TSLP production by dendritic cells is modulated by IL-1ß and components of the endoplasmic reticulum stress response.

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of TH 2-cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and ß-glucans requires dectin-1, Syk, NF-κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL-1ß, but not TNF-α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol-requiring transmembrane kinase/endonuclease 1 and protein kinase R-like ER kinase, which are activated by dectin-1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin-1, the IL-1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC-controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue.

Relationship of CD146 expression to secretion of interleukin (IL)-17, IL-22 and interferon-γ by CD4(+) T cells in patients with inflammatory arthritis.

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4(+) T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146(+) CD4(+) and interleukin (IL)-17(+) CD4(+) T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146(+) CD4(+) T cells were enriched for secretion of IL-17 [alone or with IL-22 or interferon (IFN)-γ] and for some putative Th17-associated surface markers (CD161 and CCR6), but not others (CD26 and IL-23 receptor). CD4(+) T cells producing IL-22 or IFN-γ without IL-17 were also present in the CD146(+) subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4(+) helper T cells.

Comparison of two referral strategies for diagnosis of axial spondyloarthritis: the Recognising and Diagnosing Ankylosing Spondylitis Reliably (RADAR) study.

OBJECTIVE: To determine which of two referral strategies, when used by referring physicians for patients with chronic back pain (CBP), is superior for diagnosing axial spondyloarthritis (SpA) by rheumatologists across several countries. METHODS: Primary care referral sites in 16 countries were randomised (1 : 1) to refer patients with CBP lasting >3 months and onset before age 45 years to a rheumatologist using either strategy 1 (any of inflammatory back pain (IBP), HLA-B27 or sacroiliitis on imaging) or strategy 2 (two of the following: IBP, HLA-B27, sacroiliitis, family history of axial SpA, good response to non-steroidal anti-inflammatory drugs, extra-articular manifestations). The rheumatologist established the diagnosis. The primary analysis compared the proportion of patients diagnosed with definite axial SpA by referral strategy. RESULTS: Patients (N=1072) were referred by 278 sites to 64 rheumatologists: 504 patients by strategy 1 and 568 patients by strategy 2. Axial SpA was diagnosed in 35.6% and 39.8% of patients referred by these respective strategies (between-group difference 4.40%; 95% CI -7.09% to 15.89%; p=0.447). IBP was the most frequently used referral criterion (94.7% of cases), showing high concordance (85.4%) with rheumatologists' assessments, and having sensitivity and a negative predictive value of >85% but a positive predictive value and specificity of <50%. Combining IBP with other criteria (eg, sacroiliitis, HLA-B27) increased the likelihood for diagnosing axial SpA. CONCLUSIONS: A referral strategy based on three criteria leads to a diagnosis of axial SpA in approximately 35% of patients with CBP and is applicable across countries and geographical locales with presumably different levels of expertise in axial SpA.

Endoplasmic reticulum stress-induced transcription factor, CHOP, is crucial for dendritic cell IL-23 expression.

The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.

PI3K p110delta regulates T-cell cytokine production during primary and secondary immune responses in mice and humans.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.

Cross-reactivity of self-HLA-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes for allo-HLA determinants.

Epstein-Barr (EB) virus-specific cytotoxic T cells, prepared from virus-immune donors by reactivation in vitro and maintained thereafter as IL-2-dependent T cell lines, have been tested against large panels of EB virus-transformed lymphoblastoid cell lines of known HLA type. Whilst the pattern of lysis of the majority of targets was always consistent with HLA-A and HLA-B antigen restriction of effector function, in several cases it was noticed that certain HLA-mismatched targets were also reproducibly lysed. When this "anomalous" lysis was investigated in detail, it was found to be directed against allodeterminants on class I HLA antigens; thus, mitogen-stimulated as well as EB virus-transformed lymphoblasts from the relevant target cell donors were sensitive to the killing, and in each case the lysis could be specifically blocked by monoclonal antibodies to class I HLA antigens. In one example the target for this alloreactive lysis could be identified as a single serologically defined antigen, HLA-Bw57, while in another example lysis was directed against a "public" epitope common to HLA-Bw35, -Bw62, and a subset of -B12 antigens. Both cold target inhibition experiments and limiting dilution analysis strongly suggested that this alloreactive lysis was being mediated by the same effector T cells that recognize EB viral antigens in the context of self-HLA. This is the first demonstration in man that alloreactive responses can be derived from within the antigen-specific, self MHC-restricted T cell repertoire.

