Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor alpha-chain (IL-4Ralpha-/-) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Ralpha-/-mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Ralpha-/- DO11.10/scid mice secreted approximately 50 pg of IL-4 per10(6) cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-gamma and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Ralpha-/- mice gave rise to a minor proportion (approximately 2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-gamma and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-gamma and IL-12, this IL-4 can induce Th2 polarization.     

The Metalloprotease ADAM12 Regulates the Effector Function of Human Th17 Cells

A key modulator of immune homeostasis, TGFβ has an important role in the differentiation of regulatory T cells (Tregs) and IL-17-secreting T cells (Th17). How TGFβ regulates these functionally opposing T cell subsets is not well understood. We determined that an ADAM family metalloprotease called ADAM12 is specifically and highly expressed in both Tregs and CCR6+ Th17 cells. ADAM12 is induced in vitro upon differentiation of naïve T cells to Th17 cells or IL-17-secreting Tregs. Remarkably, silencing ADAM12 expression in CCR6+ memory T cells enhances the production of Th17 cytokines, similar to suppressing TGFβ signaling. Further, ADAM12 knockdown in naïve human T cells polarized towards Th17/Treg cells, or ectopically expressing RORC, greatly enhances IL-17-secreting cell differentiation, more potently then inhibiting TGFβ signals. Together, our findings reveal a novel regulatory role for ADAM12 in Th17 cell differentiation or function and may have implications in regulating their aberrant responses during immune pathologies.     

Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells

Th cells can be subdivided into IFN-gamma-secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H(+)K(+)-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.     

Quantities of interleukin-12p40 in mature CD8alpha negative dendritic cells correlate with strength of TCR signal and determine Th cell development

Strength of T cell antigen receptor (TCR) signaling drives the development of Th1 and Th2 subsets from naive T helper precursors. The quantity of interleukin-12 (IL-12) from antigen presenting cells (APC) is also profoundly involved in Th development. TCR signal strength and IL-12 production from dendritic cells (DCs) are linked by CD40 ligand (CD40L) expression on activated T cells. CD40L on the activated T cells interacts with CD40 on DC, resulting in induction of IL-12 from DCs. However, the subsets of DC in spleen that produce the IL-12 have not been clearly identified. Purification of DC subsets itself may provide maturation signals to immature DCs. Thus, we used non-purified mouse spleen cells to analyze IL-12 producing cells, near to steady states, during the interaction of naive T cells either with or without agonist. Mature CD86highCD8alpha- DCs in spleen mainly produced IL-12p40 after stimulation of high dose agonist. The ratio of CD40L positive T cells and IL-12p40 secreting CD86high DCs correlated with the concentration of agonist and Th1 development. However, anti-IL-12 did not completely inhibit the Th1 development. Altogether, strength of TCR signaling directs Th cell development by regulating CD40L expression on T cells which determines production of IL-12p40 from CD86high CD8alpha- DC via CD40.     

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.     

N-acetylglucosaminyltransferase V (Mgat5)-mediated N-glycosylation negatively regulates Th1 cytokine production by T cells

The differentiation of naive CD4(+) T cells into either proinflammatory Th1 or proallergic Th2 cells strongly influences autoimmunity, allergy, and tumor immune surveillance. We previously demonstrated that beta1,6GlcNAc-branched complex-type (N-acetylglucosaminyltransferase V (Mgat5)) N-glycans on TCR are bound to galectins, an interaction that reduces TCR signaling by opposing agonist-induced TCR clustering at the immune synapse. Mgat5(-/-) mice display late-onset spontaneous autoimmune disease and enhanced resistance to tumor progression and metastasis. In this study we examined the role of beta1,6GlcNAc N-glycan expression in Th1/Th2 cytokine production and differentiation. beta1,6GlcNAc N-glycan expression is enhanced by TCR stimulation independent of cell division and declines at the end of the stimulation cycle. Anti-CD3-activated splenocytes and naive T cells from Mgat5(-/-) mice produce more IFN-gamma and less IL-4 compared with wild-type cells, the latter resulting in the loss of IL-4-dependent down-regulation of IL-4Ralpha. Swainsonine, an inhibitor of Golgi alpha-mannosidase II, blocked beta1,6GlcNAc N-glycan expression and caused a similar increase in IFN-gamma production by T cells from humans and mice, but no additional enhancement in Mgat5(-/-) T cells. Mgat5 deficiency did not alter IFN-gamma/IL-4 production by polarized Th1 cells, but caused an approximately 10-fold increase in IFN-gamma production by polarized Th2 cells. These data indicate that negative regulation of TCR signaling by beta1,6GlcNAc N-glycans promotes development of Th2 over Th1 responses, enhances polarization of Th2 cells, and suggests a mechanism for the increased autoimmune disease susceptibility observed in Mgat5(-/-) mice.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen

