Regulatory roles of IL-2 and IL-4 in H4/inducible costimulator expression on activated CD4+ T cells during Th cell development

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4(+) T cells obtained from six different murine strains. BALB/c-activated CD4(+) T cells expressed approximately 10-fold more H4/ICOS on their surfaces and produced approximately 10-fold more IL-4 upon restimulation than C57BL/6-activated CD4(+) T cells. BALB/c naive CD4(+) T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4(+) T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4(+) T cells strongly down-regulated both H4/ICOS expression on activated CD4(+) T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4(+) T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4(+) T cells from C57BL/6 or IL-4(-/-) mice increased H4/ICOS expression on activated CD4(+) T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4(+) T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4(+) T cells.     

IFN-gamma production by Th1 cells generated from naive CD4+ T cells exposed to norepinephrine

During activation in vivo, naive CD4(+) T cells are exposed to various endogenous ligands, such as cytokines and the neurotransmitter norepinephrine (NE). To determine whether NE affects naive T cell differentiation, we used naive CD4(+) T cells sort-purified from either BALB/c or DO11.10 TCR-transgenic mouse spleens and activated these cells with either anti-CD3/anti-CD28 mAbs or APC and OVA(323-329) peptide, respectively, under Th1-promoting conditions. RT-PCR and functional assays using selective adrenergic receptor (AR) subtype antagonists showed that naive CD4(+) T cells expressed only the beta 2AR subtype to bind NE and that stimulation of this receptor generated Th1 cells that produced 2- to 4-fold more IFN-gamma. This increase was due to more IFN-gamma produced per cell upon restimulation instead of more IFN-gamma-secreting cells, as determined by IFN-gamma-specific immunofluorescence and enzyme-linked immunospot. In contrast, Th1 cell differentiation was unaffected when naive T cells were exposed to NE and activated either in the presence of a neutralizing anti-IL-12 mAb or by APC from IL-12-deficient mice. Moreover, the addition of IL-12 to the IL-12-deficient APC cultures restored the ability of NE to increase Th1 differentiation. Taken together, these results indicate that a possible link may exist between the signaling pathways used by NE and IL-12 to increase naive CD4(+) T cell differentiation to a Th1 cell.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.     

Invariant NKT cells biased for IL-5 production act as crucial regulators of inflammation

Although invariant NKT (iNKT) cells play a regulatory role in the pathogenesis of autoimmune diseases and allergy, an initial trigger for their regulatory responses remains elusive. In this study, we report that a proportion of human CD4+ iNKT cell clones produce enormous amounts of IL-5 and IL-13 when cocultured with CD1d+ APC in the presence of IL-2. Such IL-5 bias was never observed when we stimulated the same clones with alpha-galactosylceramide or anti-CD3 Ab. Suboptimal TCR stimulation by plate-bound anti-CD3 Ab was found to mimic the effect of CD1d+ APC, indicating the role of TCR signaling for selective induction of IL-5. Interestingly, DNA microarray analysis identified IL-5 and IL-13 as the most highly up-regulated genes, whereas other cytokines produced by iNKT cells, such as IL-4 and IL-10, were not significantly induced. Moreover, iNKT cells from BALB/c mice showed similar IL-5 responses after stimulation with IL-2 ex vivo or in vivo. The iNKT cell subset producing IL-5 and IL-13 could play a major role in the development of allergic disease or asthma and also in the immune regulation of Th1 inflammation.     