Epstein-Barr virus-specific cytotoxic T lymphocytes as probes of HLA polymorphism. Heterogeneity of T cell-restricting determinants associated with the serologically defined HLA-A2 antigen.

Epstein-Barr (EB) virus-specific effector T cell lines were established from nine virus-immune donors positive for the serologically defined HLA-A2 antigen; of these, four lines contained a demonstrable A2-restricted cytotoxic component. When these four effector populations were each tested on the same panel of EB virus-transformed lines from 20 HLA-A2-positive individuals, 16 of the target cell lines were consistently killed at levels above 25% of the relevant autologous cell lysis. Cytotoxicity appeared to be mediated through a restricting determinant associated with the 'common A2' antigen that these lines shared; indeed the lysis could be specifically blocked by high concentrations of an HLA-A2-specific monoclonal antibody. In contrast, 4 out of 20 target cell lines were not killed by HLA-A2-restricted effector cells, even though they did express the serologically defined A2 antigen and were found in other tests to be susceptible to EB virus-specific cytolysis restricted through other HLA-A or -B antigens on their surface. These results suggest that EB virus-specific cytotoxic T cells can distinguish between serologically identical HLA-A2 molecules via the heterogeneity of their T cell-restricting determinants. Data from one of the effector cell populations further suggested that a serologically defined cross-reaction between the otherwise distinct HLA-A2 and -Bw57 antigens might also be reflected in a cross-reactivity of T cell-restricting determinants.

Recognition of a mycobacteria-specific epitope in the 65-kD heat-shock protein by synovial fluid-derived T cell clones.

Adjuvant arthritis in rats is induced by a T cell clone specific for amino acids 180-188 of the mycobacterial 65-kD heat-shock protein, and synovial T cell responses to this same Ag have been noted in human arthritis. We have isolated 65-kD Ag-specific T cell clones from synovial fluid mononuclear cells of a patient with acute arthritis, which, unlike the corresponding PBMC, showed a marked proliferative response to the 65-kD Ag. Using synthetic peptides corresponding to the whole sequence of the 65-kD Ag, all the clones were shown to recognize an epitope present in the first NH2-terminal peptide (amino acids 1-15), with no response to the adjacent peptide (amino acids 6-22) or to any other peptide. The complete dominance of this epitope in the response to the 65-kD Ag was shown by documenting responses to the peptide in PBMC obtained after recovery from the arthritis. This epitope, like that recognized by the rat arthritogenic T cell clone, is in a portion of the 65-kD sequence that is not conserved between bacteria and eukaryotes, so that in this case, joint inflammation could not be attributed to bacteria-induced T cell clones cross-reacting with the self 65-kD Ag.

Regulatory IL4+CD8+ T cells in patients with ankylosing spondylitis and healthy controls.

OBJECTIVES: Interleukin (IL)4+CD8+ regulatory T cells (Treg) obtained from peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) by coculture with autologous dendritic cells (DC) have been previously described. In the present work, the proportions of IL4+CD8+ T cells in PB from patients with AS and controls are examined; in addition the conditions required for the generation of IL4+CD8+ Treg cells and their frequency in T cell lines from patients with AS and controls are investigated. METHODS: CD8+ T cells were either stimulated non-specifically ex vivo and intracellular cytokines examined, or cocultured with DC and other stimuli, for 2 weeks. The resulting lines were analysed for cytokine expression. Clones derived from these lines were tested for regulatory function. RESULTS: PBMCs from patients with AS and from human leukocyte antigen (HLA)-B27+ healthy controls contained a higher frequency of IL4+CD8+ T cells than those from HLA-B27- controls. Likewise, CD8+ T cell lines obtained by coculture with DC contained a higher ratio of IL4+ to interferon (IFN)gamma+ cells when obtained from patients with AS or HLA-B27+ controls. T cell clones obtained from these lines showed regulatory activity. Outgrowth of IL4+ CD8+ T cells required contact with DC, but not maturation with lipopolysaccharide (LPS); allogeneic DC were also effective. Coculture with lymphoblastoid cells, or anti-CD3/CD28 microbeads, produced only expansion of IFN gamma-producing CD8+ T cells. CONCLUSIONS: The higher proportion of CD8+ cells which can produce IL4 in PB and in expanded CD8+ T cell lines suggests an altered pattern of CD8+ T cell differentiation in AS and in HLA-B27+ healthy individuals. This predisposition to generate IL4+CD8+ T cells may play a role in pathogenesis of spondyloarthritis.

