CD154 Costimulation Shifts the Local T-Cell Receptor Repertoire Not Only During Thymic Selection but Also During Peripheral T-Dependent Humoral Immune Responses.

CD154 is a transmembrane cytokine expressed transiently on activated CD4 T cells upon T-cell receptor (TCR) stimulation that interacts with CD40 on antigen-presenting cells. The signaling via CD154:CD40 is essential for B-cell maturation and germinal center formation and also for the final differentiation of CD4 T cells during T-dependent humoral immune responses. Recent data demonstrate that CD154 is critically involved in the selection of T-cell clones during the negative selection process in the thymus. Whether CD154 signaling influences the TCR repertoire during peripheral T-dependent humoral immune responses has not yet been elucidated. To find out, we used CD154-deficient mice and assessed the global TCRß repertoire in T-cell zones (TCZ) of spleens by high-throughput sequencing after induction of a Th2 response to the multiepitopic antigen sheep red blood cells. Qualitative and quantitative comparison of the splenic TCZ-specific TCRß repertoires revealed that CD154 deficiency shifts the distribution of Vß-Jß genes after antigen exposure. This data led to the conclusion that costimulation via CD154:CD40 during the interaction of T cells with CD40-matured B cells contributes to the recruitment of T-cell clones into the immune response and thereby shapes the peripheral TCR repertoire.

The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR.

The mechanisms by which the sensitivity of naive CD4+ T cells to stimulation by the cognate antigen via the T cell antigen receptor (TCR) determines their differentiation into distinct helper T cell subsets remain elusive. Here we demonstrate functional collaboration of the ubiquitin E3 ligases Itch and WWP2 in regulating the strength of the TCR signal. Mice lacking both Itch and WWP2 in T cells showed spontaneous autoimmunity and lung inflammation. CD4+ T cells deficient in Itch and WWP2 exhibited hypo-responsiveness to TCR stimulation and a bias toward differentiation into the TH2 subset of helper T cells. Itch and WWP2 formed a complex and cooperated to enhance TCR-proximal signaling by catalyzing the conjugation of atypical ubiquitin chains to the phosphatase SHP-1 and reducing the association of SHP-1 with the tyrosine kinase Lck. These findings indicate that targeted ubiquitination regulates the strength of the TCR signal and differentiation toward the TH2 lineage.

PARP is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice.

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.

Serine protease inhibition attenuates rIL-12-induced GZMA activity and proinflammatory events by modulating the Th2 profile from estrogen-treated mice.

Estrogen has potent immunomodulatory effects on proinflammatory responses, which can be mediated by serine proteases. We now demonstrate that estrogen increased the extracellular expression and IL-12-induced activity of a critical member of serine protease family Granzyme A, which has been shown to possess a novel inflammatory persona. The inhibition of serine protease activity with inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride significantly diminished enhanced production of proinflammatory interferon-γ, IL-1ß, IL-1α, and Granzyme A activity even in the presence of a Th1-inducing cytokine, IL-12 from splenocytes from in vivo estrogen-treated mice. Inhibition of serine protease activity selectively promoted secretion of Th2-specific IL-4, nuclear phosphorylated STAT6A, signal transducer and activator of transcription (STAT)6A translocation, and STAT6A DNA binding in IL-12-stimulated splenocytes from estrogen-treated mice. Inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride reversed the down-regulation of Th2 transcription factors, GATA3 and c-Maf in splenocytes from estrogen-exposed mice. Although serine protease inactivation enhanced the expression of Th2-polarizing factors, it did not reverse estrogen-modulated decrease of phosphorylated STAT5, a key factor in Th2 development. Collectively, data suggest that serine protease inactivity augments the skew toward a Th2-like profile while down-regulating IL-12-induced proinflammatory Th1 biomolecules upon in vivo estrogen exposure, which implies serine proteases as potential regulators of inflammation. Thus, these studies may provide a potential mechanism underlying the immunomodulatory effect of estrogen and insight into new therapeutic strategies for proinflammatory and female-predominant autoimmune diseases.

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes.

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis.

