Immunization with live and dead Chlamydia muridarum induces different levels of protective immunity in a murine genital tract model: correlation with MHC class II peptide presentation and multifunctional Th1 cells

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.     

Antigen-specific CD4+ T cells produce sufficient IFN-gamma to mediate robust protective immunity against genital Chlamydia muridarum infection

Chlamydia has been shown to evade host-specific IFN-gamma-mediated bacterial killing; however, IFN-gamma-deficient mice exhibit suboptimal late phase vaginal Chlamydia muridarum clearance, greater dissemination, and oviduct pathology. These findings introduce constraints in understanding results from murine chlamydial vaccination studies in context of potential implications to humans. In this study, we used mice deficient in either IFN-gamma or the IFN-gamma receptor for intranasal vaccination with a defined secreted chlamydial Ag, chlamydial protease-like activity factor (CPAF), plus CpG and examined the role of IFN-gamma derived from adoptively transferred Ag-specific CD4+ T cells in protective immunity against genital C. muridarum infection. We found that early Ag-specific IFN-gamma induction and CD4+ T cell infiltration correlates with the onset of genital chlamydial clearance. Adoptively transferred IFN-gamma competent CPAF-specific CD4+ T cells failed to enhance the resolution of genital chlamydial infection within recipient IFN-gamma receptor-deficient mice. Conversely, IFN-gamma production from adoptively transferred CPAF-specific CD4+ T cells was sufficient in IFN-gamma-deficient mice to induce early resolution of infection and reduction of subsequent pathology. These results provide the first direct evidence that enhanced anti-C. muridarum protective immunity induced by Ag-specific CD4+ T cells is dependent upon IFN-gamma signaling and that such cells produce sufficient IFN-gamma to mediate the protective effects. Additionally, MHC class II pathway was sufficient for induction of robust protective anti-C. muridarum immunity. Thus, targeting soluble candidate Ags via MHC class II to CD4+ T cells may be a viable vaccine strategy to induce optimal IFN-gamma production for effective protective immunity against human genital chlamydial infection.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-gamma production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb-/-/Db-/- mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb-/-/Db-/- mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.     

Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity

Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.     

Quantitation of CD8+ T cell expansion,memory, and protective immunity after immunization with peptide-coated dendritic cells

Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells.     

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4⁺ T cell-dependent process. Activation of FVIII-specific CD4⁺ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.     

Suppression of endogenous IL-10 gene expression in dendritic cells enhances antigen presentation for specific Th1 induction: potential for cellular vaccine development

A new paradigm for designing vaccines against certain microbial pathogens, including Chlamydia trachomatis, is based on the induction of local mucosal Th1 response. IL-10 is an anti-inflammatory cytokine that exerts negative immunoregulatory influence on Th1 response. This study investigated whether biochemical modulation of endogenous IL-10 expression at the level of APCs is a practical strategy for enhancing the specific Th1 response against pathogens controlled by Th1 immunity. The results revealed that the high resistance of genetically engineered IL-10-/- (IL-10KO) mice to genital chlamydial infection is a function of the predilection of their APCs to rapidly and preferentially activate a high Th1 response. Thus, in microbiological analysis, IL-10KO mice suffered a shorter duration of infection, less microbial burden, and limited ascending infection than immunocompetent wild-type mice. Also, IL-10KO were resistant to reinfection after 8 wk of the primary infection. Cellular and molecular immunologic evaluation indicated that IL-10KO mice induced greater frequency of chlamydial-specific Th1 response following C. trachomatis infection. Moreover, IL-10KO APCs or antisense IL-10 oligonucleotide-treated wild-type APCs were potent activators of Th1 response from naive or immune T cells. Furthermore, both Ag-pulsed dendritic cells from IL-10KO mice and IL-10 antisense-treated dendritic cells from wild-type mice were efficient cellular vaccines in adoptive immunotherapeutic vaccination against genital chlamydial infection. These findings may furnish a novel immunotherapeutic strategy for boosting the Th1 response against T cell-controlled pathogens and tumors, using IL-10-deficient APCs as vaccine delivery agents.     

