thyroid disease, both IFN-AT(Eu) and IFN-AT(Dy) patients showed a significant increase of IFNgamma expression in CD3+CD56+ and CD3-CD56+ cells but not in CD4+ and CD8+ cells. At this time point, IFN-AT(Eu) but not IFN-AT(Dy) patients also showed an increase of IL-4 expression in CD3+CD56+ cells and CD4+ cells. Six months later, IFN-AT(Eu) patients maintained high expression of IL-4 in CD4+ and CD3+CD56+ cells without any further increase in IFNgamma expression. By contrast, IFN-AT(Dy) patients showed an increase of IFNgamma expression in CD4+ and CD8+ cells, with a concomitant decrease of IL-4 expression in CD4+ cells. Type 2 immune response is activated early and specifically in patients with IFN-AT who remain euthyroid throughout the follow-up. Predominant in patients developing thyroid dysfunction, by contrast, is the type 1 immune response that seems to occur earlier in innate than acquired immune system.

Type-1 response in peripheral CD4+ and CD8+ T cells from patients with Hashimoto's thyroiditis.

In this study we performed single-cell analysis of the intracellular cytokine expression in peripheral CD4+ and CD8+ lymphocytes from patients with Hashimoto's thyroiditis (HT) to investigate the type-1 response separately for the two lymphocyte sub-populations. Twenty-nine patients affected by HT and 20 healthy subjects, matched for sex and age, were enrolled. After the analysis of the lymphocyte sub-populations, the intracellular content of interferon-gamma (IFN-gamma) in phorbolmyristate acetate (PMA)-stimulated CD4+ and CD8+ lymphocytes was assayed. Moreover, the CD4+ lymphocytes were also evaluated for the intracellular expression of IL-4. No significant differences in CD3+, CD4+ and CD8+ lymphocytes were found between HT patients and control subjects. However, the HT patients showed higher numbers of CD4+ IFN-gamma+, CD4+ IL-4+ and CD8+ IFN-gamma+ (t-test, P< or =0.001) cells than the control subjects. Analysing the intracellular expression of IFN-gamma and IL-4 in relation to thyroid function, we found that the euthyroid patients (18 cases) showed more expression of IL-4 in CD4+ lymphocytes than the control subjects, without any significant modification of IFN-gamma expression in CD4+ and CD8+ lymphocytes. However, the hypothyroid patients (11 cases) showed an increase of IFN-gamma expression in both CD4+ and CD8+ lymphocytes with respect to the control subjects and the euthyroid patients. Moreover, the expression of IL-4 in CD4+ cells from hypothyroid patients was significantly lower than that seen in the euthyroid cases and comparable to that found in the control subjects. Our study has demonstrated that the peripheral CD4+ and CD8+ T lymphocytes from the HT patients show a type-1 activation strictly correlated to the occurrence of hypothyroidism. Further studies will be needed to clarify the exact role of peripheral lymphocytes in HT and whether they could provide a reliable marker of thyroid immune involvement.

No Effect of Transfusion Transmitted Virus Viremia on the Distribution and Activation of Peripheral Lymphocytes in Hemodialyzed Patients

