IL-21 receptor signalling partially mediates Th2-mediated allergic airway responses.

BACKGROUND: Interleukin-21 (IL-21) has been implicated in the development of Th2-mediated immune responses; however, the exact role it plays in allergic diseases is not well understood. OBJECTIVE: To elucidate the contribution of IL-21 receptor signalling to Th2-dependent immune responses in the lung. METHODS: We compared allergic airway responses in wild-type BALB/c and Il21r-deficient mice exposed to local airway challenge with house dust mite (HDM). RESULTS: We demonstrate that IL-21R-deficiency reduces HDM-driven airway hyperresponsiveness (AHR) with only partial effects on airway inflammation. Concomitant with the reduction in AHR in Il21r-deficient mice, significant suppression was observed in protein levels of the Th2 cytokines IL-4, and IL-13. In contrast, IL-21R-deficiency was associated with an increase in PBS- and allergen-driven IgE levels, while IgG1 and IgG2a levels were decreased. Moreover, our results suggest that IL-21 may contribute to AHR through its ability to both directly induce Th2 cell survival and to impair regulatory T-cell suppression of Th2 cytokine production. Importantly, we show that IL-21-positive cells are increased in the bronchial mucosa of asthmatics compared with non-asthmatics. CONCLUSION: These results suggest that IL-21 plays an important role in the allergic diathesis by enhancing Th2 cytokine production through multiple mechanisms including the suppression of Treg inhibitory effects on Th2 cell cytokine production.

IL-12, STAT4-dependent up-regulation of CD4(+) T cell core 2 beta-1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands.

TCR activation of naive T cells in the presence of IL-12 drives polarization toward a Th1 phenotype and synthesis of P- and E-selectin ligands. Fucosyltransferase VII (Fuc-T VII) and core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) are critical for biosynthesis of selectin ligands. P-selectin glycoprotein ligand-1 is the best characterized ligand for P-selectin and also binds E-selectin. The contributions of TCR and cytokine signaling pathways to up-regulate Fuc-T VII and C2GnT during biosynthesis of E- and P-selectin ligands, such as P-selectin glycoprotein ligand 1, are unknown. IL-12 signals via the STAT4 pathway. Here, naive DO11.10 TCR transgenic and STAT4(-/-) TCR transgenic CD4(+) T cells were stimulated with Ag and IL-12 (Th1 condition), IL-4 (Th2), or neutralizing anti-IL-4 mAb only (Th0). The levels of Fuc-T VII and C2GnT mRNA in these cells were compared with their adhesive interactions with P- and E-selectin in vitro under flow. The data show IL-12/STAT4 signaling is necessary for induction of C2GnT, but not Fuc-TVII mRNA, and that STAT4(-/-) Th1 cells do not traffic normally to sites of inflammation in vivo, do not interact with P-selectin, and exhibit a partial reduction of E-selectin interactions under shear stress in vitro. Ag-specific TCR activation in CD4(+) T cells was sufficient to trigger induction of Fuc-TVII, but not C2GnT, mRNA and expression of E-selectin, but not P-selectin, ligands. Thus, Fuc-T VII and C2GnT are regulated by different signals during Th cell differentiation, and both cytokine and TCR signals are necessary for the expression of E- and P-selectin ligands.

Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4(+) T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.

Increased severity of local and systemic anaphylactic reactions in gp49B1-deficient mice.

gp49B1 is an immunoglobulin (Ig) superfamily member that inhibits FcstraightepsilonRI-induced mast cell activation when the two receptors are coligated with antibodies in vitro. The critical question of in vivo function of gp49B1 is now addressed in gene-disrupted mice. gp49B1-deficient mice exhibited a significantly increased sensitivity to IgE-dependent passive cutaneous anaphylaxis as assessed by greater tissue swelling and mast cell degranulation in situ. Importantly, by the same criteria, the absence of gp49B1 also resulted in a lower threshold for antigen challenge in active cutaneous anaphylaxis, in which the antigen-specific antibody levels were comparable in gp49B1-deficient and sufficient mice. Moreover, the absence of gp49B1 resulted in a significantly greater and faster death rate in active systemic anaphylaxis. These results indicate that gp49B1 innately dampens adaptive immediate hypersensitivity responses by suppressing mast cell activation in vivo. In addition, this study provides a new concept and target for regulation of allergic disease susceptibility and severity.

