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We recently showed that Tiam1 expression is induced in pro-inflammatory T helper 17 (Th17) cells differentiated with interleukin (IL)-6 and TGF-β1, and together with Rac1 promote Th17 cell development and autoimmunity in a mouse model of multiple sclerosis. Here we found that STAT3 and Smad3, downstream transcription factors of IL-6 and TGF-β1, respectively, play opposing roles in regulating Tiam1 transcription in CD4+ T-cells. While IL-6-STAT3 signaling promotes Tiam1 expression, TGF-β1-Smad3 induces the opposite outcome. At the Tiam1 promoter, both STAT3 and Smad3 bind to the Tiam1 promoter in Th17 cells. However, STAT3 induces Tiam1 promoter activity whereas Smad3 competes with STAT3 and inhibits its activity. Our findings uncover the complexity of STAT3/Smad3 signaling in regulating Tiam1 expression and Th17 cells. [ABSTRACT FROM AUTHOR]

Interleukin-9 (IL-9) is a T cell cytokine that acts through a ?C-family receptor on target cells and is associated with inflammation and allergy. We determined that T cells from mice deficient in the T helper type 17 (TH17) pathway genes encoding retinoid-related orphan receptor ? (ROR-?) and IL-23 receptor (IL-23R) produced abundant IL-9, and we found substantial growth inhibition of B16F10 melanoma in these mice. IL-9-blocking antibodies reversed this tumor growth inhibition and enhanced tumor growth in wild-type (WT) mice. Il9r?/? mice showed accelerated tumor growth, and administration of recombinant IL-9 (rIL-9) to tumor-bearing WT and Rag1?/? mice inhibited melanoma as well as lung carcinoma growth. Adoptive transfer of tumor-antigen-specific TH9 cells into both WT and Rag1?/? mice suppressed melanoma growth; this effect was abrogated by treatment with neutralizing antibodies to IL-9. Exogenous rIL-9 inhibited tumor growth in Rag1?/? mice but not in mast-cell-deficient mice, suggesting that the targets of IL-9 in this setting include mast cells but not T or B cells. In addition, we found higher numbers of TH9 cells in normal human skin and blood compared to metastatic lesions of subjects with progressive stage IV melanoma. These results suggest a role for IL-9 in tumor immunity and offer insight into potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Parenteral OKT3 is used to treat transplant rejection and a humanized anti-CD3 Mab has shown positive clinical effects in new onset diabetes. Oral administration of anti-CD3 has not been tested in humans, but suppresses autoimmunity in animal models. Beta-glucosylceramide enhances NKT cell and regulatory T cell activity and enhances the effects of oral anti-CD3 in animals. Fifteen healthy volunteers (three per group) received orally administered OKT3 over a dose range of 0.2 to 5.0 mg daily with or without beta-glucosylceramide 7.5 mg for 5 days. Safety and immune parameters were measured on days 5, 10, and 30. Oral OKT3 enhanced T cell proliferation, suppressed Th1 and Th17 responses by 43% and 41%, respectively, increased TGF-β/IL-10 expression and decreased IL-23/IL-6 expression by dendritic cells, and affected the IgG repertoire as measured by antigen arrays. Co-administration of oral beta-glucosylceramide induced similar effects. No side effects were observed and no subjects developed human anti-mouse antibodies. These findings demonstrate that oral anti-CD3 monoclonal antibody is safe and biologically active in humans and presents a new avenue for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Summary: The modulation of co-stimulatory pathways represents a novel therapeutic strategy to regulate autoimmune diseases. Auto-reactive CD4+ T cells play a critical role in initiating the immune response leading to inflammation and autoimmune diseases. Blocking co-stimulatory signals prevents T-cell activation, thus diminishing autoimmune responses and possibly preventing the progression of autoimmune disease. Blockade of several co-stimulatory pathways has been investigated in animal models and has led to clinical trials testing specific blocking agents in humans. In this review we will describe the role of co-stimulatory pathways, primarily the CD28-B7 pathway, in autoimmune diseases, and we will present in vivo and in vitro studies supporting the efficacy of co-stimulation blockade in animal models of autoimmune disease. Finally, we will discuss the clinical therapeutic efficacy of blocking monoclonal antibodies in preventing or reducing auto-antigen driven T-cell activation in humans with particular attention to the CD28/B7 pathway. Inhibiting co-stimulatory molecule interactions by using monoclonal antibodies seems to be an original approach to regulate autoimmune diseases in humans. [Copyright &y& Elsevier]