BACKGROUND: Cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone. Cyp11a1 expression is increased in activated T cells. OBJECTIVES: We sought to determine the role of Cyp11a1 activation in the development of peanut allergy and TH cell functional differentiation. METHODS: A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on T(H)1, T(H)2, and T(H)17 differentiation were determined. Cyp11a1 gene silencing was performed with Cyp11a1-targeted short hairpin RNA. RESULTS: Peanut sensitization and challenge resulted in diarrhea, inflammation, and increased levels of Cyp11a1, IL13, and IL17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG treatment decreased IL13 and IL17A mRNA expression in the small intestine without affecting Cyp11a1 mRNA or protein levels. In vitro the inhibitor decreased IL13 and IL17A mRNA and protein levels in differentiated T(H)2 and T(H)17 CD4 T cells, respectively, without affecting GATA3, retinoic acid-related orphan receptor γt (RORγt), or T(H)1 cells and IFNG and T-bet expression. Short hairpin RNA-mediated silencing of Cyp11a1 in polarized T(H)2 CD4 T cells significantly decreased pregnenolone and IL13 mRNA and protein levels. CONCLUSION: Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T(H)2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.

Shp1 regulates T cell homeostasis by limiting IL-4 signals.

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell­intrinsic role of Shp1, we characterized mice with a T cell­specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1(fl/fl) CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1- deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.

Indoleamine 2,3-dioxygenase is differentially expressed by different white blood cell populations of rhesus macaques (Macaca mulatta).

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is involved in immune processes such as transplant and fetal rejection, autoimmunity, cancer, and infection; however, its expression in rhesus macaques has not been fully addressed. METHODS: Indoleamine 2,3-dioxygenase mRNA and protein in the white blood cells (WBCs) of Chinese rhesus macaques were examined by RT-PCR, western blotting, real-time RT-PCR, and flow cytometry. RESULTS: Both IDO protein and mRNA could be readily detected in WBCs or peripheral blood mononuclear cells (PBMCs) of normal rhesus macaques. IDO+ cell frequency was the highest among CD14(+) mononuclear cells, followed by CD56(+) cells and DCs. No difference in the frequency of IDO+ cells between CD4(+) and CD8(+) T cells; however, Th17 cells have higher frequency of IDO+ cells than Th1 cells, with Th2 cells the lowest. Toll-like receptor (TLR) stimulation significantly increased IDO protein level in CD14(+) , CD56(+) , CD1c(+) , CD11c(+) , and CD123(+) myeloid cells. CONCLUSION: Rhesus macaques express IDO differentially in their leukocyte subsets and are suitable for IDO-related pathophysiological studies.

Effect of tyrosine hydroxylase gene silencing in CD4+ T lymphocytes on differentiation and function of helper T cells.

OBJECTIVES: We explored effect of gene silencing of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines (CAs), in CD4+ T cells on differentiation and function of helper T (Th) cells to provide more evidence for functional significance of lymphocyte-derived CAs. METHODS: CD4+ T lymphocytes were isolated and purified from the mesenteric lymph nodes of mice. Recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) was constructed and transfected into concanavalin A (Con A)-activated CD4+ T lymphocytes using nucleofection technology. After incubated for 48 h, these cells were detected for TH gene and protein expression and CA content. Simultaneously, percentage of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-producing cells and levels of IL-2, IFN-γ, tumor necrosis factor (TNF), IL-4 and IL-5 in culture supernatants of Con A-stimulated CD4+ T cells were examined by flow cytometric analysis. RESULTS: CD4+ T lymphocytes with TH RNAi expressed less TH mRNA and protein and synthesized less CAs including norepinephrine, epinephrine and dopamine than control cells with mock transfection. The silencing of TH gene in CD4+ T lymphocytes reduced percentage of IL-4-producing cells and elevated ratio of IFN-γ-producing cells to IL-4-producing cells, although it did not alter proportion of IFN-γ-producing cells. The Th1 cytokines, IL-2, IFN-γ and TNF, were increased, but the Th2 cytokines, IL-4 and IL-5, were decreased in the culture supernatants of Con A-stimulated CD4+ T lymphocytes that were transfected with TH miRNA. CONCLUSION: TH gene silencing attenuates TH expression and CA synthesis in CD4+ T lymphocytes and promotes polarization of differentiation and function towards Th1 cells.