Plasma cell differentiation requires the transcription factor XBP-1.

Considerable progress has been made in identifying the transcription factors involved in the early specification of the B-lymphocyte lineage. However, little is known about factors that control the transition of mature activated B cells to antibody-secreting plasma cells. Here we report that the transcription factor XBP-1 is required for the generation of plasma cells. XBP-1 transcripts were rapidly upregulated in vitro by stimuli that induce plasma-cell differentiation, and were found at high levels in plasma cells from rheumatoid synovium. When introduced into B-lineage cells, XBP-1 initiated plasma-cell differentiation. Mouse lymphoid chimaeras deficient in XBP-1 possessed normal numbers of activated B lymphocytes that proliferated, secreted cytokines and formed normal germinal centres. However, they secreted very little immunoglobulin of any isotype and failed to control infection with the B-cell-dependent polyoma virus, because plasma cells were markedly absent. XBP-1 is the only transcription factor known to be selectively and specifically required for the terminal differentiation of B lymphocytes to plasma cells.

CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.

Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways.

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.

Role of STAT4 and STAT6 signaling in allograft rejection and CTLA4-Ig-mediated tolerance.

STAT4(-/-) mice have impaired type 1 T cell differentiation, whereas STAT6(-/-) mice fail to generate type 2 responses. The role of type 1 and type 2 T cell differentiation in acute cardiac allograft rejection and in the induction of tolerance was examined in wild-type, STAT4(-/-), and STAT6(-/-) recipients. All recipients rejected the grafts promptly. Analysis of in situ cytokine gene expression in the allografts confirmed decreased levels of IFN-gamma in STAT4(-/-) recipients and undetectable levels of IL-4 and IL-5 in STAT6(-/-) mice. Blockade of the CD28/B7 costimulatory pathway prolonged cardiac graft survival for >100 days in 100% of wild-type and STAT4(-/-) mice. However, 14% of CTLA4-Ig-treated STAT6(-/-) mice rejected their grafts between 20 and 100 days. Moreover, of those animals followed past 100 days, 60% of the STAT6(-/-) mice rejected their grafts. Splenocytes harvested on day 145 posttransplant from CTLA4-Ig-treated rejecting STAT6(-/-) recipients were transfused into syngeneic SCID mice transplanted with donor or third party cardiac allografts. Both donor and third party grafts were rejected, indicating that the initial graft loss may be due to an immunological rejection. In contrast, when splenocytes from CTLA4-Ig-treated wild-type or nonrejecting STAT6(-/-) mice were transferred into SCID recipients, donor allografts were accepted, but third party hearts were rejected. Thus, long-term prolongation of cardiac allograft survival by CTLA4-Ig is STAT4-independent but, at least in part, STAT6-dependent. These data suggest that the balance of type 1 and type 2 T lymphocyte differentiation is not critical for acute rejection but influences the robust tolerance induced by CD28/B7 blockade in this model.

Cutting edge: STAT6-deficient mice have enhanced tumor immunity to primary and metastatic mammary carcinoma.

STAT4 and STAT6 are essential for the development of CD4(+) Th1 and Th2 development, respectively. Tumor immunologists have hypothesized that Th1 cells are critical in tumor immunity because they facilitate differentiation of CD8(+) T cells, which are potent anti-tumor effectors. We have used STAT4(-/-) and STAT6(-/-) mice to test this hypothesis. BALB/c and knockout mice were challenged in the mammary gland with the highly malignant and spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma. Primary tumor growth and metastatic disease are reduced in STAT6(-/-) mice relative to BALB/c and STAT4(-/-) mice. Ab depletions demonstrate that the effect is mediated by CD8(+) T cells, and immunized STAT6(-/-) mice have higher levels of 4T1-specific CTL than BALB/c or STAT4(-/-) mice. Surprisingly, Th1 or Th2 cells are not involved, because CD4 depletion does not diminish the anti-tumor effect. Therefore, deletion of the STAT6 gene facilitates development of potent anti-tumor immunity via a CD4(+)-independent pathway.

The role of CD154-CD40 versus CD28-B7 costimulatory pathways in regulating allogeneic Th1 and Th2 responses in vivo.

