Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors. T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.     

Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells was validated for all 18 epitopes. Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α revealed mono-, dual-, as well as triple-positive CD8(+) T cells, indicating these M. tuberculosis peptide-specific CD8(+) T cells were (poly)functional. Moreover, these T cells were primed during natural infection, because they were absent from M. tuberculosis-noninfected individuals. Control CMV peptide/HLA-A*0201 tetramers stained CD8(+) T cells in M. tuberculosis-infected and noninfected individuals equally, whereas Ebola peptide/HLA-A*0201 tetramers were negative. In conclusion, the M. tuberculosis-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly)functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification.     

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8(+) T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-gamma and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.     

Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection

The elucidation of the molecular and immunological mechanisms mediating maintenance of latency in human tuberculosis aids to develop more effective vaccines and to define biologically meaningful markers for immune protection. We analyzed granuloma-associated lymphocytes (GALs) from human lung biopsies of five patients with latent Mycobacterium tuberculosis (MTB) infection. MTB CD4+ and CD8+ T cell response was highly focused in the lung, distinct from PBL, as assessed by TCR-CDR3 spectratyping coupled with a quantitative analysis of TCR VB frequencies. GALs produced IFN-gamma in response to autologous macrophages infected with MTB and to defined MTB-derived HLA-A2-presented peptides Ag85a242-250, Ag85b199-207, early secreted antigenic target 6 (ESAT-6)28-36, 19-kDa Ag88-97, or the HLA-DR-presented ESAT-6(1-20) epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-6(1-20) could be linked to specific TCR VB families, and in two patients to unique T cell clones that constituted 19 and 27%, respectively, of the CD4+ and 17% of the CD8+ GAL population. In situ examination of MTB-reactive GALs by tetramer in situ staining and confocal laser-scanning microscopy consolidates the presence of MHC class I-restricted CD8+ T cells in MTB granuloma lesions and supports the notion that clonally expanded T cells are crucial in immune surveillance against MTB.     

Multiple Chlamydia pneumoniae antigens prime CD8+ Tc1 responses that inhibit intracellular growth of this vacuolar pathogen

CD8(+) T cells play an essential role in immunity to Chlamydia pneumoniae (Cpn). However, the target Ags recognized by Cpn-specific CD8(+) T cells have not been identified, and the mechanisms by which this T cell subset contributes to protection remain unknown. In this work we demonstrate that Cpn infection primes a pathogen-specific CD8(+) T cell response in mice. Eighteen H-2(b) binding peptides representing sequences from 12 Cpn Ags sensitized target cells for MHC class I-restricted lysis by CD8(+) CTL generated from the spleens and lungs of infected mice. Peptide-specific IFN-gamma-secreting CD8(+) T cells were present in local and systemic compartments after primary infection, and these cells expanded after pathogen re-exposure. CD8(+) T cell lines to the 18 Cpn epitope-bearing peptides were cytotoxic, displayed a memory phenotype, and secreted IFN-gamma and TNF-alpha, but not IL-4. These CTL lines lysed Cpn-infected macrophages, and the lytic activity was inhibited by brefeldin A, indicating endogenous processing of CTL Ags. Finally, Cpn peptide-specific CD8(+) CTL suppressed chlamydial growth in vitro by direct lysis of infected cells and by secretion of IFN-gamma and other soluble factors. These studies provide information on the mechanisms by which CD8(+) CTL protect against Cpn, furnish the tools to investigate their possible role in immunopathology, and lay the foundation for future work to develop vaccines against acute and chronic Cpn infections.     

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.     

ML1419c peptide immunization induces Mycobacterium leprae-specific HLA-A*0201-restricted CTL in vivo with potential to kill live mycobacteria

MHC class I-restricted CD8(+) T cells play an important role in protective immunity against mycobacteria. Previously, we showed that p113-121, derived from Mycobacterium leprae protein ML1419c, induced significant IFN-γ production by CD8(+) T cells in 90% of paucibacillary leprosy patients and in 80% of multibacillary patients' contacts, demonstrating induction of M. leprae-specific CD8(+) T cell immunity. In this work, we studied the in vivo role and functional profile of ML1419c p113-121-induced T cells in HLA-A*0201 transgenic mice. Immunization with 9mer or 30mer covering the p113-121 sequence combined with TLR9 agonist CpG induced HLA-A*0201-restricted, M. leprae-specific CD8(+) T cells as visualized by p113-121/HLA-A*0201 tetramers. Most CD8(+) T cells produced IFN-γ, but distinct IFN-γ(+)/TNF-α(+) populations were detected simultaneously with significant secretion of CXCL10/IFN-γ-induced protein 10, CXCL9/MIG, and VEGF. Strikingly, peptide immunization also induced high ML1419c-specific IgG levels, strongly suggesting that peptide-specific CD8(+) T cells provide help to B cells in vivo, as CD4(+) T cells were undetectable. An additional important characteristic of p113-121-specific CD8(+) T cells was their capacity for in vivo killing of p113-121-labeled, HLA-A*0201(+) splenocytes. The cytotoxic function of p113-121/HLA-A*0201-specific CD8(+) T cells extended into direct killing of splenocytes infected with live Mycobacterium smegmatis expressing ML1419c: both 9mer and 30mer induced CD8(+) T cells that reduced the number of ML1419c-expressing mycobacteria by 95%, whereas no reduction occurred using wild-type M. smegmatis. These data, combined with previous observations in Brazilian cohorts, show that ML1419c p113-121 induces potent CD8(+) T cells that provide protective immunity against M. leprae and B cell help for induction of specific IgG, suggesting its potential use in diagnostics and as a subunit (vaccine) for M. leprae infection.     

