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.

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.

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.

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.

Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules.

Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles.

IRE1α mediates PKR activation in response to Chlamydia trachomatis infection.

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-ß production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.

The recognition of abnormal forms of HLA-B27 by CD4+ T cells.

The MHC class I molecule, HLA-B27 can be expressed as a number of non-conventional forms, in addition to conventional HLA-B27 heterodimers presenting peptide. This has lead to new avenues of research to explain the association of this molecule with SpA. Surprisingly, HLA-B27 transgenic animal models implicated CD4+ T cells, which conventionally interact with MHC class II molecules, not MHC class I molecules, in the pathogenesis of SpA. One hypothesis to explain these finding is that non-conventional forms of HLA-B27, specifically HLA-B27 homodimers, might mimic MHC class II molecules and be recognised by CD4+ T cells. We investigated whether CD4+ T cells from AS patients can interact with HLA-B27, discovering that indeed CD4+ T cells can interact with various forms of HLA-B27. Here we discuss how such interactions between HLA-B27 and CD4+ T cells could occur in vivo and potential contributions of such interactions to the pathogenesis of SpA.

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility.

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10(-8) in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10(-6) overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Frequency and phenotype of T helper 17 cells in peripheral blood and synovial fluid of patients with reactive arthritis.

OBJECTIVE: To analyze the frequency and phenotype of peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cell (SFMC) T helper (Th)17 cells in reactive arthritis (ReA). METHODS: T cell surface phenotype and cytokine production were measured following stimulation, using 8-color flow cytometry. RESULTS: The percentages of interleukin 17 (IL-17)-positive CD4+ T cells were increased in SFMC of patients with ReA compared with PBMC. All IL-17+ cells were CD4+CD45RO+; in SFMC most expressed CCR6, but only 50% expressed CCR4. IL-17+ cells sometimes coexpressed IL-22 and/or interferon-γ, but not IL-10. CONCLUSION: These data support the hypothesis that Th17 cells are involved in ReA pathogenesis.

Vaccination of children in low-resource countries against Shigella is unlikely to present an undue risk of reactive arthritis.

Shigellosis is a major cause of morbidity and mortality among children in low-resource countries. Promising vaccine strategies in development include genetically attenuated Shigella, killed whole cell vaccines, subcellular vaccines, and O-polysaccharide-protein conjugates. There is a concern that Shigella vaccines could either induce reactive arthritis or could prime vaccinees for arthritis after a subsequent exposure to the pathogen because shigellosis is associated with reactive arthritis, especially in patients expressing the HLA B27 histocompatibility antigen. Our understanding of the pathogenesis of reactive arthritis is incomplete, and even surrogate biomarkers of bacterial arthritogenic activity have not yet been identified. Nonetheless, all of the Shigella vaccine strategies currently in development are designed to limit inflammation and intracellular antigen persistence that could trigger arthritogenic sequelae. The relatively low occurrence of the HLA B27 phenotype in most Shigella endemic areas, and the rarity of reported reactive arthritis in these populations, suggests that vaccination with attenuated, killed, or subcellular vaccines may not increase the background incidence of arthritic sequelae. More importantly, incidence rates of shigellosis in children living in low-resource countries suggest that, during maturation, the entire pediatric population may be infected with Shigella-possibly with devastating consequences. Therefore, clinical trials of candidate Shigella vaccines should be pursued aggressively in the developing world, beginning with a Phase 1 in HLA B27-negative volunteers, but proceeding to Phase 2 and Phase 3 in unscreened volunteers. Post-vaccination monitoring for possible reactive arthritis should be included in all clinical protocols.

CD27 expression discriminates between regulatory and non-regulatory cells after expansion of human peripheral blood CD4+ CD25+ cells.

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25- cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25(high) and CD25(intermediate) CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T-cell populations such as those found in joints.

Autoreactive human peripheral blood CD8+ T cells with a regulatory phenotype and function.

Despite substantial advances in our understanding of CD4+ CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCR alphabeta+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS-activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I-restricted fashion, by proliferation and production of IL-4, IL-5, IL-13 and TGFbeta1, but not IFN-gamma. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+ CD25+ regulatory T cells, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Foxp3. They suppressed IFN-gamma production and proliferation by CD4+ T cells in vitro in a cell contact-dependent manner, which could be blocked using a CTLA-4-specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up-regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+ CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft-versus-host disease. Likewise, human HLA-restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.