Hepatitis C virus-specific Th17 cells are suppressed by virus-induced TGF-beta

IL-17-secreting T (Th17) cells play a protective role in certain bacterial infections, but they are major mediators of inflammation and are pathogenic in organ-specific autoimmune diseases. However, human Th17 cells appear to be resistant to suppression by CD4(+)CD25(+)FoxP3(+) regulatory T cells, suggesting that they may be regulated by alternative mechanisms. Herein we show that IL-10 and TGF-beta suppressed IL-17 production by anti-CD3-stimulated PBMC from normal individuals. TGF-beta also suppressed IL-17 production by purified CD4(+) T cells, whereas the inhibitory effect of IL-10 on IL-17 production appears to be mediated predominantly by its effect on APC. An examination of patients infected with hepatitis C virus (HCV) demonstrated that Ag-specific Th17 cells are induced during infection and that these cells are regulated by IL-10 and TGF-beta. PBMC from HCV Ab-positive donors secreted IL-17, IFN-gamma, IL-10, and TGF-beta in response to stimulation with the HCV nonstructural protein 4 (NS4). Furthermore, NS4 induced innate TGF-beta and IL-10 expression by monocytes from normal donors and at higher levels from HCV-infected patients. Neutralization of TGF-beta, and to a lesser extent IL-10, significantly enhanced NS4-specific IL-17 and IFN-gamma production by T cells from HCV-infected donors. Our findings suggest that both HCV-specific Th1 and Th17 cells are suppressed by NS4-induced production of the innate anti-inflammatory cytokines IL-10 and TGF-beta. This may represent a novel immune subversion mechanism by the virus to evade host-protective immune responses. Our findings also suggest that TGF-beta and IL-10 play important roles in constraining the function of Th17 cells in general.     

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.     

IL-9 promotes Th17 cell migration into the central nervous system via CC chemokine ligand-20 produced by astrocytes

Newly discovered IL-9-producing helper T cells (Th9) reportedly exert both aggravating and suppressive roles on experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, it is still unclear whether Th9 is a distinct Th cell subset and how IL-9 functions in the CNS. In this study, we show that IL-9 is produced by naive CD4(+) T cells that were stimulated with anti-CD3 and anti-CD28 Abs under the conditions of Th2-, inducible regulatory T cell-, Th17-, and Th9-polarizing conditions and that IL-9 production is significantly suppressed in the absence of IL-4, suggesting that IL-4 is critical for the induction of IL-9 by each producing cell. The IL-9 receptor complex, IL-9R and IL-2Rγ, is constitutively expressed on astrocytes. IL-9 induces astrocytes to produce CCL-20 but not other chemokines, including CCL-2, CCL-3, and CXCL-2 by astrocytes. The conditioned medium of IL-9-stimulated astrocytes induces Th17 cell migration in vitro, which is cancelled by adding anti-CCL-20 neutralizing Abs. Treating with anti-IL-9 neutralizing Abs attenuates experimental autoimmune encephalomyelitis, decreases the number of infiltrating Th17 cells, and reduces CCL-20 expression in astrocytes. These results suggest that IL-9 is produced by several Th cell subsets in the presence of IL-4 and induces CCL-20 production by astrocytes to induce the migration of Th17 cells into the CNS.     

Akt is a neutral amplifier for Th cell differentiation

Both CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4(+) T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4(+) T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4(+) T cells. E40K also facilitated Th1 differentiation in CD4(+) T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation.     

Role of CD25+ dendritic cells in the generation of Th17 autoreactive T cells in autoimmune experimental uveitis

In the current study, we showed that in vivo administration of an anti-CD25 Ab (PC61) decreased the Th17 response in C57BL/6 mice immunized with the uveitogenic peptide interphotoreceptor retinoid-binding protein, while enhancing the autoreactive Th1 response. The depressed Th17 response was closely associated with decreased numbers of a splenic dendritic cell (DC) subset expressing CD11c(+)CD3(-)CD25(+) and decreased expansion of γδ T cells. We demonstrated that ablation of the CD25(+) DC subset accounted for the decreased activation and the expansion of γδ T cells, leading to decreased activation of IL-17(+) interphotoreceptor retinoid-binding protein-specific T cells. Our results show that an enhanced Th17 response in an autoimmune disease is associated with the appearance of a DC subset expressing CD25 and that treatment of mice with anti-CD25 Ab causes functional alterations in a number of immune cell types, namely DCs and γδ T cells, in addition to CD25(+)αβTCR(+) regulatory T cells.     