The involvement of specific accessory/costimulatory molecules in differentiation to Th1 and Th2 phenotypes is controversial. Reports suggest that molecules such as CD4, CD28, and Ox-40 support Th2 differentiation and suppress Th1 differentiation, whereas others such as LFA-1 support Th1 responses and suppress Th2 responses. We have previously defined an in vitro model of differentiation that is absolutely dependent on the initial dose and affinity of peptide presented to a naive CD4 cell. The dose and affinity of Ag regulate autocrine production of IL-2, IL-4, and IFN-gamma, which in turn govern differentiation to Th1 and Th2 phenotypes. We have used this system to confirm that CD4, CD28, and Ox-40 interactions can promote, and LFA-1 interactions can suppress, differentiation of cells secreting the Th2 cytokines IL-5 and IL-13. However, for CD4 and LFA-1, this is only seen over a certain range of peptide doses. In addition, CD28 and Ox-40 interactions also promote Th1 differentiation. In general, agonist Abs to accessory molecules shifted the response curves for IFN-gamma, IL-5, and IL-13 to lower doses, whereas antagonist reagents resulted in similar curves shifted toward the higher doses. We conclude that ligation of cell surface accessory receptors enables low doses of Ag to promote responses normally induced only by higher doses. Individual receptors do not intrinsically regulate one cytokine phenotype or another, suggesting that differentiation is controlled by the level of expression of multiple accessory molecule pairs integrated with the number and affinity of peptide/MHC complexes.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

Distinctly different sensitivity in the induction and reversal of anergy of Th1 and Th2 cells

T cell anergy is one of the mechanisms of immunological tolerance. We examined in this study the distinct responses of Th1 and Th2 cells to in vitro anergic stimulation using Th1 and Th2 cells from two strains of T cell receptor transgenic mice. Proliferation of the Th2 cells was difficult to suppress by anergic stimulation, while that of Th1 cells was significantly inhibited even by weak stimulation. However, IL-4 production by Th2 cells was definitely reduced by anergic stimulation, although the inhibition level of IL-4 was lower than that of IFN-gamma production by Th1 cells. We also examined the reversal of anergy in both subsets. While both the anergized Th1 and Th2 cells responded to IL-2 stimulation, only the anergy of the Th2 cells could be reversed. This result indicates that progression of the cell cycle was not sufficient for anergy reversal in Th1 cells. Our findings indicate that the induction and reversal of T cell anergy might be affected by the distinct signaling features of Th1 and Th2 cells.     

Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation

The intrinsic features of naive CD4 T cells that affect their ability to respond to polarizing signals for Th cell differentiation are not well understood. In this study, we show that naive CD4 T cells from mice transgenic for the Hlx gene expressed lower levels of IL-4Ralpha. The down-regulation of IL-4Ralpha diminished IL-4 signaling and the Th2 response and enhanced the Th1 response under suboptimal polarizing conditions. In nontransgenic CD4 T cells, blocking IL-4Ralpha with Abs had the same effect in an Ab dose-dependent manner. Conversely, Hlx haploinsufficiency caused higher expression of IL-4Ralpha to favor Th2 cell differentiation. Thus, the IL-4Ralpha level on naive CD4 T cells is genetically controlled by Hlx and determines the ratio of Th1 and Th2 cell differentiation.     

Stat signals release activated naive Th cells from an anergic checkpoint

Activation of naive Th lymphocytes by the TCR and the costimulatory molecule, CD28, is believed to provide competent signals for differentiation to effector cells. Such activated cells proliferated and expressed IL-2, but arrested in an immature state maintained by CTLA-4. Although unresponsive to restimulation by TCR/CD28 alone, restimulation with TCR/CD28 and either Stat4- or Stat6-mediated cytokine signals rescued cells to proliferate and differentiate to the appropriately matched canonical Th subsets. Addition of IL-4 at defined periods revealed that naive T cells were receptive to IL-4-mediated differentiation for up to 3 days after their initial priming. A Stat-dependent anergic checkpoint between clonal expansion and effector cell differentiation may defer the cytokine profile to be instructed at the site of infection, thus preventing the unregulated development of potentially damaging effector cells.