Epitope clusters in the major outer membrane protein of Chlamydia trachomatis.

Immunopathology that is caused by re-infection with Chlamydia trachomatis is very common in humans despite regular responses to multiple, often conserved, antibody and T cell epitopes. Recurrent mutations that disrupt T cell epitopes in the major outer membrane protein in clinical isolates and the reduced transcription of HLA genes by infected cells may be evidence for pathogen evasion of protective immune responses. Subunit vaccines containing recently discovered clusters of T cell epitopes in the major outer membrane protein that are presented with diverse HLA allotypes may allow widespread protective immunization while avoiding the suppression of lasting immunity that occurs by unknown mechanisms associated with infection.

Direct detection and magnetic isolation of Chlamydia trachomatis major outer membrane protein-specific CD8+ CTLs with HLA class I tetramers.

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8(+) T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01-0.20% of CD8(+) T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8(+) T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8(+) CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.

Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression.

Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.

T-cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein elicit serovar-specific immune responses in infected humans.

We previously identified 18 stimulatory Chlamydia trachomatis major outer membrane protein (MOMP) peptides containing at least 23 epitopes presented with various HLA class II allotypes. Only one peptide contained an epitope localized in a variable segment (VS2). Continued studies reported here identified a total of five VS peptides containing T-cell epitopes that are distributed among MOMPs VS1, VS2, and VS4. Only MOMP-primed T-cell cultures from subjects infected with serovar E responded to the serovar E VS peptides, while the response of such cultures to constant-segment peptides was independent of the infecting serovar. Furthermore, MOMP-primed T cells proliferated in response only to the VS peptides encoded in serovar E but not to the corresponding peptides derived from serovar F, I, or J, confirming that these responses were serovar specific.

T-Cell epitopes and human leukocyte antigen restriction elements of an immunodominant antigen of Blastomyces dermatitidis.

Humans infected with the dimorphic fungus Blastomyces dermatitidis develop strong T-lymphocyte responses to WI-1, an immunodominant antigen that has been shown to elicit protective immunity in mice. In the present study, the T-cell epitopes of WI-1 and human leukocyte antigen (HLA) restricting elements that display them were investigated. Peripheral blood mononuclear cells (PBMC) from 37 patients with a confirmed history of blastomycosis were tested for a response to WI-1 in primary proliferation assays; PBMC from 35 (95%) responded. Six patients whose PBMC proliferated strongly in response to WI-1 (defined as a stimulation index greater than 50) were tested further for responses to subcloned, recombinant fragments of the antigen. These patients responded chiefly to sequences within the N terminus and the 25-amino-acid tandem repeat. Cloned CD4(+) T cells from an infected individual were used to delineate more precisely the peptide epitopes in the fragments and HLA restricting elements that present them. A majority of the T-cell clones recognized an epitope spanning amino acids 149 to 172 within the N terminus, displayed by HLA-DR 15. A minority of the clones, which have been shown to perform a cytolytic function in vitro, recognized an epitope in the tandem repeat displayed by HLA-DPw4, an uncommon restricting element. Tandem repeat epitopes required display by the beta chain of DPw4 heterodimers. Thus, human T cells with different functions in vitro also recognize distinct regions of WI-1, raising the possibility that HLA restricting elements that present them could modulate immunity during blastomycosis by selection and display of WI-1 peptides.

Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef.

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.

Induction of HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein of Chlamydia trachomatis in human genital tract infections.

HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein (MOMP) of Chlamydia trachomatis are present in the peripheral blood of humans who acquired genital tract infections with the organism. Three HLA-A2-restricted epitopes and two HLA-B51-restricted epitopes were identified in serovar E-MOMP. One of the five epitopes spans a variable segment of MOMP and is likely a serovar E-specific epitope. The other four epitopes are localized in constant segments and are C. trachomatis species specific. CTL populations specific for one or more of the four constant segment epitopes were isolated from all 10 infected subjects tested, regardless of infecting serovars, but from only one of seven uninfected subjects tested. The CTLs failed to recognize corresponding peptides derived from Chlamydia pneumoniae MOMP, further suggesting that they indeed resulted from genital tract infections with C. trachomatis. Significantly, ME180 human cervical epithelial cells productively infected with C. trachomatis were killed by the MOMP peptide-specific CTLs. Further investigations of the ability of such CTLs to lyse normal infected epithelial cells and their presence at inflamed sites in the genital tract will help understand the protective or pathological role of CTLs in chlamydial infections. The MOMP CTL epitopes may be explored as potential components of a subunit vaccine against sexually transmitted diseases caused by C. trachomatis. Moreover, the knowledge provided here will facilitate studies of HLA class I pathways of chlamydial Ag processing and presentation in physiologically relevant human APCs.

Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus.

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.

Chlamydia trachomatis major outer membrane protein (MOMP) epitopes that activate HLA class II-restricted T cells from infected humans.