CD8 T cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized HLA-A*0201-transgenic mice

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.     

Langerhans-type dendritic cells genetically modified to express full-length antigen optimally stimulate CTLs in a CD4-dependent manner

Oncoretroviral vectors encoding either full-length Ag or a corresponding immunodominant peptide were expressed in Langerhans-type dendritic cells (LCs) differentiated from CD34(+) progenitors. We used human CMV as a model Ag restricted by HLA-A*0201 to define parameters for eventual expression of cancer Ags by LCs for active immunization against tumors. Stimulation by CMVpp65(495-503)-pulsed LCs, CMVpp65(495-503)-transduced LCs, and full-length CMVpp65-transduced LCs respectively increased tetramer-reactive T cells with an effector memory phenotype by 10 +/- 11, 34 +/- 21, and 51 +/- 24-fold (p < 0.05) from CMV-seropositive donors. CMV-specific CD8(+) CTLs achieved respective frequencies of 231 +/- 102, 583 +/- 219, and 714 +/- 281 spot-forming cells per 10(5) input cells (p < 0.01) in ELISPOT assays for IFN-gamma secretion. LCs expressing full-length Ag stimulated greater lytic activity than either peptide-transduced or peptide-pulsed LCs (p < 0.05), all in the absence of exogenous cytokines. pp65-transduced LCs presenting class I and II MHC-restricted epitopes expanded IFN-gamma-secreting CD4(+) T cells, whereas pp65(495-503)-transduced LCs did not. CD4(+) T cell numbers even declined after stimulation by pp65(495-503) peptide-pulsed LCs. CD4(+) T cell depletion confirmed their contribution to the more robust CTL responses. LCs, transduced with a retroviral vector encoding full-length Ag, stimulate potent CTLs directed against multiple epitopes in a CD4(+) Th cell-dependent manner.     

Absence of immunodominant anti-Gag p17 (SL9) responses among Gag CTL-positive, HIV-uninfected vaccine recipients expressing the HLA-A*0201 allele

According to a number of previous reports, control of HIV replication in humans appears to be linked to the presence of anti-HIV-1 Gag-specific CD8 responses. During the chronic phase of HIV-1 infection, up to 75% of the HIV-infected individuals who express the histocompatibility leukocyte Ag (HLA)-A*0201 recognize the Gag p17 SLYNTVATL (aa residues 77-85) epitope (SL9). However, the role of the anti-SL9 CD8 CTL in controlling HIV-1 infection remains controversial. In this study we determined whether the pattern of SL9 immunodominance in uninfected, HLA-A*0201 HIV vaccine recipients is similar to that seen in chronically HIV-infected subjects. The presence of anti-SL9 responses was determined using a panel of highly sensitive cellular immunoassays, including peptide:MHC tetramer binding, IFN-gamma ELISPOT, and cytokine flow cytometry. Thirteen HLA-A*0201 vaccinees with documented anti-Gag CD8 CTL reactivities were tested, and none had a detectable anti-SL9 response. These findings strongly suggest that the pattern of SL9 epitope immunodominance previously reported among chronically infected, HLA-A*0201-positive patients is not recapitulated in noninfected recipients of Gag-containing canarypox-based candidate vaccines and may be influenced by the relative immunogenicity of these constructs.     

Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guérin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara

Heterologous prime-boost immunization strategies can evoke powerful T cell immune responses and may be of value in developing an improved tuberculosis vaccine. We show that recombinant modified vaccinia virus Ankara, expressing Mycobacterium tuberculosis Ag 85A (M.85A), strongly boosts bacille Calmette-Guérin (BCG)-induced Ag 85A specific CD4(+) and CD8(+) T cell responses in mice. A comparison of intranasal (i.n.) and parenteral immunization of BCG showed that while both routes elicited comparable T cell responses in the spleen, only i.n. delivery elicited specific T cell responses in the lung lymph nodes, and these responses were further boosted by i.n. delivery of M.85A. Following aerosol challenge with M. tuberculosis, i.n. boosting of BCG with either BCG or M.85A afforded unprecedented levels of protection in both the lungs (2.5 log) and spleens (1.5 log) compared with naive controls. Protection in the lung correlated with the induction of Ag 85A-specific, IFN-gamma-secreting T cells in lung lymph nodes. These findings support further evaluation of mucosally targeted prime-boost vaccination approaches for tuberculosis.     

Investigation of peptide involvement in T cell allorecognition using recombinant HLA class I multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Identification of multiple HLA-A*0201-restricted cruzipain and FL-160 CD8+ epitopes recognized by T cells from chronically Trypanosoma cruzi-infected patients

Chronic Chagas disease occurs in 16 million individuals chronically infected by the protozoan Trypanosoma cruzi in Latin America, and may lead to a dilated cardiomyopathy in 10-30% of patients. A vigorous cellular immune response holds parasitism in check. However, up to now, few T. cruzi proteins have been shown to be recognized by CD8+ T cells from Chagas disease patients. In this study, we designed 94 peptides derived from T. cruzi proteins cruzipain and FL-160, predicted to bind to HLA-A2 molcules. After in vitro binding assays to HLA-A*0201, 26 peptides were selected, and their recognition by PBMC from Chagas disease patients was tested with the IFN-gamma ELISPOT assay. All 26 peptides were recognized by PBMC from at least one patient. Furthermore, a tetrameric HLA-A*0201 complex built with the cruzipain 60-68 peptide that was frequently recognized in the periphery also bound to CD8+ T cells from a heart-infiltrating T cell line obtained from a single patient with Chagas disease cardiomyopathy. Thus, our results suggest that the recognition of CD8+ T cell epitopes in cruzipain and FL-160 may have a pathogenic or protective role in chronic Chagas disease.