IMMUNOLOGY
T- helper cell epitopes
Antigen presenting cells (APC) takes up extracellular complexes as protein aggregates in phagosoms which fuses with lyzosomes and the up-taken proteins are fragmented by proteases and eventually presented in the MHC class II molecule at the cell surface for T-helper cells. In blood samples APC are present so that exogenous antigens can be presented for T-helper cells. Therefore full-length protein, recombinant fragments and synthetic peptides of proteins can be used for narrowing down T-helper cell epitopes.
This has been done for human T-helper cell epitopes on C. trachomatis serovar E MOMP.
All epitopes were localized to constant regions, except for one that was localized to VS2 (Ortiz et al. 1996). The epitope localized to VS2 was only recognized by persons that had had an infection with C. trachomatis serotype E, and corresponding peptides to VS2 form serovar F, I and J were not responding showing that the epitope was serovar specific (Ortiz et al. 2000).
Omp2 which is also an outer membrane protein was found to have a proliferative response on CD4+ cells from patients with C.
trachomatis induced reactive arthritis. A peptide of 15 amino acids (aa400-414) was identified byjuse of deletion mutants of Omp2 and overlapping peptides. This peptide was conserved in all C. trachomatis sequences but
not in C. pneumoniae Omp2, so the peptide was specific for C. trachomatis infections (Goodall et al. 2001a).
By screening a C. trachomatis library with three human C. trachomatis reactive T cell clones of unknown specificity PmpD was identified as the target for one of the clones, enolase and CT580 for the two others (Goodall et al. 2001b). In the outer membrane beside MOMP, Omp2 and Omp3 a family of polymorphic membrane proteins (Pmp) is present. In C. trachomatis the genome encodes 9 proteins and in C. pneumoniae the family is expanded to 21 genes. These proteins have the C-terminal ß-barrel embedded in the outer membrane and the N-terminal part, probably in the form of a parallel ß-tube protruding on the surface of the Chlamydia.
CD4+ and CD8+ T-cell epitopes to C.
pneumoniae.
The cellular immunity to C. pneumoniae is not so extensively investigated as to C.
trachomatis.
Halme et al. (1997) analyzed T-cell clones obtained by stimulation with C. pneumoniae, by separating C. pneumoniae antigens with SDS-PAGE and transferring the antigens to nitrocellulose and used the size fractions antigens for stimulation of the clones.
Antigens with sizes of 92-98, 51-55, 43-46 and 31.5-33 kDa were found to be C. pneumoniae specific (Halme et al. 1997). Several of the Pmps in C. pneumoniae has a size of approximately 98 kDa. Therefore all of the 21 Pmp genes from C. pneumoniae were cloned, expressed in E. coli and purified. Spleen cells from mice intranasally infected with C.
pneumoniae were investigated with ELISpot assay for reaction to the Pmps. A significant response was found to Pmp8, Pmp20 and Pmp21, but no response was found to MOMP, which in C. pneumoniae is invariant (Mygind et al. 2004). There thus seems to be a difference in the response to homologous
proteins. The direction of the immune response was also investigated; it was evident that the mice reacted with a Thl response.
Conclusion
New knowledge concerning the specific cellular immune response to chlamydial infections has the potential of defining protective subcomponents, which may be used for vaccines.
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GENOMICS SEARCH FOR HOST CELL GENES THAT CAN INHIBIT CHLAMYDIA GROWTH IN PRIMARY EPITHELIAL CELLS
David E. Nelson1, Dezso Peter Virok1, Heidi Wood2, Grant McClarty2, Harlan D.
Caldwell1
laboratory of Intracellular Parasites, National Institutes of Health, National Institute of Allergy and Infectious Disease, Rocky Mountain Laboratories, Hamilton, department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
IFN-gamma plays critical roles in orchestrating the innate and acquired immune responses to bacterial pathogens. Studies of animal models of chlamydial infection have demonstrated that resistance of mice to genital tract infection with human adapted strains of C. trachomatis is IFN-gamma dependent.
Recently, we determined that primary mouse urogenital epithelial cells (MEC) resist infection with human serovars in an IFN-gamma dependent fashion, and with a pattern similar to that seen in mouse models of experimental chlamydial infection. Therefore, the goal of this work was to use comparative transcriptome analysis of MEC cells to identify the IFN-gamma mediated factor(s) responsible for control of chlamydial growth in vivo.
DNA microrarray analysis was used to identify IFN-gamma regulated genes that might function in controlling infection of human strains by MEC. We analyzed and correlated transcriptional changes of MEC under several experimental conditions; (i) infection alone, (ii) IFN-gamma treatment alone, and (iii) infection plus IFN-gamma treatment. Amplified cRNA from the different groups was hybridized to Affymetrix 430A mouse chips. The array contains -20.000 probes covering the majority of known mouse genes. Array results for specific genes and timepoints were validated and expanded upon using Taqman RT-PCR.