Tim-3 is a T helper type 1 (TH1)–specific cell surface molecule that seems to regulate TH1 responses and the induction of peripheral tolerance. However, the identity of the Tim-3 ligand and the mechanism by which this ligand inhibits the function of effector TH1 cells remain unknown. Here we show that galectin-9 is the Tim-3 ligand. Galectin-9-induced intracellular calcium flux, aggregation and death of TH1 cells were Tim-3-dependent in vitro, and administration of galectin-9 in vivo resulted in selective loss of interferon-γ-producing cells and suppression of TH1 autoimmunity. These data suggest that the Tim-3–galectin-9 pathway may have evolved to ensure effective termination of effector TH1 cells. [ABSTRACT FROM AUTHOR]

Fetal survival during gestation implies that tolerance mechanisms suppress the maternal immune response to paternally inherited alloantigens. Here we show that the inhibitory T cell costimulatory molecule, programmed death ligand 1 (PDL1), has an important role in conferring fetomaternal tolerance in an allogeneic pregnancy model. Blockade of PDL1 signaling during murine pregnancy resulted in increased rejection rates of allogeneic concepti but not syngeneic concepti. Fetal rejection was T cell- but not B cell-dependent because PDL1-specific antibody treatment caused fetal rejection in B cell-deficient but not in RAG-1-deficient females. Blockade of PDL1 also resulted in a significant increase in the frequency of IFN-γ-producing lymphocytes in response to alloantigen in an ELISPOT assay and higher IFN-γ levels in placental homogenates by ELISA. Finally, PDL1-deficient females exhibited decreased allogeneic fetal survival rates as compared with littermate and heterozygote controls and showed evidence of expansion of T helper type 1 immune responses in vivo. These results provide the first evidence that PDL1 is involved in fetomaternal tolerance. [ABSTRACT FROM AUTHOR]

Background We have previously shown that the inducible kinin B1 receptor is expressed on T lymphocytes during relapses and progression in multiple sclerosis. Objective To evaluate the correlation between the expression of B1 receptor on peripheral blood mononuclear cells derived from patients who have multiple sclerosis with serial, clinical magnetic resonance imaging and immunological study–derived measures. Design Using frozen samples obtained from a high-frequency magnetic resonance imaging–immunological study, we analyzed B1 receptor messenger RNA (mRNA) expression in peripheral blood–derived mononuclear cells serially collected from 6 patients with multiple sclerosis and 8 healthy control subjects by semiquantitative radioactive duplex reverse transcriptase–polymerase chain reaction amplification. Time-course kinin B1–actin mRNA ratios were subsequently compared with corresponding clinical magnetic resonance imaging and immune parameters. Results The time-course kinin B1–actin mRNA ratio correlated positively with the Expanded Disability Status Scale index (P<.001), occurrence of clinical relapse (P = .02), volume of lesion on T2-weighted images (P<.003) and interleukin 2 receptor and major histocompatibility complex class II expression on CD4+ lymphocytes, but not with gadolinium-enhancing lesions. The time-course kinin B1–actin mRNA ratios were 5 to 25 times lower in samples derived from healthy controls. Conclusion The correlation of kinin B1 receptor mRNA levels with dynamic clinical and magnetic resonance imaging measures suggests that expression of this receptor can serve as an index of disease activity in multiple sclerosis. [ABSTRACT FROM AUTHOR]

The B7 family of T cell costimulatory molecules has recently acquired several new members. Some of these are activating while others are inhibitory. In this review, we will focus on the novel inhibitory pathways with particular emphasis on the PD-1:PD-L pathway. Understanding the mechanisms of these pathways has implications for development of novel treatment strategies for autoimmune disease, transplantation, tumor immunotherapy, and vaccine development. [Copyright &y& Elsevier]