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4(-/-) mice but was only marginally effective in STAT6(-/-) recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28(-/-) BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6(-/-) mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

Increased susceptibility of Stat4-deficient and enhanced resistance in Stat6-deficient mice to infection with Trypanosoma cruzi.

Although Th1-type responses tend to be associated with resistance to Trypanosoma cruzi infection, mixed Th1 and Th2 cytokine responses are generally observed in both resistant and susceptible mice. To help clarify the role of type 1 and type 2 cytokine responses in immunity to T. cruzi, mice with induced deficiencies in the Stat4 or Stat6 genes were infected with T. cruzi. As expected, Stat4-/- mice deficient in type 1 cytokine responses were highly susceptible to infection, exhibiting increased parasitemia levels relative to wild-type mice and 100% mortality. In contrast, parasitemia levels and survival in Stat6-deficient mice were not different from wild type. The type 1 and type 2 cytokine bias of Stat6- and Stat4-deficient mice, respectively, was confirmed by in situ immunocytochemical analysis of cytokine-producing cells in the tissues of infected mice and by subclass analysis of anti-T. cruzi serum Abs. Notably, both Stat4- and Stat6-deficient mice produced substantial amounts of anti-T. cruzi Abs. Tissues from chronically infected Stat6-deficient mice had little to no evidence of inflammation in the heart and skeletal muscle in contrast to wild-type mice, which exhibited substantial inflammation. In situ PCR analysis of these tissues provided evidence of the persistence of T. cruzi in wild-type mice, but no evidence of parasite persistence in Stat6-deficient mice. These data suggest that type 1 T cells are required for the development of immune control to T. cruzi, but that type 2 T cells contribute to parasite persistence and increased severity of disease.

The biology of Stat4 and Stat6.

IL-4 and IL-12 are cytokines that are important regulators of the proliferation, differentiation and functional capacity of lymphocytes. STATs (signal transducers and activators of transcription) are transcription factors that provide a direct link between the cytokine receptors and cytokine induced gene transcription. Stat6 and Stat4 are two STAT family members that specifically mediate signals that emanate from the IL-4 and IL-12 receptors, respectively. Recently a great deal of progress has been made in understanding the specific roles that Stat6 and Stat4 play in lymphocyte function through in vitro as well as in vivo studies using Stat6 and Stat4-deficient mice. This report will summarize and describe the recent advances made in understanding the activation and regulation of Stat6 and Stat4 as well as their roles in the development of an immune response. Oncogene (2000).

Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets.

STAT4 and STAT6 are transcription factors that play crucial roles in responding to IL-12 and IL-4, respectively. STAT4 gene knockout (STAT4(-/-)) mice have markedly reduced Th1 responses and enhanced Th2 responses. STAT6(-/-) mice show the inverse phenotype. We compared the ability of bone marrow transplantation (BMT) with the inclusion of spleen cells from STAT6(-/-), STAT4(-/-), and wild-type (WT) mice to produce graft-versus-host disease (GVHD) in lethally irradiated MHC-mismatched recipients. Acute GVHD mortality was more rapid when induced by cells from STAT6(-/-) mice than when induced by STAT4(-/-) cells. However, cells from STAT4(-/-) and STAT6(-/-) donors both induced delayed GVHD mortality compared with WT controls, or compared with combined STAT4(-/-) and STAT6(-/-) cells, indicating a contribution of both Th1 cells and Th2 cells to acute GVHD. Recipients of STAT6(-/-) BMT showed evidence of acute GVHD with severe diarrhea and marked weight loss. Recipients of STAT4(-/-) BMT showed signs of GVHD with only initial transient weight loss and later development of severe skin GVHD. Histopathology showed that Th2 responses were required for the induction of both hepatic and severe skin GVHD. In contrast, both Th1 cells and Th2 cells were capable of causing intestinal pathology of GVHD. Our studies demonstrate an additive role for Th1 and Th2 cells in producing acute GVHD, and suggest a cytokine-directed approach to treating end-organ manifestations of GVHD.

An essential role in liver development for transcription factor XBP-1.