Dendritic cell: T-cell interactions in spondyloarthritis.

The discovery of the association between spondyloarthritis (SpA) and HLA-B27 inevitably turned the spotlight on T-lymphocytes as the cells which recognize peptide antigens within the binding groove of the HLA-B27 molecule and then carry out effector functions. These include cytolysis, cytokine and chemokine production and activation of other effector cells, such as those which could destroy joints or drive new bone formation. In this view the T-cell assumed the role of "director" of the immune response and therefore, in inflammatory diseases such as SpA, of immuno-pathology. The important research questions under this paradigm were the identity of the peptides recognized by T-cells in disease, including whether they were derived from self proteins or from micro-organisms, the influence of HLA-B27 in selecting antigenic peptides for recognition by T-cells, the T-cell receptors used in recognition and the effector programmes which the T-cells initiated. Whilst these questions continue to be explored-many have not yet been answered-attention has shifted to a new "master regulator" of the immune response, namely the dendritic cell and the possibility that the genetic influences which contribute to susceptibility to SpA do so at the level of the dendritic cell (DC).

Dendritic Cell-Derived TSLP Negatively Regulates HIF-1α and IL-1ß During Dectin-1 Signaling.

Thymic stromal lymphopoietin (TSLP) is a functionally pleotropic cytokine important in immune regulation, and TSLP dysregulation is associated with numerous diseases. TSLP is produced by many cell types, but has predominantly been characterized as a secreted factor from epithelial cells which activates dendritic cells (DC) that subsequently prime T helper (TH) 2 immunity. However, DC themselves make significant amounts of TSLP in response to microbial products, but the functional role of DC-derived TSLP remains unclear. We show that TSLPR signaling negatively regulates IL-1ß production during dectin-1 stimulation of human DC. This regulatory mechanism functions by dampening Syk phosphorylation and is mediated via NADPH oxidase-derived ROS, HIF-1α and pro-IL-1ß expression. Considering the profound effect TSLPR signaling has on the metabolic status and the secretome of dectin-1 stimulated DC, these data suggest that autocrine TSLPR signaling could have a fundamental role in modulating immunological effector responses at sites removed from epithelial cell production of TSLP.

Human leukocyte antigen class I-restricted immunosuppression by human CD8+ regulatory T cells requires CTLA-4-mediated interaction with dendritic cells.

We previously reported autoreactive CD8(+) regulatory T cells (Tregs) that were expanded and cloned from human peripheral blood by coculture with autologous dendritic cells (DC). Here we demonstrate that these CD8(+) Tregs require human leukocyte antigen (HLA)-class I restricted activation and then mediate cell-contact-dependent suppression of CD4(+) T cells. CD8(+) Tregs interacted with DC to suppress T-cell responses but DC were not irreversibly altered by this interaction because they could subsequently stimulate CD4(+) T cells normally. The ability of DC to form conjugates with CD4(+) T cells was reduced in the presence of CD8(+) Tregs. Suppression was blocked by Abs to CD80 and CTLA-4, implicating CTLA-4:CD80 interactions in the function of CD8(+) Tregs. CD8(+) Tregs rapidly express very high levels of surface CTLA-4 following activation compared with conventional T cells. Related to this, the expression of TRAT1 mRNA (T-cell receptor interacting molecule, or TRIM) was highly upregulated in microarray analysis of CD8(+) Tregs compared with conventional cytotoxic or nonregulatory CD8(+) T cells. TRIM acts to chaperone CTLA-4 transport to the cell surface; this function would be required to account for the phenotypic and functional properties of CD8(+) Tregs.

Cytokines in arthritis--the 'big numbers' move centre stage.

More than 20 yrs ago, T-helper lymphocytes were divided into Th1 and Th2 subsets on the basis of their cytokine production. The pro-inflammatory Th1 subset was considered predominant in inflammatory arthritis, but evidence for this notion was incomplete, and some called into question the role of helper T cells. The identification of a novel T cell subset, Th17 cells, which appears to be critical for several forms of autoimmune inflammation, including arthritis, requires a reconsideration of arthritis pathogenesis and the role of T cells. This review deals with several of the newly described ('big number') cytokines which are involved in the differentiation and action of Th17 cells, and pays particular attention to the pathogenesis of spondyloarthritis because of the implication of the same cytokine networks in psoriasis and inflammatory bowel disease. The role of dendritic cells as coordinators of T cell differentiation in response to pathogen-derived signals in also emphasized.