Chlamydia trachomatis-specific human CD8+ T cells show two patterns of antigen recognition.

Chlamydia trachomatis is an intracellular gram-negative bacteria which causes several clinically important diseases. T-cell-mediated immunity and the production of gamma interferon (IFN-gamma) are known to be essential for the clearance of the bacteria in vivo. Here we have investigated CD8(+)-T-cell responses to C. trachomatis in patients with previous episodes of chlamydia infection. To isolate C. trachomatis-specific CD8(+)-T-cell lines, dendritic cells (DC) were infected with C. trachomatis and cocultured with purified CD8(+) T cells to generate C. trachomatis-specific CD8(+)-T-cell lines which were then cloned. Two patterns of recognition of C. trachomatis-infected cells by CD8(+)-T-cell clones were identified. In the first, C. trachomatis antigens were recognized in association with classical class I HLA antigens, and responses were inhibited by class I HLA-specific monoclonal antibodies. The second set of clones was unrestricted by classical HLA class I, and further studies showed that CD1 molecules were also not the restriction element for those clones. Both types of clones produced IFN-gamma in response to C. trachomatis and were able to lyse C. trachomatis-infected target cells. However, unrestricted clones recognized C. trachomatis-infected cells at much earlier time points postinfection than HLA-restricted clones. Coculture of C. trachomatis-infected DC with the C. trachomatis-specific clones induced DC activation and a rapid enhancement of interleukin-12 (IL-12) production. Early production of IL-12 during C. trachomatis infection, facilitated by unrestricted CD8(+)-T-cell clones, may be important in ensuring a subsequent Th1 T-cell-mediated response by classical major histocompatibility complex-restricted CD4(+) and CD8(+) T cells.

Major histocompatibility complex class I-restricted alloreactive CD4+ T cells.

Although it is well established that CD4+ T cells generally recognize major histocompatibility complex (MHC) class II molecules, MHC class I-reactive CD4+ T cells have occasionally been reported. Here we describe the isolation and characterization of six MHC class I-reactive CD4+ T-cell lines, obtained by co-culture of CD4+ peripheral blood T cells with the MHC class II-negative, transporter associated with antigen processing (TAP)-negative cell line, T2, transfected with human leucocyte antigen (HLA)-B27. Responses were inhibited by the MHC class I-specific monoclonal antibody (mAb), W6/32, demonstrating the direct recognition of MHC class I molecules. In four cases, the restriction element was positively identified as HLA-A2, as responses by these clones were completely inhibited by MA2.1, an HLA-A2-specific mAb. Interestingly, three of the CD4+ T-cell lines only responded to cells expressing HLA-B27, irrespective of their restricting allele, implicating HLA-B27 as a possible source of peptides presented by the stimulatory MHC class I alleles. In addition, these CD4+ MHC class I alloreactive T-cell lines could recognize TAP-deficient cells and therefore may have particular clinical relevance to situations where the expression of TAP molecules is decreased, such as viral infection and transformation of cells.

T cell recognition of a highly conserved epitope in heat shock protein 60: self-tolerance maintained by TCR distinguishing between asparagine and aspartic acid.

Cross-reactive T cell recognition of self-heat shock proteins (hsp) has been ascribed a regulatory role in inflammatory arthritis in both animal models and human disease. The previous work implies that a repertoire for epitopes in self-hsp60 should exist in normal subjects. Accordingly, we sought to generate self-hsp60-reactive T cell clones from a healthy individual using a highly purified preparation of recombinant human (Hu) hsp60. Epitope mapping using synthetic peptides and truncated constructs indicated that the T cell clones obtained actually recognized hsp60 derived from Escherichia coli. Using a series of alanine-substituted peptides and additional appropriate synthetic peptides, it was demonstrated that the clones maintain self-tolerance because of their sensitivity to an asparagine to aspartic acid sequence difference between E. coli and HuHsp60 in the epitope-containing peptide. In addition, despite substantial conservation of sequence, the homologous peptide from HuHsp60 did not compete with the E. coli-derived peptide for recognition or antagonize responses by acting as an altered peptide ligand. The results suggest that, even when the immune system targets a highly conserved epitope in bacterial hsp60, self-tolerance is maintained. Furthermore, the finding that T cell clones specific for minor contaminant proteins in HuHsp60 preparations can readily be isolated raises the possibility that the HuHsp60 facilitates presentation of antigenic proteins to the immune system.