TH17 cells express ST2 and are controlled by the alarmin IL-33 in the small intestine.

TH17 cells are major drivers of inflammation and involved in several autoimmune diseases. Tissue inflammation is a beneficial host response to infection, but it can also contribute to autoimmunity. The crosstalk between a tissue and the immune system during an inflammatory response is key for preserving tissue integrity and restoring physiological processes. However, how the inflamed tissue regulates the magnitude of an immune response by controlling pro-inflammatory T cells is not well characterized so far. Here we show that TH17 cells accumulating in the small intestine upon inflammation express the IL-33 receptor (ST2) and intestinal epithelial cells (IEC) are the main source of the alarmin interleukin-33 (IL-33). We show that pro-inflammatory TH17 cells acquire a regulatory phenotype with immunosuppressive properties in response to IL-33. Absence of ST2 signaling promotes the secretion of pro-inflammatory cytokines by TH17 cells and dampens the secretion of IL-10. Our results provide new insights into the mechanisms by which IEC, via IL-33/ST2 axis, may control pro-inflammatory TH17 cells in the small intestine to sustain homeostasis.

T-bet expression by Th cells promotes type 1 inflammation but is dispensable for colitis.

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.

An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Regulation and function of T1/ST2 expression on CD4+ T cells: induction of type 2 cytokine production by T1/ST2 cross-linking.

The orphan receptor T1/ST2, a member of the IL-1R family, is preferentially expressed on the surface of murine Th2 cells. In this study, we analyzed the kinetics and function of T1/ST2 expression on Th2 cells in vitro. Whereas naive CD4(+) cells did not express T1/ST2, most CD4(+) cells became T1/ST2(+) upon repeated antigenic stimulation under Th2-polarizing conditions. Flow cytometric analyses revealed that the kinetics of T1/ST2 expression on Th2 cells was delayed compared with the kinetics of type 2 cytokine production. Exogenous IL-6, IL-5, IL-1, and TNF-alpha enhanced the expression of T1/ST2 on Th2 cells, and IL-6 was by far most effective in this regard. However, the expression of T1/ST2 did not depend on the presence of IL-6 and was also detected in IL-6-deficient mice. Most important, cross-linking of T1/ST2 provided a costimulatory signal for Th2 but not Th1 cells and directly induced proliferation and type 2 cytokine production. Thus, T1/ST2 is not only a Th2 cell marker but also plays an important role in the activation of Th2 cells.

Regulation of expression of IL-4 alleles: analysis using a chimeric GFP/IL-4 gene.

CD4 cells from mice heterozygous for an IL-4 and a GFP/IL-4 gene frequently express a single allele. Analysis of IL-4 or GFP production by cells from recently primed Th2 cells indicates that essentially all are competent to transcribe either allele but have a low probability of doing so. By contrast, long-term Th2 clones show distinct and heritable ratios in the proportion of cells that express IL-4 or GFP. We conclude that in the course of Th2 priming an early efficient event renders both alleles capable of being inefficiently transcribed; a second, less frequent event occurs that renders one allele more competent, accounting for the differential expression of IL-4 and GFP in different clones.

Reversible expression of tryptases in continuous L138.8A mast cells.

It has been established that mast cells can alter their expression of granule chymases and tryptases in vivo. In vitro, a reversible cytokine regulation has so far only been demonstrated for chymases. We now show a reversible and cytokine-regulated expression of the tryptases MMCP-6 and MMCP-7 and of the chymases MMCP-1, MMCP-2 and MMCP-4 in the continuous murine mast cell line L138.8A. The L138.8A mast cells lacked expression of mRNA for mast cell-specific proteases when cultured in IL-3, and only 49% and 41% of the cells were c-kit+ and FcepsilonRI+, respectively, by flow cytometry. Kit-ligand/stem cell factor induced synthesis of the chymase MMCP-4 and the tryptases MMCP-6 and MMCP-7 and increased the fraction of c-kit+ and FcepsilonRI+ L138.8A cells to >70%. Kit-ligand-induced tryptase expression was suppressed in the presence of IL-3 or IL-9, and reversed after withdrawal of kit-ligand. IL-9 or IL-3/IL-10 promoted the formation of Alcian blue+ granules and increased the fraction of c-kit+ and FcepsilonRI+ L138.8A cells to >90%. IL-9 further induced the expression of the chymases MMCP-1, MMCP-2 and MMCP-4. Thus, the immature mast cell line L138.8A has the capacity to modulate both tryptase and chymase expression and represents the first model system to analyze the molecular regulation of tryptase expression in vitro.

Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment.

The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.

Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell cycle progression.

T helper (Th) lymphocytes, when reactivated, recall expression of those cytokines they had been instructed to express in earlier activations, even in the absence of specific cytokine-inducing factors. In cells that memorize their expression, the cytokine genes are modified by chromatin rearrangement and demethylation, suggesting that they have been somatically imprinted. Here we show, by using inhibitors blocking the cell cycle in various stages, that for the instruction of a Th cell to express interleukin (IL)-4 or IL-10 upon restimulation, entry of the cell into the S phase of the first cell cycle after initial activation is required. Separation of the IL-4 receptor (IL-4R) and T cell antigen receptor (TCR) signals in time, demonstrates that this instruction is dependent on concomitant signaling from both receptors. In Th cells, inhibited to progress into the first S phase after activation, the IL-4R and TCR signals can be memorized for at least 1 d, priming the T cell to become instructed for expression of IL-4 upon restimulation, when entering the S phase after release of the cell cycle block. The requirement of the initial S phase of T cell activation, for instruction of Th cells to express IL-4 or IL-10 upon restimulation points to the decisive role of epigenetic modification of cytokine genes as a molecular correlate of the memory to express particular cytokines.

T1/ST2 expression is enhanced on CD4+ T cells from schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo.

Th cells are categorized into subsets based on the cytokine production of in vitro-differentiated Th populations. For in vivo-differentiated Th subsets, little is known about the heterogeneity of cytokine production in single cells. We recently described a molecule, T1/ST2, that is preferentially expressed on the surface of Th2 cells. Here we combined high-gradient magnetic cell separation with four-color single-cell cytometry to analyze simultaneously three intracellular cytokines and T1/ST2 surface expression on CD4+ cells from lungs containing granulomas induced by Schistosoma mansoni eggs. T1/ST2 was highly up-regulated on CD4+ T cells from hepatic granulomas and granulomatous lungs. T1/ST2+ cells from granulomatous lungs preferentially produced type 2 cytokines ex vivo. In the total CD4+ population, coexpression of type 1 and type 2 cytokines occurred frequently. However, such coproduction was drastically reduced in T1/ST2+ cells compared with T1/ST2- cells. Coexpression of type 1 and type 2 cytokines was also rare in cells simultaneously producing two cytokines of one type. These findings indicate that individual CD4+ T cells in vivo have different levels of commitment to a certain Th phenotype. Coexpression of two type 2 cytokines or production of one type 2 cytokine together with surface expression of T1/ST2 indicate advanced commitment to the Th2 phenotype.

Survival of long-lived plasma cells is independent of antigen.

Recent studies have shown that persistent specific antibody titer is provided by long-lived plasma cells (PC) which constitute a new kind of 'memory-providing cells'. In the present study, we examine the role of antigen for the long-term survival of PC and the maintenance of specific serum antibody titers. Using a novel cytometric technology, to identify and isolate antigen-specific PC, we analyzed long-lived PC of BALB/c mice, during their development (between day 1 and 10) after secondary immunization with ovalbumin (OVA) and in the phase of the established immune reaction. Most if not all OVA-specific PC were generated within a few days after immunization. Within approximately 3 weeks, they matured, as indicated by down-regulation of expression of MHC class II. These PC are long lived and located in spleen and bone marrow. Upon adoptive transfer, OVA-specific PC from bone marrow, but not memory B cells, conferred specific and long-lasting antibody titers to antigen-free IgH syngeneic recipients. In response to antigenic challenge, new OVA-specific antibody-secreting cells were generated from transferred memory B cells. Antibody secretion by long-lived PC was not affected. Our results confirm that persistent antibody titers are provided by long-lived PC, independent of memory B cells and demonstrate that this humoral memory is inert to antigen.