Objective: To determine whether changes in activation markers on peripheral blood T cells correlate with disease activity in patients with multiple sclerosis. Design: In a prospective longitudinal study during 1 year, we analyzed the change in percentage of activated T lymphocytes in the peripheral blood of 40 patients with multiple sclerosis in relation to clinical findings and changes on brain magnetic resonance imaging (MRI) scans. The patients underwent repeated imaging of the brain (mean number of MRIs for each patient, 22) at the time blood samples were obtained as well as at monthly neurological examinations, and at the time of scoring on the Kurtzke Expanded Disability Status Scale (EDSS) and ambulation index scale. Results: A change in the percentage of cells expressing the activation markers interleukin 2 receptor (CD25), class II major histocompatibility complex (MHC) (I3) or surface dipeptidyl peptidase (CD26) correlated significantly with a change in lesion volume or a change in number of gadolinium-enhancing lesions as detected on MRI. Changes in CD25[sup +] cells and in CD4[sup +] cells expressing class II MHC also correlated with changes in disability as measured by EDSS in patients with relapsing-remitting disease, and changes in CD4[sup +]CD25[sup +] cells correlated with the occurrence of attacks in patients with relapsing-remitting disease. These correlations are dependent on measurement of changes between time points sampled at 1- or 2-week intervals. Conclusion: There is a linkage between peripheral T-lymphocyte activation as measured by cell surface markers and disease activity in patients with multiple sclerosis. [ABSTRACT FROM AUTHOR]

Abstract: Cytokines play an important role in the pathogenesis of inflammatory diseases including multiple sclerosis (MS). Experimental models have played a critical role in unraveling the roles of individual cytokines in this disease; however, these studies occasionally yield conflicting results, highlighting the complex role cytokines play in the disease process. Efforts to modulate cytokine function in MS have shown that effective treatments alter cytokine expression in the central nervous system (CNS) and in activated mononuclear cells, indicating that they are important therapeutic targets. In this review, we will summarize the current knowledge on the role of cytokine pathways in MS and what we learned from investigation of its animal model: experimental autoimmune encephalomyelitis (EAE). [Copyright &y& Elsevier]

We aimed to investigate the immunologic effects of vitamin D replacement in RRMS patients. In a controlled single center study, patients deficient in 25-hydroxyvitamin D (serum level < 25 ng/ml) received 10,000 IU/week cholecalciferol for 3 months. Sufficient vitamin D patients (serum level > 35 ng/ml) were followed for the same period. Assessments were performed at baseline and at 3 months. 25-hydroxyvitamin D levels increased significantly from baseline to month-3 in the deficient group after treatment and remained stable in the sufficient group. We observed a decreased interferon-γ (IFNγ) secretion by CD4 + T cells in vitamin D deficient group but not in the sufficient group, and a negative correlation between baseline serum vitamin D and IFNγ production. There was no change in the frequency of T helper or regulatory T cell subsets in either group. Increasing serum levels of 25-hydroxyvitamin D are associated with decreased production of IFNγ by CD4 + T cells. [ABSTRACT FROM AUTHOR]

Abstract: Studies of the underlying immune mechanisms of multiple sclerosis (MS) in children may shed light on the initial events of MS pathogenesis. We studied T cell responses to myelin peptides in 10 pediatric MS patients (PMS), 10 pediatric healthy controls (PHC), 10 adult MS patients (AMS) and 10 adult healthy controls (AHC). A significantly higher proportion of divided CD4+ T cell responses in response to myelin peptides by the CFSE assay in PMS compared to PHC at both concentrations of myelin peptide tested (t test, 95% CI, p=0.0067 for MP1; p=0.0086 for MP10), and between PMS and AMS (p=0.0012 at 1μg/mL of myelin peptides, p<0.0001 at 10μg/mL) was found. In addition, T cells with a central memory phenotype producing IL-17 were increased in PMS compared to PHC (p<0.05). IL-7 levels in culture supernatants were elevated in PMS compared to PHC and AMS (t test<0.01). [Copyright &y& Elsevier]

Summary: Interleukin 9 (IL-9) is a pleiotropic cytokine that can regulate autoimmune responses by enhancing regulatory CD4+FoxP3+ T regulatory (Treg) cell survival and T helper 17 (Th17) cell proliferation. Here, we analyzed the costimulatory requirements for the induction of Th9 cells, and demonstrated that Notch pathway cooperated with TGF-β signaling to induce IL-9. Conditional ablation of Notch1 and Notch2 receptors inhibited the development of Th9 cells. Notch1 intracellular domain (NICD1) recruited Smad3, downstream of TGF-β cytokine signaling, and together with recombining binding protein (RBP)-Jκ bound the Il9 promoter and induced its transactivation. In experimental autoimmune encephalomyelitis (EAE), Jagged2 ligation regulated clinical disease in an IL-9-dependent fashion. Signaling through Jagged2 expanded Treg cells and suppressed EAE when administered before antigen immunization, but worsened EAE when administered concurrently with immunization by favoring Th17 cell expansion. We propose that Notch and Smad3 cooperate to induce IL-9 and participate in regulating the immune response. [Copyright &y& Elsevier]