IFN-gamma treatment, chlamydial infection, and the combination of each had a significant impact on the transcriptome of MEC altering the expression -800-1200 genes.
Although similarities between the
transcriptional responses of MEC and the previously reported response of human epithelial cells to chlamydial infection and interferon were identified, a number of important distinctions between the mouse and human responses were evident. Unlike human epithelial cells, MEC failed to significantly alter tryptophan metabolism in response to IFN-gamma or infection, suggesting that this critical mechanism of chlamydial control in human epithelium may not be as relevant to murine infection. Further, MEC responded to IFN-gamma and chlamydial infection by highly up-regulating expression of a murine specifc family of innate immune effectors (p47 GTPases); members of which have been implicated as predominate players in the IFN-gamma mediated control of multiple intracellular bacterial pathogens.
Comparative transcriptome analysis in MEC suggests that early innate immune control of chlamydial infection in mouse and human epithelial cells by IFN-gamma may be mediated by distinct mechanisms. The transcriptome results suggest that IFN-gamma mediated control of human adapted chlamydial strains in mouse epithelium is potentially regulated by a mouse specific family of innate immune effectors, and likely does not depend upon heightened tryptophan metabolism, as occurs in human epithelium. The findings have important implications for rational design of murine models of human chlamydial infection.
CHLAMYDIA PNEUMONIAE INDUCES EXPRESSION OF SUPPRESSOR OF CYTOKINE SIGNALING-3 (SOCS-3) IN HUMAN LUNG EPITHELIAL CELLS Vjera Magdalenic1'2, Sean Wattegedera2, Gwen Wathne1'2, Sarah E.M. Howie1 and Gary Entrican2*
1 Department of Pathology, University of Edinburgh Medical School, Edinburgh, & 2 Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh, UK
Interferon-gamma (IFN-y) is an important effector cytokine for host control of Chlamydia pneumoniae infection. However, human lung-derived epithelial cell lines differ in their ability to control C. pneumoniae in response to IFN-y treatment. HEp-2 cells restrict C. pneumoniae growth whereas A459 cells do not. The objective of this work was to identify the factors that influence IFN-y responsiveness in epithelial cells with a view to understanding host immunity and disease control.
Hep-2 and A459 cells were treated with recombinant human IFN-y then infected with
C. pneumoniae (AR39). Cells were collected at various time points after infection and either lysed for analysis of mRNA expression or fixed for immunocytochemical studies.
Expression of cell-surface molecules was performed by flow cytometry.
Both HEp-2 and A549 cells were found to express CD119, the a chain of the IFN-y receptor. This suggested that intracellular events downstream of receptor ligation were responsible for the differences in responsiveness to IFN-y and prompted investigation of molecules associated with regulation of IFN-y signaling. Both suppressor of cytokine signaling-1 (SOCS-1) and SOCS-3 are known to modulate IFN-y signaling.
Immunocytochemical analysis of unstimulated Hep-2 and A549 cells failed to detect SOCS-1 expression, but SOCS-3 was found in both cell lines. However, A549 cells expressed higher levels of SOCS-3, which may account for the difference observed between the cells. To ascertain if IFN-y treatment could alter the
expression of SOCS-3, levels of mRNA encoding SOCS-3 were measured by real-time RT-PCR. There was a marked, significant increase in expression of mRNA encoding SOCS-3 in Hep-2 following IFN-y treatment, but not in A549 cells. This may be a normal counter-inflammatory mechanism of epithelial cells to regulate IFN-y-mediated cell activation. Interestingly, C. pneumoniae infection resulted in upregulation of mRNA encoding SOCS-3 in both cell lines irrespective of IFN-y treatment. This increase was confirmed by confocal microscopy, which revealed that SOCS-3 protein was concentrated in the vicinity of the chlamydial inclusion. As a first step to elucidating the chlamydial components responsible for this, uninfected cells were treated with lipopolysaccharide (LPS) derived from Salmonella minnesota (rough chemotype similar to chlamydial LPS) and LPS from Escherichia coli. S. minnesota LPS did not affect SOCS-3 expression whereas E. coli LPS induced SOCS-3 expression, suggesting that chlamydial LPS on its own is solely responsible for the upregulation in SOCS-3 expression following C. pneumoniae infection.
Chlamydiae form complex relationships with their hosts that include the establishment of persistent infections and chronic disease. The data presented here demonstrate that C. pneumoniae can induce the expression, of molecules that interfere with responsiveness of host cells to IFN-y. This adds another dimension to the relationship of C. pneumoniae with human lung epithelial cells.