We localized peptide epitopes within the Chlamydia trachomatis (Ct) major outer membrane protein (MOMP) that activate human T cells. T-MOMP' cells were prepared by culturing PBL from 38 humans who had Ct infections in the presence of Ct serovar E MOMP. Some epitopes were first localized by quantifying proliferative responses of T-MOMP' cells to overlapping MOMP segments (sixths) that were produced in Escherichia coli. Further localization was achieved by using overlapping synthetic 16-22 mers that spanned stimulatory MOMP sixths as Ags. The APCs used were human B cell lines (LCL) that were matched or mismatched with respect to HLA class II alleles of the T-MOMP' cells. T cell epitopes were detected in 18 Ct serovar E MOMP 16-22 mers and were presented in association with HLA-DR1, -7, -13, -17, HLA-DRw52, HLA-DQ3 and at least two from among HLA-DR4, -8, -11, -14, -18, in probable addition to HLA-DP4. Peptide 249-265 stimulated T-MOMP' cells from 83% of the subjects; studies with overlapping 11-13 mers spanning peptide 249-265 revealed at least six epitopes presented with different HLA-class II allotypes. Diverse T-MOMP' cultures responded to between 2 and 7 MOMP epitopes. All but 1 of the 23 epitopes localized to date are distributed among four MOMP constant segments. T-MOMP' cells from subjects infected with serovars other than E also responded.

The intracellular transport of MHC class II molecules in the absence of HLA-DM.

The HLA-DM alpha and HLA-DM beta genes encode a nonpolymorphic, class II-like molecule that functions by an as yet undefined mechanism in the assembly of processed antigen-HLA class II complexes. Mutant cells that fail to express HLA-DM are deficient in Ag processing. We previously isolated a subcellular compartment in mouse B cells in which functional processed Ag-class II complexes are first formed, referred to as the peptide-loading compartment. Here, evidence is provided that HLA-DM resides in a subcellular compartment with the characteristics of a peptide-loading compartment in a human B lymphoblastoid cell line, but is not required for the intracellular transport of HLA-DR3 molecules to a corresponding compartment in HLA-DM-deficient cells. Thus, the primary defect in HLA-DM-deficient cells does not appear to be a failure in the intracellular trafficking of class II molecules.

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

DMA and DMB are the only genes in the class II region of the human MHC needed for class II-associated antigen processing.

Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0 degree, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the approximately 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy.

Novel allele-specific, post-translational reduction in HLA class I surface expression in a mutant human B cell line.

Human B cell line .220 has a novel defect in HLA class I cell surface expression. Mutant .220 was derived from .184TGr, from which both copies of HLA-A and -B are deleted, and has less surface HLA-C than .184TGr. Transfer of class I genes into .220 revealed allele-specific reductions in surface expression: HLA-A1 and -B8 were 1 to 21% of normal; HLA-A11, -A24, and -B5 were moderately reduced; and HLA-A2, -A3, and -B7 were reduced little, if at all. Class I mRNA in .220(A1) and .220(B8) transferents is normal in size and at least normal in quantity. Surface expression of class I molecules was restored by fusing .220 transferents with mutant .174, which lacks the TAP-1 and -2 genes needed for transport of class I-binding peptides. Fusion of .220(A1) cells with beta 2-microglobulin-deficient Daudi cells also fully restored surface expression of class I molecules encoded by both parental cells, indicating beta 2-microglobulin is functional in .220. Pulse-chase experiments showed transgene-encoded HLA-A1 and -B8 alpha-chains are made in apparently normal amounts and associate with beta 2-microglobulin in .220. However, post-translational processing of the HLA-A1 and -B8 molecules is retarded in or before the Golgi apparatus, and immunoprecipitable HLA-A1 molecules disappear after their synthesis. The effects of these abnormalities on surface expression of class I molecules were reversed by incubating .220(A1) and .220(B8) cells at 21 degrees C, which greatly increased the amounts of cell surface HLA-A1 and -B8.

Induction of anti-tumor cytotoxic T lymphocytes in normal humans using primary cultures and synthetic peptide epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptide antigens associated with cell surface major histocompatibility complex (MHC) molecules. The identification of tumor cell-derived peptides capable of eliciting anti-tumor CTL responses would enable the design of antigen-specific immunotherapies. Our strategy to identify such potentially therapeutic peptides relies on selecting high-affinity MHC binders from known tumor-associated antigens. These peptides are subsequently tested for their ability to induce CTLs capable of killing tumor cells. With this strategy, we have identified a nine-residue epitope, derived from the product of the tumor-associated gene MAGE-3, which has the capacity to induce in vitro CTLs that kill melanoma and other tumor cell lines. These results show the primary in vitro induction of tumor-specific human CTLs and illustrate the feasibility of ex vivo antigen-specific approaches to the immunological therapy of cancer.

