Cuiyan Tan

Phenotype Switching by Inflammation-Inducing Polarized Th17 Cells, but not by Th1 Cells

Th1 and Th17 cells are characterized by their expression of IFN-γ or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-γ suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-γ was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-γ or both IFN-γ and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-γ or IFN-γ/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20–25% nonpolarized cells than in recipients of 35–40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-γ or IFN-γ/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-γt was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-γ or IFN-γ/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.

Antigen-Specific Th9 Cells Exhibit Uniqueness in Their Kinetics of Cytokine Production and Short Retention at the Inflammatory Site

Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-β and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the “natural mode” (i.e., exposure of CD4 cells to their target Ag, hen egg lysozyme [HEL] and APCs). Main observations are the following: 1) both IL-9 and IL-10 were expressed by the line cells, but with strikingly different kinetics, with IL-9 being produced rapidly, reaching a peak on day 3 in culture and declining sharply thereafter, whereas IL-10 production increased gradually, resembling IL-4 and IL-17 production by their corresponding lineage cells; 2) reactivation of Th9, following expansion, triggered faster and higher production of both IL-9 and IL-10; 3) incubating Th9 cells in polarizing media specific for other phenotypes stimulated moderate levels of phenotype switching to Th1 or Th17 but a massive switching to Th2; 4) Th9 cells induced moderate inflammation in HEL-expressing recipient eyes but only when producing high levels of IL-9; and 5) IL-9–producing donor cells were detected in the blood of Th9 recipients but not in their inflamed eyes, suggesting that similar to findings in culture, exposure to HEL in these eyes arrested the IL-9 production in Th9 cells. Collectively, these data provide new information concerning Th9 cells and reveal their uniqueness, in particular with regard to the unusual production kinetics of IL-9 and the short retention of these cells in affected target tissues.

Unlike Th1, Th17 Cells Mediate Sustained Autoimmune Inflammation and Are Highly Resistant to Restimulation-Induced Cell Death

Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4+ cells in the eye for as long as 25 days after transfer, mediating more long-lasting pathological changes. Unlike Th1, Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced Fas ligand production and resistance to Fas-induced apoptosis, relative to Th1 cells, despite similar surface expression of Fas. Th17-induced ocular inflammation also differed from Th1-induced inflammation by consisting of more neutrophils, whereas Th1-induced disease had higher proportions of CD8 cells. Taken together, our data show that pathogenic processes triggered by Th17 lag behind those induced by Th1, but then persist remarkably longer, apparently due to the relative resistance of Th17 cells to restimulation-induced cell death. The long-lasting inflammation induced by Th17 cells is in accord with these cells being involved in chronic conditions in humans.

The TNF-Family Ligand TL1A and Its Receptor DR3 Promote T Cell–Mediated Allergic Immunopathology by Enhancing Differentiation and Pathogenicity of IL-9–Producing T Cells

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A–DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

Differential Involvement of Th1 and Th17 in Pathogenic Autoimmune Processes Triggered by Different TLR Ligands

The interaction between TLRs and their cognate ligands triggers both the innate and adaptive immune systems, and thus can play a pivotal role in the defense against pathogen invasion. This work investigates the differentiation of naive CD4 cells into Th1 or Th17 phenotypes in mice treated with different TLR ligands. We use a model system in which naive transgenic cells specific to hen egg lysozyme are adoptively transferred into recipients that express hen egg lysozyme in the lens of the eye. The transferred naive T cells induce ocular inflammation only in recipients treated with TLR ligands. Treatment with LPS preferentially stimulated IL-17 production, whereas CpG oligodeoxynucleotide and polyinosinic:polycytidylic acid primarily stimulated Th1 cells. Peptidoglycan stimulated the two Th subpopulations equally. The preferential induction of Th1 or Th17 by the four ligands was detected in the spleen (where a major portion of the adoptively transferred cells homed) and in the eyes, where activated Th cells initiate inflammation. Analysis of the cytokines present in recipient mice suggests that Th1 induction is elicited by IL-12 and/or IFN-α, whereas Th17 generation is preferentially mediated by IL-6. Importantly, we show in this article that treatment with LPS selectively promoted in the recipient mice the generation of IL-6–producing activated B cells. An inverse correlation was found between the level of regulatory T cells and severity of inflammation induced by the donor cells. Taken together, our data show that specific TLR ligands differentially activate the immune system as evidenced by the generation of distinct Th phenotypes from naive CD4 cells.

Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response

Background Mutations in LRRK2 are related to certain forms of Parkinson’s disease and, possibly, to the pathogenesis of Crohn’s disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. Methods Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). Results The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. Conclusions Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.

Cell–Cell Interaction with APC, not IL-23, Is Required for Naive CD4 Cells To Acquire Pathogenicity during Th17 Lineage Commitment

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23–dependent Th17 cells.

Phenotypes of Th lineages generated by the commonly used activation with anti-CD3/CD28 antibodies differ from those generated by the physiological activation with the specific antigen

T-helper (Th) lineages have been generated in vitro by activating CD4 cells with anti-CD3/CD28 antibodies during polarization. Physiologically, however, the generation of Th lineages is by activation with the specific antigen presented by antigen-presenting cells (APC). Here, we used T-cell receptor (TCR)-transgenic mice to compare the phenotypes of Th1, Th9 and Th17 lineages when generated by either one of the two activation modes. Lineage Th cells specific against hen egg lysozyme (HEL), were adoptively transferred into recipient mice transgenically expressing HEL in their lens. Remarkable differences were found between lineages of Th1, Th9 or Th17, generated by either one of the two modes in their capacities to migrate to and proliferate in the recipient spleen and, importantly, to induce inflammation in the recipient mouse eyes. Substantial differences were also observed between the lineage pairs in their transcript expression profiles of certain chemokines and chemokine receptors. Surprisingly, however, close similarities were observed between the transcript expression profiles of lineages of the three phenotypes, activated by the same mode. Furthermore, Th cell lineages generated by the two activation modes differed considerably in their pattern of gene expression, as monitored by microarray analysis, but exhibited commonality with lineages of other phenotypes generated by the same activation mode. This study thus shows that (i) Th lineages generated by activation with anti-CD3/CD28 antibodies differ from lineages generated by antigen/APC; and (ii) the mode of activation determines to a large extent the expression profile of major transcripts.

Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC

Regulatory T-cells (Tregs) are responsible for homeostasis of the immune system, as well as for inhibition of pathogenic autoimmune processes. Induced-(i)-Tregs, can be generated in vitro by activation of CD4 cells in the presence of TGF-β. A commonly used activation mechanism is by antibodies against CD3 and CD28. The physiological-like activation of T-cells, however, is with the specific target antigen presented by antigen-presenting cells (APC). The two modes of activation have been considered to yield the same populations of iTregs. Here, we compared between iTreg populations generated by either one of the two methods and found differences between their capacities to inhibit T-lymphocyte proliferative response, their expression of cell surface antigens and particularly, in their transcript expression profiles of certain chemokines and chemokine receptors. Our data thus indicate that iTregs generated by activation with anti-CD3/CD28 antibodies cannot be considered identical to iTregs generated by antigen/APC.

Inflammation-inducing Th1 and Th17 cells differ in their expression patterns of apoptosis-related molecules

Th1 cells are remarkably more susceptible to activation induced cell death than Th17. Here, we compared cultures of these two cell subpopulations for their expression of apoptosis-related molecules when re-exposed to their specific antigen. We also compared the expression of apoptosis-related molecules in the mouse eye with inflammation induced by Th1 or Th17 cells. Using qPCR we found that the mRNA transcript levels of the majority of tested apoptosis-related molecules were higher in the Th1 cultures, and in eyes with Th1-induced inflammation. Apoptotic intrinsic pathway molecules played minor roles in the processes in vitro or in vivo, whereas extrinsic pathway molecules, as well as PD-1, its ligands and Tim3, were heavily involved.