CD11b+Ly-6Chi cells, including inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs), are important in infectious, autoimmune, and tumor models. However, their role in T cell regulation is controversial. In this article, we show that T cell regulation by IMCs and IDCs is determined by their activation state and is plastic during an immune response. Nonactivated IMCs and IDCs function as APCs, but activated IMCs and IDCs suppress T cells through NO production. Suppressive IMCs are induced by IFN-γ?, GM-CSF, TNF-α, and CD154 derived from activated T cells during their interaction. In experimental autoimmune encephalomyelitis, CD11b+Ly-6Chi cells in the CNS are increasingly activated from disease onset to peak and switch their function from Ag presentation to T cell suppression. Furthermore, transfer of activated IMCs or IDCs enhances T cell apoptosis in the CNS and suppresses experimental autoimmune encephalomyelitis. These data highlight the interplay between innate and adaptive immunity: immunization leads to the expansion of Ly-6Chi myeloid cells initially promoting T cell function. As T cells become highly activated in the target tissue, they induce activation and NO production in Ly-6Chi myeloid cells, which in turn suppress T cells and lead to the contraction of local immune response. [ABSTRACT FROM AUTHOR]

Notch signaling pathway plays an important role in T cell differentiation. Delta-like ligand (Dll)4, one of five known Notch ligands, has been implicated in regulating Th2 cell differentiation in animal models of human diseases. However, the role of Dll4 in Th1/Th17-mediated autoimmune diseases remains largely unknown. Using an anti-Dll4 blocking mAb, we show that neutralizing Dll4 during the induction phase of experimental autoimmune encephalomyelitis in C57BL/6 mice significantly increased the pool of CD4+Foxp3+ regulatory T cells (Treg) in the periphery and in the CNS, and decreased the severity of clinical disease and CNS inflammation. Dll4 blockade promoted induction of myelin-specific Th2/Treg immune responses and impaired Th1/Th17 responses compared with IgG-treated mice. In vitro, we show that signaling with recombinant Dll4 inhibits the TGF-β-induced Treg development, and inhibits Janus kinase 3-induced STAT5 phosphorylation, a transcription factor known to play a key role in Foxp3 expression and maintenance. Depletion of natural Treg using anti-CD25 Ab reversed the protective effects of anti-Dll4 Ab. These findings outline a novel role for Dll4-Notch signaling in regulating Treg development in EAE, making it an encouraging target for Treg-mediated immunotherapy in autoimmune diseases, such as multiple sclerosis. [ABSTRACT FROM AUTHOR]

Abstract: T helper 2 (Th2) cytokines are known to be important in protection against experimental autoimmune encephalomyelitis (EAE). To investigate the role of the signal transducer and activator of transcription factor 6 (STAT6) in EAE we used mice with two different targeted disruptions of the STAT6 gene. In this report, we show that mice with a targeted deletion of the first coding exon of the SH2 domain of STAT6 induce Th2 cell differentiation and are resistant to EAE induction. By contrast, STAT6−/− mice generated by deletion of amino acids 505 to 584 encoding the SH2 domain of STAT6 are defective in Th2 cell differentiation and develop very severe EAE. These results suggest that an altered STAT6 gene can be more efficient than wild type STAT6 in regulating the autoimmune response in EAE. [Copyright &y& Elsevier]

Abstract: A significant skewing of the peripheral T cell repertoire has been shown in relapsing–remitting multiple sclerosis (MS). Most of the studies already performed in this field are cross-sectional and therefore, little is known of the T cell repertoire evolution over time in MS and the correlation of T cell repertoire variation with clinical and MRI parameters. This study was performed on serially harvested frozen PBMC from nine untreated MS patients (27 samples) and 14 healthy individuals. The blood T cell repertoire of each patient was analysed at the complementarity determining region 3 (CDR3) level and compared with a monthly MRI scan performed over a six month period with assessment of T2 lesion load and gadolinium enhancing lesions. A highly significant blood T cell repertoire skewing was observed in MS patients as compared with healthy controls (p <0.01). In addition, the number of altered Vβ families correlated significantly with both the T2 lesion volume and the number of gadolinium enhancing lesions as assessed by MRI (Spearman correlation tests, r =0.51 and r =0.44, p <0.01 and p <0.05 respectively). Furthermore, the variation of the number of altered Vβ families over time also correlated with the appearance of new gadolinium enhancing lesions (r =0.36, p =0.05). These findings which need confirmation on larger serial cohorts, suggest an association between the magnitude of TCRBV CDR3 length distribution alterations in the peripheral blood of MS patients and the disease process. [Copyright &y& Elsevier]