Gene required for normal MHC class II expression and function is localized to approximately 45 kb of DNA in the class II region of the MHC.

In certain mutant human B cell lines, MHC-encoded class II molecules displayed at the cell surface have an abnormal conformation and are unstable in the presence of SDS. The mutants cannot present exogenous protein Ags to T cells but elicit responses with exogenous antigenic peptides; thus, formation of intracellular complexes between antigenic peptides and class II molecules is impaired. Previous analysis of LCL deletion mutants, .82, .174, and 5.2.4, showed that genes needed for this function must be present in approximately 230 kb of DNA in the class II region of the MHC. We now describe a new deletion mutant, .61, which has normal class II-mediated Ag processing/presentation. The TAP1, TAP2, LMP2, and LMP7 genes are deleted from .61, demonstrating that those genes are not needed for normal formation of intracellular class II/peptide complexes. The genes in question must be located in DNA that is present in .61 and .82 (both normal) and absent from .174 and 5.2.4. (both defective). Mapping of the deletion breakpoints indicates that genes needed for normal class II-associated Ag processing/presentation are either: 1) in an approximately 40 kb L DNA segment located between the DMB and LMP2 loci or 2) in an R region between the DQA2 and DQB1 loci and are completely included on a 5.1-kb fragment formed by joining of DNA that flanks the deletion in .61. The evidence favors location of the genes in the L DNA segment.

Presentation of cytosolic antigen by HLA-DR requires a function encoded in the class II region of the MHC.

The processing pathway for the MHC class II-restricted presentation of endogenous cytosolic Ag is distinct from the class I pathway since a cytosolic form of the influenza virus A hemagglutinin, expressed by a recombinant vaccinia virus, was presented by HLA-DR in a B cell mutant lacking the TAP1 subunit of the transporter for Ag presentation (TAP). In this report, two additional B cell mutants have been used to define the requirements of this TAP1-independent processing pathway. The first mutant, .61, lacks expression of both TAP1 and TAP2 genes, and of both LMP2 and LMP7 genes encoding proteasome subunits. As expected, class I-restricted presentation of the influenza virus matrix protein was totally deficient in mutant .61. In contrast, class II-restricted presentation of both the natural cytosolic matrix and the engineered cytosolic hemagglutinin proteins was functional in mutant .61. Thus, presentation of cytosolic Ag by class II molecules is independent of both TAP subunits and of the two MHC-encoded proteasome subunits. However, this endogenous processing pathway is dependent on at least one other function encoded in the class II region of the MHC as demonstrated with the second mutant, .174, in which a large deletion eliminates all expressed class II genes. Mutant .174 transfected with HLA-DR1 genes was previously shown to be defective in the presentation of exogenous Ag but normal in the presentation of short exogenous peptides. We show here that .174(DR1) is also defective in the presentation of cytosolic matrix and hemagglutinin proteins. This similar requirement for the class II-restricted presentation of either cytosolic Ag or internalized exogenous Ag suggests that both forms of Ag are ultimately targeted to the same cellular compartment for association with class II molecules.

MHC class II deletion mutant expresses normal levels of transgene encoded class II molecules that have abnormal conformation and impaired antigen presentation ability.

Successive transfers of HLA-DR alpha and beta genes restored expression of HLA-DR antigens to human B-lymphoblastoid cell line, LCL .174, from which all known expressible class II genes are deleted. While transferent cells displayed large amounts of DR on their surfaces, transgene-encoded DR3 molecules lacked a conformation-dependent epitope. DR1-restricted CTL lysis of DR1-expressing transferents pulsed with native influenza virus proteins was greatly reduced; the same cells were efficiently lysed in the presence of CTL-recognized influenza peptides. The properties of DR-expressing transferents of .174 suggest they are defective in producing peptides from exogenous proteins or in forming DR/peptide complexes. Comparison with other DR-expressing deletion mutants indicates that at least one gene in an approximately 230 kb DNA segment between the DQ1 and Ring 7 loci is needed for normal DR-mediated processing and presentation. Production of DR3 molecules having the conformation-dependent 16.23 epitope and efficient DR1-restricted presentation of influenza viral epitopes occurred in a B cell line that has a mutation specifically eliminating expression of the TAP1 transporter gene, which is in the approximately 230 kb interval and is needed for production of HLA class I/peptide complexes.

Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.

No evidence for germline mutations in exons 5-9 of the p53 gene in 25 breast cancer families.

Recent studies have demonstrated that families with the Li-Fraumeni syndrome carry inherited point mutations of the p53 gene. In the present study 25 families with strong histories of breast cancer were screened for the presence of such mutations. Polymerase chain reaction products of exons 5-9 of the p53 gene were examined by single-stranded conformational polymorphism analysis and, in addition, exon 7 was further screened by direct sequencing. No mutations were detected in constitutive DNA by either method. These results indicate that familial breast cancer does not usually result from germline point mutations in the p53 gene.