XBP-1 is a CREB/ATF family transcription factor highly expressed in hepatocellular carcinomas. Here we report that XBP-1 is essential for liver growth. Mice lacking XBP-1 displayed hypoplastic fetal livers, whose reduced hematopoiesis resulted in death from anemia. Nevertheless, XBP-1-deficient hematopoietic progenitors had no cell-autonomous defect in differentiation. Rather, hepatocyte development itself was severely impaired by two measures: diminished growth rate and prominent apoptosis. Specific target genes of XBP-1 in the liver were identified as alphaFP, which may be a regulator of hepatocyte growth, and three acute phase protein family members. Therefore, XBP-1 is a transcription factor essential for hepatocyte growth.

Positive selection of NKT cells by CD1(+), CD11c(+) non-lymphoid cells residing in the extrathymic organs.

Previously, we found that NK1.1(+), TCRalpha beta(+) natural killer T (NKT) cells develop in cytokine-supplemented suspension cultures of fetal liver established from normal, but not from beta2 microglobulin-deficient [beta2m(- / -)] mice, and that recombination-deficient SCID fetal liver can reconstiute NKT cell development in beta2m(- / -) fetal liver cultures. We found here that cells of SCID adult liver, bone marrow, spleen and thymus were able to reconstitute NKT cell development in the former culture system with efficiency comparable to normal thymic cells. The reconstitution of NKT cells was also seen in the bone marrow chimeras that had been administered a combination of beta2m(- / -) and Rag-2(- / -) bone marrow cells. Development of NKT cells was hampered by depletion of CD11c(+) or CD11b(+) cells, but not by removal of B220(+) or Gr-1(+) cells from cultures of normal fetal liver cells. Furthermore CD11c(+), CD11b(+) and / or CD11c(+) CD11b(-) cells (both populations were CD1-dull positive) enriched from Rag-2-deficient fetal livers and pulsed with alpha-galactosylceramide, a possible antigen for NKT cells, were shown to reconstitute the NKT cell development in beta2m(- / -) fetal liver cultures. Collectively, our findings suggest that non-lymphoid cells, presumably CD11c(+), CD11b(+) and / or CD11c(+), CD11b(-) dendritic cells, are involved in the mechanism of positive selection of NKT cells in the thymus and extrathymic organs.

Consequences of Stat6 deletion on Sis/PDGF- and IL-4-induced proliferation and transcriptional activation in murine fibroblasts.

Aberrant communication among growth factors and cytokines that regulate tissue homeostasis often results in malignancy. Among the many cell types that participate in this process, stromal fibroblasts communicate in a paracrine and juxtracrine manner with cells of epithelial, endothelial, and hematopoietic origin. For fibroblasts, platelet-derived growth factor (PDGF) is a major proliferative and differentiation agent. Interleukin-4 (IL-4), however, possesses only modulating functions in this cell type. Here, we investigated the consequences of deleting Stat6 on PDGF and IL-4 signaling, proliferation, and transcriptional activation by establishing and characterizing early passage fibroblasts from wild-type and Stat6 null mice. Both wild-type and Stat6-/- fibroblasts showed nearly identical PDGFR and IL-4R activation, gross substrate tyrosine phosphorylation, PI 3-kinase activation, as well as Stat1, 3 and 5 DNA binding activities. Unexpectedly, IL-4's enhancement of PDGF-induced [3H]thymidine incorporation was greatly diminished in Stat6-/-, but not wild-type fibroblasts. PDGF-induced [3H]thymidine uptake was largely unaffected. Strikingly, IL-4, but not PDGF induction of the proinflammatory gene products, IL-6 and MCP-1 was markedly reduced in Stat6-/- fibroblasts. Thus, Stat6 is an important and specific mediator of IL-4-enhanced PDGF-induced proliferation as well as IL-4's transcriptional activation of IL-6 and MCP-1.

Stat6-dependent and -independent pathways for IL-4 production.