Human Th17 cells express high levels of enzymatically active dipeptidylpeptidase IV (CD26).

Dipeptidylpeptidase IV (CD26) is a multifunctional ectoenzyme involved in T cell activation that has been implicated in autoimmune pathophysiology. Because IL-17-producing CD4(+) T cells (Th17 cells) are important mediators of autoimmune disease, we analyzed the expression of CD26 and its enzymatic function on human Th17 cells. Analysis of CD26 expression on different CD4(+) T helper subsets showed that CD26 expression is highest on CD4(+) T cells producing type 17 cytokines (e.g., IL-22, IL-17, GM-CSF, or TNF) compared with Th1, Th2, and regulatory T cells. Phenotypic analysis revealed that CD26(++)CD4(+) T cells express the type 17 differentiation molecules CD161, CCR6, lL-23R, and retinoic acid-related orphan receptor-γt. Furthermore, sorted CD26(++)CD4(+) T cells contain >90-98% of Th17 cells, indicating that CD26(++) T cells harbor the Th17 lineage. A comparison with CD161 and CCR6 indicated that analysis of CD26 coexpression may improve the phenotypic characterization of Th17 cells. Of note, CD26(++) Th17 cells are enriched in the inflamed tissue of patients with hepatitis and inflammatory bowel disease. Functional analysis in migration assays revealed that CD26 expressed on Th17 cells is enzymatically active. Indeed, CD26 negatively regulates the chemotactic CD4(+) T cell response to the inflammatory chemokines CXCL9-12 that can be restored by pharmacological blockade of the enzymatic center of CD26. In summary, these results strongly suggest that CD26 may contribute to the orchestration of the immune response by Th17 cells in human inflammatory diseases. They also suggest that the phenotypic analysis of Th17 cells may be facilitated by determination of CD26 expression.

Functional involvement of dual specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, in the regulation of T helper cell differentiation.

Naïve CD4(+) T helper (Th) cells differentiate into distinct subsets of effector cells (Th1, Th2, Th17, and induced regulatory T cells (iTreg)) expressing different sets of cytokines upon encounter with presented foreign antigens. It has been well established that Th1/Th2 balance is critical for the nature of the following immune responses. Previous reports have demonstrated important roles of c-Jun N-terminal kinase (JNK) in Th1/Th2 balance, whereas the regulatory mechanisms of JNK activity in Th cells have not been elucidated. Here, we show that dual specificity phosphatase 16 (DUSP16, also referred to as MKP-M or MKP-7), which preferentially inactivates JNK, is selectively expressed in Th2 cells. In the in vitro differentiation assay of naïve CD4(+) cells, DUSP16 expression is up-regulated during Th2 differentiation and down-regulated during Th1 differentiation. Chromatin immunoprecipitation revealed the increased acetylation of histone H3/H4 at the dusp16 gene promoter in CD4(+) T cells under the Th2 condition. Adenoviral transduction of naïve CD4(+) T cells with DUSP16 resulted in increased mRNA expression of IL-4 and GATA-3 in Th2 and decreased expression of IFNγ and T-bet in Th1 differentiation. In contrast, transduction of a dominant negative form of DUSP16 had the reverse effects. Furthermore, upon immunization, T cell-specific dusp16 transgenic mice produced antigen-specific IgG2a at lower amounts, whereas DN dusp16 transgenic mice produced higher amounts of antigen-specific IgG2a accompanied by decreased amounts of antigen-specific IgG1 and IgE than those of control mice. Together, these data suggest the functional role of DUSP16 in Th1/Th2 balance.

Distinct proteinase 3-induced cytokine patterns in Wegener´s granulomatosis, Churg-Strauss syndrome, and healthy controls.