Increased IL-4+ CD8+ T cells in peripheral blood and autoreactive CD8+ T cell lines of patients with inflammatory arthritis.

OBJECTIVE: To measure the frequencies of IL-4+ CD8+ T cells from patients with AS and RA, and to assess their clinical relevance and properties. METHODS: Peripheral blood (PB) and clinical data were obtained from 37 AS, 36 RA patients and 37 healthy controls. We also generated IL-4-producing CD8+ T cell lines and clones by co-culture with autologous dendritic cells. Using flow cytometry, we evaluated intracellular cytokine expression by T cells following stimulation with PMA and calcium ionophore. The phenotype and ability of the IL-4-producing CD8+ T cell clones to suppress IFN-gamma production were examined. RESULTS: The percentages of IL-4+ CD8+ T cells were higher in PB of patients with AS and RA than controls (medians 0.90 and 0.84% vs 0.30%). In RA, patients with active inflammation had an increased percentage of IL-4+ CD8+ T cells. Higher frequencies of IL-4+ CD8+ T cells were also found in CD8+ T cell lines established from patients with arthritis. Interestingly, most IL-4+ CD8+ T cells produced TNF-alpha. Cloning the CD8+ T cell lines yielded more IL-4-producing clones from AS (23%) and RA patients (14%) than from controls (7%). The ability to suppress IFN-gamma production was observed in 56% (AS) and 85% (RA) of IL-4-producing clones. Suppressive IL-4+ CD8+ T cell clones from RA patients showed a similar regulatory phenotype to the clones previously isolated from AS patients. CONCLUSIONS: Expansion of IL-4+ CD8+ T cells, which may include precursors of a regulatory CD8+ T cell subset, may represent a general response to chronic joint inflammation.

Analysis of antigen reactive T-cells.

Because antigen-specific cells are the central coordinators of the immune response to infectious organisms, and the principal effector cells in autoimmune disease, there are many circumstances in which investigators may wish to examine the T-cell responses to particular antigens. This chapter outlines techniques for assessing the responses of polyclonal populations of T-lymphocytes by measuring a variety of outputs each of which gives different kinds of information about the response. The outputs discussed are proliferation and cytokine production, with methods for measuring cytokine secretion by the whole population together with techniques for making an estimate of the numbers of cells producing a cytokine in response to antigen, and examining the phenotype of the responsive cells. In many cases detailed information about responses to particular antigens requires the isolation and characterization of antigen-responsive T-cell clones, and this is also described together with methods of identifying unknown antigens by screening recombinant expression libraries. Lastly, because the techniques differ in many respects, methods for isolating antigen-specific CD8+ T-cells, particularly those which recognize bacteria, are also included.

Recent advances in understanding spondyloarthritis.

This review is concerned with a number of recent publications that contribute to current thinking on the pathogenesis of spondyloarthritis. The areas covered include the lymphocyte population in the enthesis, which is thought to drive enthesitis, and hence clinical manifestations. The debate on how HLA-B27 is implicated in inflammation is also considered, together with recent and contradictory evidence on the effects of the peptide-trimming enzyme ERAP1 on B27 expression and hence susceptibility to spondylitis. Lastly, a recent report on the role of the gut microbiome in an important model of spondyloarthritis is considered.

Th17 cell responses in spondyloarthritis.

Targeting IL-17 has become an important option in the current treatment of spondyloarthritis (SpA). To place this therapeutic advancement in context, we review the discovery and properties of this cytokine, noting those which predispose to inflammation and led to it being considered as an attractive target for the treatment of arthritis, especially SpA. The processes that regulate the differentiation of IL-17-producing cells, particularly Th17 CD4+ T cells, have been investigated thoroughly, including the role of IL-23, as these point to additional potential therapies as alternatives to direct IL-17 blockade. IL-17 is a critical cytokine in combatting infection, particularly caused by fungi, but it also has an important role in maintaining epithelial barrier functions, especially in the gut. Both these functions help predict possible adverse effects of IL-17 blockade. Finally, we review the current evidence for the use of IL-17 blockade in various forms of SpA and briefly speculate on future developments.

ß-Glucan Size Controls Dectin-1-Mediated Immune Responses in Human Dendritic Cells by Regulating IL-1ß Production.

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes ß-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. ß-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate ß-glucans are thought to be poor activators of innate immunity. We show that ß-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large ß-glucan-stimulated DC generate significantly more IL-1ß, IL-6, and IL-23 compared to those stimulated with the smaller ß-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to ß-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1ß production determined by ß-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that ß-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.