Aim: We aimed to examine the distribution and activation of peripheral T cells in TTV positive (n = 32) and negative (n = 17) hemodialyzed patients. The control group (n = 20) consisted of healthy blood donors. Method: TTV-DNA was detected by seminested PCR. CD3, CD4, CD8, CD19, CD56, CD3/HLA-DR, CD3/CD69 and the Th1/Th2 ratio of T cells were analyzed by flow cytometry. Circulating IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-13, TNF-α, TGF-β levels were measured by ELISA in the sera. Results: There was no difference between the CD3, CD4, CD8 and CD19 values of HD subjects. In addition, the expression of both activation markers, HLA-DR and CD69, was significantly elevated in the TTV-positive and -negative HD groups compared to the controls, but not showing any difference from each other. The measurements of intracellular cytokines showed the enhanced occurrence of INF-γ + CD4 T cells, and decreased appearance of IL-4 + CD4 lymphocytes in the HD groups without any significant difference between the TTV virus positive and negative patients. In addition, HD also elevated the expression of IL-10 in CD4 and CD8 (Th2) cells. There were only two significant changes in the levels of circulating cytokines: (a) IL-2 increased; (b) IL-13 decreased in both groups of HD patients compared to the controls, independently of TTV positvity or negativity. Conclusions: We assume that transfusion-transmitted virus does not cause any specific change in the distribution and activation of lymphocytes in the peripheral blood of hemodialyzed patients. Hemodialysis itself, however, results in a significant activation of peripheral T cells with the domination of increased production of Th1 type cytokines, IFN-γ, IL-2, in contrast to the decreased synthesis of Th2 type cytokines, IL-4 and IL-13. Furthermore, the increased expression of IL-10 in the CD4 and CD8 cells of HD patients can be the sign of a contraregulatory Th2 activation as an answer on the Th1 effect.

Single-cell analysis of lymphokine imbalance in asymptomatic HIV-1 infection: evidence for a major alteration within the CD8+ T cell subset.

In this study we investigated at single-cell level by flow cytometry the potential of T cell cytokine production in asymptomatic HIV-1-infected subjects with > 200 CD4 counts and possible correlation with T helper cell depletion and viral load. Mitogen-stimulated peripheral blood mononuclear cells from 32 HIV-1+ patients and 16 healthy subjects were intracytoplasmically stained for IL-2, interferon-gamma (IFN-gamma), IL-4 or IL-10, and the frequency of cytokine-producing cells was assessed in total T cells, CD4, CD8 and CD45RO subsets as well as in CD69+CD3+ gated lymphocytes. HIV-1+ patients, irrespective of their degree of CD4 depletion, exhibited a major increase in IFN-gamma+ CD8 T cells, largely due to CD28- cells, as well as a decrease in the capacity of CD8 T cells to produce IL-2. Patients with > 500 CD4 counts showed a diminished frequency of IL-4 expression in CD4 T cells and a negative correlation was found between this parameter and the ex vivo CD4 counts in the 32 patients. Analysis of patients stratified according to viral load revealed a significantly higher proportion of IL-2-producing CD4 cells in the group with < 5000 RNA copies/ml. In short, using single-cell analysis and an antigen-presenting cell-independent stimulus, we have not been able to find any significant cytokine imbalances in the CD4 subset, suggesting that the well described T helper defects are not due to intrinsic alterations in the potential of CD4 T cells to produce cytokines. On the other hand, the major disturbances in the CD8 T lymphocytes agree with the marked activation and possible replicative senescence of CD8 T cells and emphasize the role of this subset in HIV immunopathogenesis.

Differential effect of corticosteroid therapy on the cytoplasmic cytokine expression in CD4 and CD8 positive T cells from lupus patients

Due to their different antiinflammatory and immunmodulatory effects corticosteroids are widely used in the treatment of SLE. Literature data support both Th1 and Th2 dominance in lupus. There are only few reports about cytokine profile of CD8+ T cells in SLE. In the present study we examined by flow cytometry the cytoplasmic IFNγ, IL-4 and IL-10 expression in CD4+ and CD8+ T cells from six active, untreated, newly diagnosed SLE patients, who received after that 1 mg/kg methylprednisolon. Pretreatment expression of IFNγ was lower, while IL-4 and IL-10 expressions were higher in CD4+, but not in CD8+ T cells of patients than in control cells. Corticosteroid treatment increased IFNγ and decreased IL-4 and IL-10 expressions in patients' CD4+ cells, but had no significant effect on the cytoplasmic cytokine expression of CD8+ cells. In conclusion, present results indicate that corticosteroid therapy does not influence INFγ, IL-4 and IL-10 expression in CD8+ T cells of lupus patients, and it may have pathogenic significance.