DENDRITIC CELLS AND MACROPHAGES PULSED WITH CHLAMYDIA PNEUMONIAE ELICIT DISTINCT IMMUNE RESPONSES IN VITRO
Anne Tammiruusu* and Jenni M. Vuola
National Public Health Institute, Departments of Vaccines, Helsinki, Finland Experimental studies have indicated an
important cooperative role for both CD4+ and CD8+ T cells in adaptive immunity against Chlamydia pneumoniae. Clearance seems to be dependent on Thl-biased response characterized with the secretion of high levels of gamma interferon (IFN-y). An important factor contributing to T cell polarization is the type of antigen-presenting cell (APC).
Macrophages (Mtj)) are mononuclear leukocytes distributed throughout the body and are an important part of the first level defence mechanisms during infection. Macrophages serve as professional APCs by processing and presenting antigens and secreting chemokines and cytokines that result in boosting or limiting other immune functions. All chlamydial species are capable of infecting and surviving inside mononuclear cells. Ability of chlamydiae to infect and survive in macrophages may provide a mechanism by which chlamydiae establish a chronic infection and disseminate to various tissues. Dendritic cells (DCs) represent specialized migratory derivatives of mononuclear APCs that efficiently uptake and process antigen for presentation. Dendritic cells unlike the other APCs are able to prime naive T cells, initiating the T cell response. A central importance of DCs is their role in linking innate immunity and antigen-specific adaptive immunity.
To understand better the interaction between APCs and C. pneumoniae, we isolated and pulsed mouse DCs and macrophages with live or UV-inactivated C. pneumoniae and assessed their phenotypic changes and cytokine production patterns in vitro. In addition, we examined the capacities of DCs and M<() to prime syngeneic naive CD4+ T cells. DCs (CDllc+) and CD4+ T cells were isolated
from spleen using cell sorting magnetic beads.
Mouse peritoneal macrophages were isolated on the basis of adherence to plastic. The cell surface markers were detected by flow cytometry. Compared with naive DCs, many phenotypic changes of DCs pulsed with UV-inactivated or live C. pneumoniae were observed. These changes included upregulation of MHC class H, CD8, CD80 and CD86 expression. Similar results were obtained from DCs isolated from murine lungs after intranasal challenge in vivo. Preliminary studies showed that DCs pulsed with live or dead bacteria were able to prime naive CD4+
T cells very efficiently. Pulsed macrophages instead, were not able to prime naive CD4+
cells. However, primed CD4+ T cells isolated from mice after C. pneumoniae challenge were activated by macrophages pulsed with dead C.
pneumoniae but not macrophages pulsed with live bacteria. Cytokine profiles of pulsed DC and M<|> will be analysed next.
Our results indicate that live as well as UV-inactivated C. pneumoniae can stimulate the maturation process of DCs. Furthermore, the mature DCs serve as efficient APCs to both naive and primed CD4+ cells. Macrophages on the other hand were severely defective in their ability to serve as APCs.
CLASS IB RESTRICTED CD8+ T CELL RESPONSES IN MICE INFECTED WITH CHLAMYDIA PNEUMONIAE
Amy R. Tvinnereim* and Benjamin Wizel
University of Texas Health Center at Tyler, Tyler, Texas, USA Previous studies have demonstrated
that class lb restricted CD8+ T cells are important for control of infection with several intracellular pathogens prior to expansion of class la restricted CD8+ T cells. Therefore, we wished to determine if class lb restricted CD8+ T cells were generated in response to Chlamydia pneumoniae (Cpn) infection.
The well characterized class lb molecule H2-M3 has been demonstrated to preferentially bind 5-6 amino acid peptides containing a formylated methionine at residue 1. Therefore, the Cpn genome was studied to find the 6 amino acids residues that initiated each protein. Of the initial 48 sequences which fit the predicted binding motif of fM (Y, F, W, I, L)(Y, F, I, L, V)ZXX (Z, amino acids with a non-polar side chain; X any amino acid), 18 were selected for peptide synthesis. The 18 peptides were used to stabilize H2-M3 on the surface of P388-M3 transfected cell lines as detected by an H2-M3-specific monoclonal antibody. These peptides were also tested for the ability to stimulate IFN-y production from splenocytes and lung mononuclear cells from Cpn-infected mice. In addition, short term T cell cultures were tested for the ability to lyse target cells coated with H2-M3 peptides.