OBJECTIVES: To analyse whether a specific cytokine pattern is elicited in response to the autoantigen proteinase 3 (PR3) in active Wegener's granulomatosis (WG). METHODS: Six-colour flow cytometry was used to analyse cytokine production and surface markers of the total CD4+ T-cell population ex vivo and in PR3-stimulated T-cell lines of patients with active PR3-ANCA-positive WG, PR3-ANCA-negative Churg-Strauss syndrome (CSS), and healthy controls (HC). RESULTS: The cytokine response of the total PB CD4+ T cell population was skewed towards distinct pro-inflammatory cytokine patterns in WG (Th1-type) and CSS (Th17, Th1-/Th2-type). Th2-type as well as Th17 cell populations including Th17/Th1, Th17/Th2 and Th22 cells were elicited in response to PR3 stimulation in WG. In contrast, CSS patients displayed a Th2-type dominated response following PR3 stimulation. CONCLUSIONS: These data suggest that the cytokine response of the total CD4+ T-cell population and PR3-specific cells is influenced by the underlying disorder.

Lipopolysaccharide-induced expression of Th1/Th2 cytokines in whole neonatal cord and adult blood: role of nuclear factor-kappa B and p38 MAPK.

BACKGROUND: Sepsis continues to be a leading cause of morbidity and mortality in newborns. OBJECTIVE: As both nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) appear to be critical mediators in inflammatory response, we studied the effects of lipopolysaccharide (LPS) on expression and function of NF-κB and p38 MAPK in whole neonatal cord and adult blood. METHODS: Th1/Th2 cytokine concentrations and phosphorylation of NF-κB and p38 MAPK were determined by flow-cytometric analysis. RESULTS: Tumor necrosis factor-alpha (TNF-α), IL-6, and IL-10 concentrations were significantly elevated in supernatants of neonatal and adult blood after LPS stimulation for 4 h. IFN-γ, IL-4, and IL-2 showed no significant alterations. Furthermore, TNF-α concentrations were significantly higher in adult compared to neonatal blood after LPS stimulation. Stimulation with LPS resulted in significantly decreased activation of p38 MAPK in neonatal blood, whereas NF-κB showed no difference. Following inhibition of p38 MAPK with the specific inhibitor SB-202190, levels of TNF-α and IL-6 significantly decreased in neonatal and adult blood, whereas pharmacological inhibition of NF-κB with SC-514 showed no significant effect on cytokine expression. CONCLUSIONS: We conclude that p38 MAPK phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

Early induction of GAD(65)-reactive Th2 response in type 1 diabetic children treated with alum-formulated GAD(65).

BACKGROUND: We have previously shown that two injections of 20 µg alum-formulated glutamic acid decarboxylase 65 (GAD(65)) (GAD-alum; Diamyd(®)) in children with recent-onset type 1 diabetes lead to preservation of residual insulin secretion. In vitro cytokine production at the 15 months' follow-up indicated immunomodulation. In the present study, we took advantage of peripheral blood mononuclear cells, cryopreserved during early follow-ups, to investigate whether the immunomodulatory effect of GAD-alum was apparent earlier after treatment, preceding the changes previously reported at 15 months. METHODS: Peripheral blood mononuclear cells from 70 type 1 diabetic children, randomly assigned GAD-alum (n = 35) or placebo (n = 35), that had been frozen at baseline (n = 27) and after 1 (n = 58), 3 (n = 67) and 9 (n = 66) months, were stimulated in vitro with GAD(65), tyrosine phosphatase-like protein IA-2 peptide, insulin peptide, GAD-alum, alum formulation or phytohaemagglutinin. Interleukin (IL)-5, -6, -10, -12, -13, -17, tumour necrosis factor and interferon-γ were measured in cell supernatants and serum samples using Luminex. Expression of FOXP3 and transforming growth factor-ß was determined by real-time reverse transcription polymerase chain reaction. RESULTS: Already 1 month after the first injection, GAD(65)-induced IL-5 and IL-13 together with FOXP3 were enhanced in GAD-alum-treated patients compared to those with placebo. The in vitro response at 3 and 9 months was characterized by a broader range of cytokines in the treated group. Notably, only the T-helper 2-associated cytokines IL-5 and IL-13 together with FOXP3 increased continuously over time. CONCLUSIONS: Treatment with GAD-alum in type 1 diabetic children induced an early T-helper 2 immune enhanced response to GAD(65), followed by a wider spectrum of cytokines at 3 and 9 months.

