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.

Infection of epithelial and dendritic cells by Chlamydia trachomatis results in IL-18 and IL-12 production, leading to interferon-gamma production by human natural killer cells.

Control of infection by Chlamydia trachomatis usually requires the production of interferon-gamma. Whilst this can be produced by CD4+ and CD8+ T lymphocytes, natural killer (NK) cells are another important source of this cytokine, and are known to be recruited early to the infected genital tract. We show that both IL-12 and IL-18, which synergise to stimulate NK cells to produce interferon-gamma, are produced following the infection of dendritic cells and epithelial cells respectively, since supernatants from infected cells could substitute for recombinant cytokines. These results suggest that conditions, which lead to NK cell production of interferon-gamma will be present at the site of infection, where epithelial cells are the primary targets of infection and dendritic cells within the epithelium can also access the bacterium.

Bacillus Calmette-Guérin sequestered in the brain parenchyma escapes immune recognition.

We have previously shown that heat-killed bacillus Calmette-Guérin (BCG) injected into the brain parenchyma becomes sequestered behind the blood-brain barrier for months, apparently unrecognised by the immune system (Matyszak and Perry, 1995, 1996a,b). In this paper we have studied T-cell and antibody responses to purified protein derivative (PPD) at different times after intracranial injection of BCG or after the same dose of BCG was injected intradermally. We detected no antibody to PPD in the sera of animals which received intracranial injection, although there was a clear antibody response in the sera of animals injected intradermally, as shown using immunoblot analysis. The skin contact sensitivity to PPD was robust in animals which had received a previous intradermal injection of BCG. 72 h after a PPD injection, the injected site showed many MHC class II + macrophages and T-cells. However, the response in skin following PPD challenge, in animals injected intracranially (i.c.), was comparable with that of naive animals which had received no previous BCG challenge. The skin lesions in animals injected i.c. and in naive animals, were characterised by a small number of MHC class II + cells and rare T-cells. T-cell responses were also studied in an in vitro proliferation assay. The proliferative response was measured for cells isolated from the cervical lymph nodes and the spleen. Cells purified from the spleen and the cervical lymph nodes of animals injected with BCG i.c. showed no specific proliferative response to PPD. The response was comparable to that found in naive, uninjected animals. However, spleen and cervical lymph node cells from animals injected intradermally with BCG showed a significant proliferative response to PPD. These results show that a dose of bacteria injected into the brain parenchyma fails to prime the immune system even though the same dose injected subcutaneously will do so. This response to bacteria in the CNS differs from that previously reported for soluble proteins.

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.

Uptake and processing of Chlamydia trachomatis by human dendritic cells.

Chlamydia trachomatis (CT) causes several sexually transmitted diseases. In 2-5% of cases, CT infection leads to the development of reactive arthritis. Dendritic cells (DC) are central in T cell priming and the induction of antigen specific immunity. Here we have studied the uptake and processing of CT serovar L2 by human DC, and their ability to present CT antigens to both CD4(+) and CD8(+) T cells. We show that the entry of CT was mediated by the attachment of CT to heparan sulfates and could be inhibited by heparin. There was no inhibition of uptake by an agent which blocks micropinocytosis. Infecting DC with CT led to their activation and the production of IL-12 and TNF-alpha but not IL-10. Following invasion, CT was confined to distinct vacuoles which were visualized with anti-CT antibodies using confocal microscopy. Unlike with epithelial cells, these vacuoles did not develop into characteristic inclusion bodies. In the first 48 h, CT(+) vacuoles were negative for Lamp-1 and MHC class II. Despite no obvious co-localization between CT vacuoles and MHC loading compartments, infected DC efficiently presented CT antigens to CD4(+) T cells. Infected DC also expanded CT specific CD8(+) T cells, allowing us to generate a number of CT-reactive CD8(+) T cell clones. There is still controversy about the importance of chlamydia-specific CD8(+) T cell responses in patients with arthritis. This is largely due to the difficulties in studying CTL responses at the clonal level. The use of DC as antigen-presenting cells should enable more detailed characterization of these CTL responses.