Jane C. Goodall

Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis

OBJECTIVEnTo analyze the frequency, surface phenotype, and cytokine secretion of CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with both healthy control subjects and patients with rheumatoid arthritis (RA).nnnMETHODSnEight-color flow cytometry was used to analyze the surface phenotype and cytokine production of PBMCs from 20 patients with AS, 12 patients with RA, and 16 healthy control subjects, following stimulation ex vivo with phorbol myristate acetate and ionomycin for 5 hours. Secretion of interleukin-17 (IL-17) by PBMCs was measured by enzyme-linked immunosorbent assay, following stimulation with anti-CD3/CD28 for 4 days.nnnRESULTSnThe percentages of IL-17-positive CD4+ T cells and IL-22-positive CD4+ T cells were increased in the PBMCs of both patients with AS and patients with RA compared with healthy control subjects, whereas there were no differences in the percentages of interferon-gamma (IFNgamma)-positive or IL-10-positive CD4+ T cells. Likewise, concentrations of IL-17 in supernatants from patients with AS were significantly higher compared with those from healthy control subjects. In patients with RA, the concentrations of IL-17 were increased but not significantly. There was a correlation between the percentages of IL-17-positive CD4+ T cells detected in PBMCs and the amounts of IL-17 in culture supernatants (r=0.414, P=0.0034). All IL-17-producing cells were CD4+CD45RO+; most expressed both CCR6 and CCR4, but only 50% expressed the IL-23 receptor (IL-23R). Nevertheless, there was a positive relationship between the percentage of IL-23R-positive CD4+ T cells and the frequency of IL-17-positive CD4+ T cells or IL-22-positive CD4+ T cells (r=0.57, P<0.0001 and r=0.46, P=0.001, respectively). A significant proportion of cells that produced IL-17 also produced IL-22 and IFNgamma, but none produced IL-10.nnnCONCLUSIONnThe frequencies of IL-17-positive and IL-22-positive CD4+ T cells were increased in PBMCs from patients with AS and patients with RA, resulting in secretion of higher quantities of IL-17 by PBMCs following stimulation. These data support the hypothesis that Th17 cells, particularly when present in excess of IL-10-producing cells, are involved in the pathogenesis of inflammatory arthritis.

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.

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

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

Despite substantial advances in our understanding of CD4+CD25+ regulatory Tu2004cells, a possible equivalent regulatory subset within the CD8+ Tu2004cell population has received less attention. We now describe novel human CD8+/TCRαβ+ Tu2004cells 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 classu2004I‐restricted fashion, by proliferation and production of IL‐4, IL‐5, IL‐13 and TGFβ1, but not IFN‐γ. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+CD25+ regulatory Tu2004cells, including cytotoxic Tu2004lymphocyte‐associated antigen‐4 (CTLA‐4) and Foxp3. They suppressed IFN‐γ production and proliferation by CD4+ Tu2004cells 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 Tu2004cells. 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 Tu2004cells that can be cloned and expanded in vitro may have therapeutic applications.

The Recognition of HLA-B27 by Human CD4+ T Lymphocytes

HLA-B27 transgenic animal models suggest a role for CD4+ T lymphocytes in the pathogenesis of the spondyloarthropathies, and murine studies have raised the possibility that unusual forms of B27 may be involved in disease. We demonstrate that CD4+ T cells capable of recognizing B27 can be isolated from humans by coculture with the MHC class II-negative cell line T2 transfected with B27. These CD4+ T cells recognize a panel of B27-transfected cell lines that are defective in Ag-processing pathways, but not the nontransfected parental cell lines, in a CD4-dependent fashion. Inhibition of responses by the MHC class I-specific mAb w6/32 and the B27 binding mAb ME1 implicates the recognition of a form of B27 recognized by both of these Abs. We suggest that B27-reactive CD4+ T cells may be pathogenic in spondyloarthropathies, particularly if factors such as infection influence expression of abnormal forms of B27.

Identification of Chlamydia trachomatis antigens recognized by human CD4+ T lymphocytes by screening an expression library.

