J. S. H. Gaston

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.

Autoimmune T cell responses to seminal plasma in chronic pelvic pain syndrome (CPPS)

The aetiology of chronic prostatitis is not understood. The aim of this study is to investigate an autoimmune hypothesis by looking for T cell proliferation in response to proteins of the seminal plasma. We studied peripheral blood mononuclear cell proliferation from 20 patients with chronic prostatitis and 20 aged‐matched controls in response to serial dilutions of seminal plasma (SP) from themselves (autologous SP) and from a healthy individual without the disease (allo‐SP). We found that the patients have a statistically greater lymphocyte proliferation to autologous SP at the 1/50 dilution on day 6 compared to controls (P = 0·01). They also have a greater proliferation to allo‐SP on both day 5 (P = 0·001) and day 6 (P = 0·01) at the same dilution. Using a stimulation index (SI) of 9 to either autologous SP or allo‐SP on day 6 at the 1/50 dilution as a definition of a proliferative response to SP, then 13/20 patients as compared to 3/20 controls showed a proliferative response to SP (P = 0·003, Fisher’s exact test). These data support an autoimmune hypothesis for chronic prostatitis.

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.

Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA‐DRB1*0401‐restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38–46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N‐terminal to the putative P1 position were markedly more stimulatory than a peptide whose N‐terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N‐ and C‐terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia‐induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli‐related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross‐reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia‐induced reactive arthritis in this patient.

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.

Heat shock proteins and innate immunity

Heat shock proteins (hsp) have attracted considerable attention from immunologists over the last 20 years, and their interest has evolved in three distinct phases. Initially hsp were investigated primarily as antigens, particularly when it was found that they were rather common targets of both the humoral and T cell-mediated responses to intracellular pathogens like mycobacteria. Their recognition by T cells in models of autoimmune disease, particularly arthritis and diabetes, gave rise to much speculation that immune responses initially directed against hsp from pathogens might cross-react with self antigens including hsp themselves [1,2]. Since hsp are often up-regulated at sites of inflammation this would provide opportunities for persistent stimulation of cross-reactive hsp-specific T cells [3]. Such speculation continues, although more recent evidence points to anti-inflammatory properties of T cells which recognize self hsp rather than their ability to induce autoimmune disease [4].

Stimulation of peripheral blood lymphocytes with Campylobacter jejuni generates a γδ T cell response in patients with Guillain-Barré syndrome

In three patients whose Guillain–Barré syndrome (GBS) was preceded by gastrointestinal infection due to Campylobacter jejuni, γδ T cells were generated from peripheral blood in response to in vitro stimulation with C. jejuni. In one of the patients, where a diagnostic sural nerve biopsy was performed, γδ T cells were also isolated following culture of the nerve tissue. Studies with healthy volunteers and C. jejuni gastroenteritis patients also showed preferential enrichment for γδ T cells in peripheral blood cells stimulated with C. jejuni, although the response was significantly lower than that seen in GBS patients. In two out of three GBS patients and all of the controls, γδ T cell receptor (TCR) gene usage was shown to be Vγ9/Vδ2+. In the GBS patient where nerve‐infiltrating γδ T cells were isolated, these and C. jejuni‐specific peripheral blood cells had similar TCR gene usage, predominantly consisting of Vγ5/Vδ1+ cells. Sequencing the Vδ1 products from nerve and peripheral blood showed similarities in CDR3 length, but the single Vδ1 sequence obtained from nerve was not identified in peripheral blood. These results suggest that the generation of γδ T cells is part of a normal immune response to C. jejuni, which, in patients with GBS, may contribute to the pathogenesis of their inflammatory neuropathy.

IFN-γ regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions

Fas and Fas Ligand (FasL) expression, activation‐induced cell death (AICD) and mycobacterial antigen‐specific cytotoxicity of peripheral T cells from patients with complete inherited IFN‐γ receptor 1 binding chain deficiency (IFN‐γR1–/–) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti‐Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN‐γ signaling, IFN‐γ induced surface FasL export and their Fas‐dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN‐γR1 (IFN‐γR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II‐matched, mycobacterial antigen‐pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN‐γR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN‐γ signaling in the control of mycobacterial infection in humans.

Alpha beta but not gamma delta T cell clones in synovial fluids of patients with reactive arthritis show active transcription of tumour necrosis factor α and interferon γ

Objective: To compare the cytokine expression profile of three CD8+, three CD4+, and three γδ+ T cell clones all derived from the synovial fluids of three patients with reactive arthritis (ReA). Methods: Complementary DNA based microarrays containing the specific sequence of 56 cytokine transcripts were used for screening. Selected genes were confirmed by reverse transcriptase-polymerase chain reaction assay. Results: Microarray showed that transcripts encoding for interferon γ and tumour necrosis factor α were expressed by all CD8+ and CD4+ T cell clones. However, γδ+ T cells predominantly expressed transforming growth factor β2 and granulocyte monocyte-colony stimulating factor. Conclusion: T lymphocyte clones from the joint of patients with ReA exhibit differential cytokine expression profiles. CD8+ and CD4+ T cells demonstrate a Th1 mediated profile, whereas γδ+ T cells show a more heterogeneous and less proinflammatory Th3 driven pattern.