Analysis and sorting of T cells according to cytokine expression.

Functional distinct populations of T helper cells can be defined according to the expression of cytokines. A remarkable diversity of cytokine expression has been demonstrated in single T cells even from clonal populations and up to now no stable surface markers have been described which on the single-cell level directly correlate with the secretion of a certain cytokine. Since cytokines are the major parameters of T cell effector function we have developed strategies which now allow to separate cells according to the specific cytokines they secrete. The "affinity matrix technology"--secreted molecules are relocated to the cell surface by an artificially created antibody matrix--allows to isolate cells according to a distinct secreted product. In addition to this universal, but laborious technology, we could demonstrate by high-sensitivity immunofluorescence that IFN-gamma and IL-10 but not IL-2 and IL-4 are specifically expressed in low copy number on the surface of cells secreting these cytokines. Surface IFN-gamma and IL-10 are the first unambiguous surface markers for pro-inflammatory IFN-gamma-secreting Th 0/1 cells and IL-10-producing anti-inflammatory Th2/3 cells. We have used purified cytokine-secreting T cells to study in individual T cells the sequential production of IL-2, IFN-gamma, and IL-10, the stability of IFN-gamma expression and the selective homing of IFN-gamma-producing cells into inflamed tissues.

Commitment of individual Th1-like lymphocytes to expression of IFN-gamma versus IL-4 and IL-10: selective induction of IL-10 by sequential stimulation of naive Th cells with IL-12 and IL-4.

Commitment of Th lymphocytes to the Th1 phenotype, as characterized by the expression of the major proinflammatory cytokine IFN-gamma, may be critically involved in the establishment of chronic inflammation and inflammatory autoimmune disease. To date, it has been shown that in IL-12-stimulated murine Th cell lines containing a major fraction of Th1 cells, Th2 cells can be induced by IL-4 until about 2 wk after initial activation, but not later. Here we analyze, based on the magnetic isolation of viable Th1 cells according to their specific expression of IFN-gamma, the cytokine commitment of individual Th1 cells. After activation of naive Th cells with Ag and IL-12 for up to 5 wk, isolated IFN-gamma-producing cells were restimulated with Ag and IL-4. Within the first 3 to 4 wk of IL-12 stimulation, some IFN-gamma+ cells stopped expression of IFN-gamma when restimulated with IL-4. However, within only 1 to 2 wk of IL-12 stimulation, few IFN-gamma+ cells could be converted to produce IL-4. Others continued to express IFN-gamma and thus were already committed to a proinflammatory, Th1-like phenotype. Surprisingly, within 3 wk of IL-12 stimulation, many of the IFN-gamma-producing cells responded to IL-4 restimulation by expression of IL-10, but neither IFN-gamma nor IL-4, i.e., by conversion to a suppressive, anti-inflammatory phenotype.

T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function.

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-gamma. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo following adoptive transfer of Th2 cells.

Sequential production of IL-2, IFN-gamma and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes.

Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-gamma and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-gamma and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-gamma, separated according to specific surface-associated IFN-gamma as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-gamma expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-gamma-inducing monokines and limiting the pro-inflammatory effects of IFN-gamma.

P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues.

When activated, T helper cells differentiate into one of two subsets, Th1 and Th2, characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms involved in protection and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation. Apart from differences in the repertoire of cytokines, no phenotypic attributes are established that distinguish the two subsets. Here we show that Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin. Moreover, only Th1 cells can efficiently enter inflamed sites in Th1-dominated models, such as sensitized skin or arthritic joints, but not in a Th2-dominated allergic response. Immigration of Th1 cells into inflamed skin can be blocked by antibodies against P- and E-selectin. These results provide evidence for adhesion mechanisms to distinguish between the two T helper subsets and mediate their differential trafficking. They indicate that selective recruitment is an additional level of regulation for both effector function profile and character of a local immune response.