Th2-specific immunity and function of peripheral T cells is regulated by the p56Lck Src homology 3 domain.

T cell activation and effector function is essential for robust immunity. Ag TCR signals are known to regulate T lymphocyte differentiation, but the mechanisms involved in this regulation remain unclear. Recent work has demonstrated that the Src family protein tyrosine kinase p56Lck specifically links TCR signaling to activation of the MAPK pathway through the function of its Src homology 3 (SH3) domain. The MAPK pathway is involved in T cell activation and has previously been implicated in Th2 immunity. We have used Lck SH3 mutant knockin mice (LckW97A) to investigate the potential role of this regulatory mechanism in T lymphocyte activation and effector function. Our results demonstrate that Lck SH3 domain function regulates activation of T lymphocytes as indicated by reduced IL-2 production, CD69 induction, and proliferation of LckW97A T cells following TCR stimulation. Biochemical studies confirm that activation of the MAPK pathway is selectively altered following TCR ligation in LckW97A T lymphocytes. Phospho-ERK induction is reduced, but phospho-phospholipase Cgamma1 induction and calcium mobilization are largely unaffected. Immunization with DNP-keyhole limpet hemocyanin, heat-killed Brucella abortus, or infection with Nippostrongylus brasiliensis demonstrates selectively impaired Th2 immunity with reduced serum levels of IgG1, IgE, and IL-4. In vitro studies show that LckW97A T cells can differentiate into Th2-type cells, but they form IFN-gamma-producing cells under conditions that normally favor Th2 development. These data indicate that the Lck SH3 domain controls T lymphocyte activation by regulating MAPK pathway induction and demonstrate a novel role for Lck in the regulation of Th2-type immunity.

The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.

The mannose receptor (MR) is a C-type lectin expressed by dendritic cells (DCs). We have investigated the ability of MR to recognize glycosylated allergens. Using a gene silencing strategy, we have specifically inhibited the expression of MR on human monocyte-derived DCs. We show that MR mediates internalization of diverse allergens from mite (Der p 1 and Der p 2), dog (Can f 1), cockroach (Bla g 2), and peanut (Ara h 1) through their carbohydrate moieties. All of these allergens bind to the C-type lectin-like carbohydrate recognition domains 4-7 of MR. We have also assessed the contribution of MR to T cell polarization after allergen exposure. We show that silencing MR expression on monocyte-derived DCs reverses the Th2 cell polarization bias, driven by Der p 1 allergen exposure, through upregulation of IDO activity. In conclusion, our work demonstrates a major role for MR in glycoallergen recognition and in the development of Th2 responses.

Lck mediates Th2 differentiation through effects on T-bet and GATA-3.

The Src family kinase Lck has been shown to be crucial in T cell signaling and development. However, its role in Th effector functions is not well understood. Lck has previously been shown to play a role in the cytokine expression of Th2 cells, but the mechanism by which Lck influences Th2 effector functions is unknown. Using a mouse model, we report that Lck is important in regulating the expression of IL-4 in Th2 skewed cells but is not as necessary for the expression of Th2 cytokines IL-5, IL-10, and IL-13. Furthermore, in the absence of Lck, T-bet and GATA-3 expression is aberrant. Moreover, this atypical expression pattern of T-bet and GATA-3 correlates with increased histone 3 acetylation at the Ifng locus and production of the Th1 cytokine IFN-gamma. We find overexpression of GATA-3 restores IL-4 expression in lck(-/-) Th2 cells; this indicates that the decreased IL-4 expression is due in part to reduced amounts of GATA-3. Taken together, these data imply that Lck mediates Th2 differentiation through effects on T-bet and GATA-3.

SHP-1 deficient mast cells are hyperresponsive to stimulation and critical in initiating allergic inflammation in the lung.

Phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1)-deficient mice display an allergic asthma phenotype that is largely IL-13 and STAT6 dependent. The cell types responsible for the Th2 phenotype have not been identified. We hypothesized that SHP-1 deficiency leads to mast cell dysregulation and increased production and release of mediators and Th2 cytokines, leading to the allergic asthma phenotype. We examined SHP-1 regulation of mast cell differentiation, survival, and functional responses to stimulation using bone marrow-derived mast cells from viable motheaten (mev) mice. We assessed pulmonary phenotypical changes in mev mice on the mast cell-deficient Kit(W-Sh) genetic background. The results showed that SHP-1 deficiency led to increased differentiation and survival, but reduced proliferation, of mast cells. SHP-1-deficient mast cells produced and released increased amounts of mediators and Th2 cytokines IL-4 and -13 spontaneously and in response to H(2)O(2), LPS, and Fc epsilonI cross-linking, involving c-Kit-dependent and -independent processes. The Fc epsilonRI signaling led to binding of SHP-1 to linker for activation of T cells 2 and enhanced linker for activation of T cells 2 phosphorylation in mev bone marrow-derived mast cells. Furthermore, the number of mast cells in the lung tissue of mev mice was increased and mast cell production and release of Th2 cytokines were distinctly increased upon Fc epsilonRI stimulation. When backcrossed to the Kit(W-Sh) background, mev mice had markedly reduced pulmonary inflammation and Th2 cytokine production. These findings demonstrate that SHP-1 is a critical regulator of mast cell development and function and that SHP-1-deficient mast cells are able to produce increased Th2 cytokines and initiate allergic inflammatory responses in the lung.

Transglutaminase II interacts with rac1, regulates production of reactive oxygen species, expression of snail, secretion of Th2 cytokines and mediates in vitro and in vivo allergic inflammation.

Transglutaminase II (TGase II) is a protein cross-linking enzyme with diverse biological functions. Here we report the role of TGase II in allergic inflammation. Antigen stimulation induced expression and activity of TGase II by activation of NF-kappaB in rat basophilic leukemia (RBL2H3) cells. This induction of TGase II was dependent on FcepsilonRI and EGFR. Interaction between TGase II and rac1 was induced following antigen stimulation. TGase II was responsible for the increased production of reactive oxygen species, expression of prostaglandin E2 synthase (PGE2 synthase) and was responsible for increased secretion of prostaglandin E2. ChIP assay showed that TGase II, through interaction with NF-kappaB, was responsible for the induction of histone deacetylase-3 (HDAC3) and snail by direct binding to promoter sequences. HDAC3 and snail induced by TGase II, exerted transcriptional repression on E-cadherin. Snail exerted negative effect on expression of MMP-2, and secretion of Th2 cytokines. Inhibition of matrix metalloproteinase-2 (MMP-2) inhibited secretion of Th2 cytokines. In vivo induction of TGase II was observed in Balb/c mouse model of IgE antibody-induced passive cutaneous anaphylaxis. Chemical inhibition of TGase II exerted negative effect on IgE-dependent passive cutaneous anaphylaxis. Chemical inhibition of TGase II by cystamine exerted negative effect on Balb/c mouse model of phorbol myristate acetate (PMA)-induced atopic dermatitis. These results suggest novel role of TGase II in allergic inflammation and TGase II can be developed as target for the development of allergy therapeutics.

Human activated T lymphocytes modulate IDO expression in tumors through Th1/Th2 balance.

Previous cancer vaccination approaches have shown some efficiency in generating measurable immune responses, but they have rarely led to tumor regression. It is therefore possible that tumors emerge with the capacity to down-regulate immune counterparts, through the local production of immunosuppressive molecules, such as IDO. Although it is known that IDO exerts suppressive effects on T cell functions, the mechanisms of IDO regulation in tumor cells remain to be characterized. Here, we demonstrate that activated T cells can induce functional IDO expression in breast and kidney tumor cell lines, and that this is partly attributable to IFN-gamma. Moreover, we found that IL-13, a Th2 cytokine, has a negative modulatory effect on IDO expression. Furthermore, we report IDO expression in the majority of breast and kidney carcinoma samples, with infiltration of activated Th1-polarized T cells in human tumors. These findings demonstrate complex control of immune activity within tumors. Future immune therapeutic interventions should thus include strategies to counteract these negative mechanisms.