Of the 18 H2-M3 peptides studied, 5 did not increase cell surface expression of H2-M3. Of the remaining 13 peptides, 7 increased mean fluorescent intensity of an H2-M3 expressing cell line by at least 2 fold. All 18 peptides were then tested for the ability to stimulate IFN-y production in lung mononuclear cells and splenocytes from infected mice. At least 4 of the H2-M3-binding peptides were able to stimulate IFN-y production from lung mononuclear cells and at
least 2 peptides did so from splenocytes. To determine if the H2-M3 restricted cells that produce IFN-y following Cpn infection were lytic, short term CTL lines were generated and tested in a 51Cr-release assay. CD8+ T cell cultures for one peptide (peptide 4) were able to lyse peptide coated P388-M3 and TR8.4A targets but were unable to lyse the same targets in the absence of peptide. It is unlikely that this lysis is due to class la restricted responses since the CD8+ T cells were generated from C57B1/6 mice that are H-2b and the P388-M3 and TR8.4A cells are derived from H-2d and H-2wl? parental cell lines, respectively. Not all peptides which stimulated IFN-y production were able to lyse peptide coated target cells as short term cultures for 2 peptides (peptide 1 and peptide 5) did not lyse targets coated with the corresponding peptides.
H2-M3 responsive cells are present in the spleen and lungs of Cpn-infected mice.
These cells produce IFN-y in response to stimulation with individual H2-M3 peptide coated cells. Furthermore, lytic cells recognize some of these H2-M3 peptides since short term cultures developed from H2-M3 stimulated
1 splenocytes are able to lyse H2-M3 coated target cells. These results demonstrate that the H2-M3-restricted CD8+ T cell response is generated during Cpn infection and may be important to control bacteria numbers early in primary infection as demonstrated for Listeria monocytogenes. Since class lb molecules are non-polymorphic, including H2-M3 epitopes in a vaccine would result in a construct with subunits that are universally recognized.
RECOGNITION OF CHLAMYDIA TRACHOMATIS BY CD8+ CYTOTOXIC T CELLS
Malgosia K. Matyszak and J. S. Hill Gaston
University of Cambridge Clinical School, Dept of Medicine, Addenbrooke's Hospital, Cambridge T cell mediated immune responses and
IFN-y production are essential for the clearance of Chlamydia trachomatis (CT) infection.
However, although the importance of CT specific CD4+ T cells has been clearly demonstrated, the importance of chlamydia specific CD8+ T cells in anti-CT responses is still poorly understood. Here we have investigated CD8+ T cell responses to CT in patients with previous episodes of chlamydia infection.
We used dendritic cells (DC) infected with live CT to generate CT specific CD8+ T cell lines and clones. DC were generated in vitro from CD 14+ monocytes obtained from patients' PBMC. After seven days of culture in the presence of GM-CSF and IL-4, DC were fully differentiated. DC were infected with live CT and co-cultured with autologous highly purified CD8+ T cells to generate CT specific CD8+ T cell lines. These lines were then used to generate CT specific CD8+ T cell clones.
The clones were tested for their ability to produce IFN-y in response to CT by ELISA.
They were also tested for their ability to lyse infected target cells in a cytotoxicity assay.
Anti-HLA class I antibodies were used to block both IFN-g production and lysis. IL-12 production was detected by ELISA. The surface phenotype of the CD8+ T cell clones was analysed by FACS using a wide range of monoclonal antibodies. Intracellular CT staining was also analysed by FACS.
Using CT specific CD8+ T cell lines we showed that they can produce a high level of IFN-y when co-cultured with CT infected autologous DC or PBMC. However, experiments in which blocking antibodies to HLA class I were used revealed that IFN-y
production can only be partially inhibited, suggesting the existence of HLA class I unrestricted responses. The two patterns of recognition of CT-infected cells were further confirmed at the clonal level: 1. CD8+ clones recognised CT antigens in association with classical class I HLA antigens and responses were inhibited by class I HLA-specific monoclonal antibodies; 2. the recognition of CT by CD8+ clones was unrestricted by classical HLA class I. Further characterisation of a number of HLA class I unrestricted clones showed that these clones were also not restricted by CD1 molecules. CD8+ clones produced high levels of IFN-y in response to CT and were able to lyse CT-infected target cells. All of these clones were CD8+/RCRa(3+
and contained perforin granules. The clones were negative for NKT receptor (CD161) but positive for some NK receptors, CD56, NKG2D. Interestingly, recognition of CT infected target cells by these clones was rapid (the lysis was detected within 6 hour post infection) when compared with CT specific HLA class I restricted clones. The latter required at least 20 hours of CT processing by DC. In addition, co-culture of CT-infected DC with the CT-specific clones induced DC activation and a rapid enhancement of IL-12 production.
CT specific CD8+ T cells can facilitate clearance of bacteria through production of IFN-y and by direct lysis of infected cells. In addition, early production of IL-12 during CT infection, facilitated by unrestricted CD8+ T cell clones, may be important in ensuring a subsequent Thl T cell mediated response by classical MHC restricted CD4+ and CD8+ T cells.