Stat6 has been shown to have a crucial role in the IL-4-dependent differentiation of Th2 cells. In this report, we explore whether in vitro Th2 differentiation driven by altered costimulatory signals or Ag dose is Stat6 dependent. We find that blocking B7-1 signaling in vitro promotes the differentiation of IL-4-secreting Th2 cells in wild-type but not Stat6-deficient T cell cultures. Additionally, stimulation with peptide Ag doses that normally result in the production of Th2 cells in vitro fails to do so in cultures of Stat6-deficient cells. We also demonstrate that Stat6 is required for the in vitro differentiation of CD8+ T cells into IL-4-secreting cytotoxic T cell type 2 cells. However, IL-4 expression is not absolutely dependent on Stat6. We demonstrate that populations of T cells that do not require IL-4 for their development, such as NK T cells, are still competent to secrete IL-4 in the absence of Stat6. These results demonstrate that Stat6 is required for the differentiation program leading to the generation of Th2 and cytotoxic T cell type 2 cells but not for IL-4 expression in cells that do not undergo differentiation in response to IL-4.

Disruption of the STAT4 signaling pathway protects from autoimmune diabetes while retaining antiviral immune competence.

The role of the STAT4 signaling pathway in autoimmune diabetes was investigated using the rat insulin promoter lymphocytic choriomeningitis virus model of virally induced autoimmune diabetes. Abrogation of STAT4 signaling significantly reduced the development of CD4+-T cell-dependent but not CD4+-T cell-independent diabetes, illustrating the fine-tuned kinetics involved in the pathogenesis of autoimmunity. However, the absence of STAT4 did not prevent the generation of autoreactive Th1/Tc1 T cell responses, as well as protective antiviral immunity. Protection from insulin-dependent diabetes mellitus was associated with decreased numbers of autoreactive CTL precursors in the pancreas and the spleen and a general as well as Ag-specific reduction of IFN-gamma secretion by T lymphocytes. A shift from Th1 to Th2 T cell immunity was not observed. Hence, our results implicate both CTL and cytokines in beta cell destruction. Selective inhibition of the STAT4 signal transduction pathway might constitute a novel and attractive approach to prevent clinical insulin-dependent diabetes mellitus in prediabetic individuals at risk.

Autoreactive CD4+ T cells protect from autoimmune diabetes via bystander suppression using the IL-4/Stat6 pathway.

Targeted immune regulation can be achieved by use of tissue-specific T cells and offers the potential for organ-specific suppression of destructive autoimmune processes. Here, we report the generation and characterization of insulin B chain-specific "autoreactive" CD4+ regulatory T cells that locally suppress diabetogenic T cell responses against an unrelated self-antigen (viral transgene) in a virus-induced model for type 1 diabetes. Interleukin 4 (IL-4) is essential for prevention of diabetes since regulatory T cells cannot be induced in the absence of IL-4 or stat6 (IL-4 signaling pathway). Our observations demonstrate that autoreactive regulatory T cells can suppress autoreactive destructive T cell activity of differential antigenic specificity locally in the pancreatic draining lymph node, probably via cytokine-mediated modulation of antigen-presenting cells.

A role for T helper 2 cells in mediating skin fibrosis in tight-skin mice.

Mice heterozygous for the tight-skin (Tsk) mutation develop skin fibrosis. Previous studies have implicated a role for the immune system and, specifically, CD4(+) T cells, in the etiology of skin fibrosis in Tsk/+ mice. We have recently shown that the administration of neutralizing anti-IL-4 antibodies to Tsk/+ mice prevented the development of skin fibrosis in these mice. Since IL-4 is a major cytokine produced by T helper 2 (Th2) cells, we investigated the role of Th2 cells in mediating skin fibrosis in Tsk/+ mice. Previous studies have shown that the development of Th2 cells in non-Tsk mice is abrogated in mice with null mutation for either the IL-4 or the Stat6 gene. In this study we showed that the polarization of CD4(+) T cells from Tsk/+ mice toward the Th2 lineage is also dependent on a functioning IL-4 or Stat6 gene. More importantly, the development of skin fibrosis in Tsk/+ mice was abrogated by the IL4(-/-) or the Stat6(-/-) mutation. We also determined whether alteration of the TCR repertoire in Tsk/+ mice, achieved by the introduction of TCR transgenes, was able to prevent the development of skin fibrosis in Tsk/+ mice. We found that the exclusive usage of the Vbeta8.2 gene segment by T cells was sufficient to prevent skin fibrosis in Tsk/+ mice. This result suggests that the exclusive use of this Vbeta gene segment by T cells may have prevented the development of fibrosis-causing Th2 cells.