Identification of the immunogenic proteins that induce Chlamydia trachomatis (CT)‐specific T cell responses is crucial to the development of protective vaccines and understanding the mechanisms of chlamydia‐induced pathology. To characterize the targets of the human T cell response we have used chlamydia‐reactive human T cell clones as cellular probes to screen a CT genomic library expressed in Escherichia coli using peripheral blood mononuclear cells to present antigens. The library was screened with three chlamydia‐reactive T cell clones of unknown specificity and three novel stimulatory chlamydia antigens were identified. These E. coli recombinants were shown to express the chlamydia proteins, enolase, pmpD and CT579. Enolase and pmpD proteins were purified and shown to induce the proliferation of synovial fluid mononuclear cells isolated from the knee joints of patients suffering from chlamydia‐associated reactive arthritis. We suggest that these stimulatory antigens are common targets of the T cell response in this group of patients. A greater understanding of T cell‐mediated immunity in uncomplicated CT infection, and in patients with CT‐induced chronic inflammatory disease (trachoma, salpingitis, arthritis) may identify the principal immune responses associated with immunopathology.

T lymphocyte lines isolated from atheromatous plaque contain cells capable of responding to Chlamydia antigens

Chlamydia pneumoniae infection is associated with atherosclerosis and the organism has been identified in arterial lesions. To determine whether T lymphocyte‐mediated immune responses to Chlamydia antigens within plaque could contribute to pathogenesis, we have derived T cell lines from atherosclerotic plaques of 32 patients. Culture with IL‐2 alone proved insufficient for cellular activation and expansion, but additional stimulation with phytohaemagglutinin (PHA) or recall antigens allowed consistent establishment of T cell lines. Furthermore, in cultures of approx. 500 tissue fragments, Chlamydia organisms proved as effective as other recall antigens in producing outgrowth of arterial T cells (20–25% wells produced T cell lines). Testing the antigen responsiveness of T cell lines showed that those derived using Chlamydia organisms were more likely to respond to Chlamydia (5/29+) than those isolated using other stimuli (6/69+ for PHA; 5/57+ for PPD and tetanus toxoid (TT)). However, lines responsive to each of the recall antigens were observed. Using recombinant Chlamydia antigens, some Chlamydia‐specific T cell lines were shown to respond to OMP2 and/or hsp60. Those recognizing Chlamydia hsp60 did not cross‐react with human hsp60, but human hsp60‐responsive lines were also observed. Thus, atherosclerotic plaque tissue contains a variety of memory T lymphocytes, and amongst these are cells capable of recognizing Chlamydia antigens. In a C. pneumoniae‐infected plaque, such T cells may be activated by local antigen and could contribute to the inflammatory process in the arterial wall through CD40 ligand expression and cytokine secretion.

Effects of Chlamydia trachomatis infection on the expression of natural killer (NK) cell ligands and susceptibility to NK cell lysis

Natural killer (NK) cells are an important component of the immediate immune response to infections, including infection by intracellular bacteria. We have investigated recognition of Chlamydia trachomatis (CT) by NK cells and show that these cells are activated to produce interferon (IFN)‐γ when peripheral blood mononuclear cells (PBMC) are stimulated with CT organisms. Furthermore, infection of epithelial cell lines with CT renders them susceptible to lysis by human NK cells. Susceptibility was observed 18–24 h following infection and required protein synthesis by the infecting chlamydiae, but not by the host cell; heat or UV inactivated chlamydiae did not induce susceptibility to NK cell lysis. CT infection was also shown to decrease the expression of classical and non‐classical major histocompatibility complex (MHC) molecules on infected cells, thus allowing recognition by NK cells when combined with an activating signal. A candidate activating signal is MICA/B, which was shown to be expressed constitutively on epithelial cells.

Recognition of the 60 kilodalton cysteine-rich outer membrane protein OMP2 by CD4(+) T cells from humans infected with Chlamydia trachomatis.

T cell‐mediated immunity is important in the control of chlamydia infection but chlamydia‐specific T cells are also implicated in the inflammation and tissue damage which characterize chlamydia associated diseases. To investigate target antigens of the T cell‐mediated immune response to chlamydia infection, Chlamydia trachomatis‐specific CD4+ T cell clones were isolated from a patient with chlamydia‐induced reactive arthritis. T cell immunoblotting indicated that an antigen of ∼60 kilodaltons molecular mass was recognized, and recombinant 60 kilodalton cysteine‐rich outer membrane 2 (OMP2) proved to be stimulatory. By using deletion constructs and synthetic peptides an epitope presented by HLA‐DRB1*0401 was defined and proved to contain the nonamer peptide within the OMP2 sequence predicted to have the greatest binding affinity for DRB1*0401 The sequence of the epitope is conserved in all C. trachomatis strains but not in C. pneumoniae. Investigation of patients with acute urethritis and additional patients with sexually acquired reactive arthritis showed that OMP2‐reactive T cells were readily detectable in peripheral blood and synovial fluid. Thus OMP2 is a target antigen of the T cell‐mediated immune response to CT infection.

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.