Dendritic cells prime in vivo alloreactive CD4 T lymphocytes toward type 2 cytokine- and TGF-beta-producing cells in the absence of CD8 T cell activation

The mechanisms that influence the polarization of CD4 T cells specific for allogeneic MHC class II molecules in vivo are still poorly understood. We have examined the pathway of alloreactive CD4 T cell differentiation in a situation in which only CD4 T cells could be activated in vivo. In this report we show that priming of adult mice with allogeneic APC, in the absence of MHC class I-T cell interactions, induces a strong expansion of type 2 cytokine-producing allohelper T cells. These alloantigen-specific CD4 T cells directly recognize native allogeneic MHC class II molecules on APC and secrete, in addition to the prototypic Th2 cytokines IL-4, IL-5, and IL-10, large amounts of TGF-beta. The default Th2-phenotype acquisition is not genetically controlled and occurred both in BALB/c and C57BL/6 mice. CD8 T cells are the principal cell type that controls CD4 T cell differentiation in vivo. Furthermore, we demonstrate that strong Th2 priming can be induced not only with allogeneic splenocytes but also with a low number of bone marrow-derived dendritic cells. Finally, using a passive transfer system, we provide direct evidence that CD8 T cell expansion in situ promotes alloreactive Th1 cell development principally by preventing their default development to the Th2 pathway in a mechanism that is largely IFN-gamma independent. Therefore, this work demonstrates that type 2 cytokine production represents a dominant pathway of alloreactive CD4 T cell differentiation in adult mice, a phenomenon that was initially thought to occur only during the neonatal period.     

Hierarchical IL-5 expression defines a subpopulation of highly differentiated human Th2 cells

Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5(+) (IL-5(+), IL-4(+), IL-13(+)) and majority IL-5(-) Th2 (IL-5(-), IL-4(+), IL-13(+)) population. IL-5(+) Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5(-) Th2, but required multiple rounds of differentiation to generate IL-5(+) Th2 cells. IL-5(+) Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5(-) Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5(+) Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5(-) Th2 cells. GATA-3 and H3K4me(3) binding to the IL5 promoter (IL5p) was greater in IL-5(+) relative to IL-5(-) Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me(3) binding to the IL5p was greater in IL-5(-) Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5(+) Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5(+) Th2 cells. These results demonstrate that IL-5(+) and IL-5(-) Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.     

Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimulated dendritic cells to differentiate into Th2 cells

Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-gamma. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.     

Despite increased CD4+Foxp3+ cells within the infection site, BALB/c IL-4 receptor-deficient mice reveal CD4+Foxp3-negative T cells as a source of IL-10 in Leishmania major susceptibility

BALB/c IL-4Ralpha(-/-) mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4(+)CD25(+) T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4(+)CD25(+) cells in vivo and reconstituted IL-4Ralpha x RAG2 recipients with purified CD4(+)CD25(-) T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Ralpha(-/-) mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Ralpha x RAG2 mice reconstituted with CD4(+) cells depleted of CD25(+) Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Ralpha(-/-) inflammatory sites revealed that the majority of IL-10 was secreted by the CD4(+)Foxp3(-) population, with a fraction of IL-10 coming from CD4(+)Foxp3(+) Treg cells. All T cell IFN-gamma production was also derived from the CD4(+)Foxp3(-) population. Nevertheless, the IL-4Ralpha(-/-)-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4(+)CD25(+) and CD4(+)Foxp3(+) Treg cells compared with the BALB/c-infected dermis. Thus, CD4(+)Foxp3(-) T cells are a major source of IL-10 that disrupts IFN-gamma activity in L. major-susceptible BALB/c mice. However, the increase in CD4(+)Foxp3(+) T cells within the IL-4Ralpha(-/-) dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.     

IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.     

Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis

Th1 and Th17 T cells are often colocalized in pathological environments, yet Th1-derived IFN-gamma inhibits Th17 cell development in vitro. We explored the physiologic basis of this paradox in humans. In this study, we demonstrate increased the number of CD4(+) and CD8(+) IL-17(+) T cells in skin lesions of psoriasis. Furthermore, we show that myeloid APCs potently support induction of IL-17(+) T cells, and that this activity is greatly increased in psoriasis. We tested stimuli that might account for this activity. Th1 cells and IFN-gamma are increased in psoriatic blood and lesional skin. We show that IFN-gamma programs myeloid APCs to induce human IL-17(+) T cells via IL-1 and IL-23. IFN-gamma also stimulates APC production of CCL20, supporting migration of IL-17(+) T cells, and synergizes with IL-17 in the production of human beta-defensin 2, an antimicrobial and chemotactic protein highly overexpressed by psoriatic keratinocytes. This study reveals a novel mechanistic interaction between Th1 and IL-17(+) T cells, challenges the view that Th1 cells suppress Th17 development through IFN-gamma, and suggests that Th1 and IL-17(+) T cells may collaboratively contribute to human autoimmune diseases.