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system considered to be mediated by T helper type-1 cells. Several agents have been found to modify the disease course of MS, including interferon-β1 (IFN-β1), glatiramer acetate mitoxantrone. We have employed pulse therapy with cyclophosphamide in a selected group of patients with actively progressive disease. Chemokine receptors have been found to differentiate between polarized T helper type-1 (Th1) and type-2 (Th2) lymphocytes. The chemokine receptors CCR5 and CXCR3 are expressed primarily on Th1 cells and CCR3, CCR4 and CCR8 on Th2 cells. Previous studies of the expression of chemokine receptors in MS have shown that active MS plaques are infiltrated by CCR5+ and CXCR3+ T cells. Some of these T cells may express both CCR5 and CXCR3. These T cells are major producers of IFN-γ, which worsens the clinical condition of patients with MS. We previously found that patients with MS had a high proportion of CXCR3+ T cells and that those with chronic progressive MS had a high proportion of CCR5+ T cells in their peripheral blood. We report here that in patients with secondary progressive MS, cyclophosphamide induces a marked increase in the percentage of CCR4+ T cells that produce high levels of IL-4 and reverses the increase in the percentages of IFN-γ-producing CCR5+ and CXCR3+ CD8+ T cells. Furthermore, therapy with cyclophosphamide increases IL-4-producing CD4+ T cells and reverses the increase in IFN-γ-producing CD8+ T cells. Our study shows that cyclophosphamide has immunomodulatory properties besides its suppressive effects, and that chemokine receptors can be important tools both for understanding the immune dysregulation in MS and for monitoring response to therapy. [Copyright &y& Elsevier]

CD8+ T cell depletion renders CD28-deficient mice susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, CD8-/-CD28-/- double-knockout mice are susceptible to EAE. These findings suggest a role for CD8+ T cells in the resistance of CD28-deficient mice to disease. Adoptive transfer of CD8+CD28- T cells into CD8-/- mice results in significant suppression of disease, while CD8+CD28+ T cells demonstrate no similar effect on the clinical course of EAE in the same recipients. In vitro, CD8+CD28- but not CD8+CD28+ T cells suppress IFN-gamma production of myelin oligodendrocyte glycoprotein-specific CD4+ T cells. This suppression requires cell-to-cell contact and is dependent on the presence of APCs. APCs cocultured with CD8+CD28- T cells become less efficient in inducing a T cell-dependent immune response. Such interaction prevents upregulation of costimulatory molecules by APCs, hence decreasing the delivery of these signals to CD4+ T cells. These are the first data establishing that regulatory CD8+CD28- T cells occur in normal mice and play a critical role in disease resistance in CD28-/- animals. [ABSTRACT FROM AUTHOR]

CD8[sup+] T cell depletion renders CD28-deficient mice susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, CD8[sup-/-]CD28[sup-/-] double-knockout mice are susceptible to EAE. These findings suggest a role for CD8[sup+] T cells in the resistance of CD28-deficient mice to disease. Adoptive transfer of CD8[sup+]CD28[sup-] T cells into CD8[-/-] mice results in significant suppression of disease, while CD8[sup+]CD28[sup+] T cells demonstrate no similar effect on the clinical course of EAE in the same recipients. In vitro, CD8[sup+]CD28[sup-] but not CD8[sup+]CD28[sup+] T cells suppress IFN-γ, production of myelin oligodendrocyte glycoprotein-specific CD4[sup+] T cells. This suppression requires cell-to-cell contact and is dependent on the presence of APCs. APCs cocultured with CD8[sup+]CD28[sup-] T cells become less efficient in inducing a T cell-dependent immune response. Such interaction prevents upregulation of costimulatory molecules by APCs, hence decreasing the delivery of these signals to CD4[sup+] T cells. These are the first data establishing that regulatory CD8[sup+]CD28[sup-] T cells occur in normal mice and play a critical role in disease resistance in CD28[sup-/-] animals. [ABSTRACT FROM AUTHOR]