An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8(+) T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen's developmental cycle. In addition to their use as candidate Ags for stimulating CD8(+) T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8(+) T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8(+) T cells specific for an H-2D(b)-restricted epitope from CrpA are elicited at a significant level (approximately 4% of splenic CD8(+) T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.     

H2-M3-restricted CD8+ T cells induced by peptide-pulsed dendritic cells confer protection against Mycobacterium tuberculosis

One of the oligopolymorphic MHC class Ib molecules, H2-M3, presents N-formylated peptides derived from bacteria. In this study, we tested the ability of an H2-M3-binding peptide, TB2, to induce protection in C57BL/6 mice against Mycobacterium tuberculosis. Immunization with bone marrow-derived dendritic cell (BMDC) pulsed with TB2 or a MHC class Ia-binding peptide, MPT64(190-198) elicited an expansion of Ag-specific CD8+ T cells in the spleen and the lung. The number of TB2-specific CD8+ T cells reached a peak on day 6, contracted with kinetics similar to MPT64(190-198)-specific CD8+ T cells and was maintained at an appreciable level for at least 60 days. The TB2-specific CD8+ T cells produced less effector cytokines but have stronger cytotoxic activity than MPT64(190-198)-specific CD8+ T cells. Mice immunized with TB2-pulsed BMDC as well as those with MPT64(190-198)-pulsed BMDC showed significant protection against an intratracheal challenge with M. tuberculosis H37Rv. However, histopathology of the lung in mice immunized with TB2-pulsed BMDC was different from mice immunized with MPT64(190-198)-pulsed BMDC. Our results suggest that immunization with BMDC pulsed with MHC class Ib-restricted peptides would be a useful vaccination strategy against M. tuberculosis.     

Diverse repertoire of the MHC class II-peptide complexes is required for presentation of viral superantigens

Among other features, peptides affect MHC class II molecules, causing changes in the binding of bacterial superantigens (b-Sag). Whether peptides can alter binding of viral superantigens (v-Sag) to MHC class II was not known. Here we addressed the question of whether mutations limiting the diversity of peptides bound by the MHC class II molecules influenced the presentation of v-Sag and, subsequently, the life cycle of the mouse mammary tumor virus (MMTV). T cells reactive to v-Sag were found in mice lacking DM molecules as well as in A(b)Ep-transgenic mice in which MHC class II binding grooves were predominantly occupied by an invariant chain fragment or Ealpha(52-68) peptide, respectively. APCs from the mutant mice failed to present v-Sag, as determined by the lack of Sag-specific T cell activation, Sag-induced T cell deletion, and by the aborted MMTV infection. In contrast, mice that express I-A(b) with a variety of bound peptides presented v-Sag and were susceptible to MMTV infection. Comparison of v-Sag and b-Sag presentation by the same mutant cells suggested that presentation of v-Sag had requirements similar to that for presentation of toxic shock syndrome toxin-1. Thus, MHC class II peptide repertoire is critical for recognition of v-Sag by the T cells and affects the outcome of infection with a retrovirus.     

Puromycin-sensitive aminopeptidase limits MHC class I presentation in dendritic cells but does not affect CD8 T cell responses during viral infections

Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice. In addition, dendritic cells from PSA-deficient mice display more MHC class I molecules on the cell surface, suggesting that PSA normally limits Ag presentation by destroying certain peptides in these key APCs. Surprisingly, MHC class I levels are not altered on other PSA-deficient cells and the processing and presentation of peptide precursors in PSA-deficient fibroblasts is normal. Moreover, PSA-deficient mice have normal numbers of T cells in the periphery, and respond as well as wild type mice to eight epitopes from three viruses. These data indicate that PSA may play a role in limiting MHC class I Ag presentation in dendritic cells in vivo but that it is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags.     

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.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.     

Antitumor cytotoxic T-cell response induced by a survivin peptide mimic

Survivin is a tumor-associated antigen with significant potential as a cancer vaccine target. We have identified a survivin peptide mimic containing human MHC class I epitopes and a potential class II ligand that induces a potent antitumor response in C57BL/6 mice with GL261 cerebral gliomas. This peptide is able to elicit both CD8+ CTL and T helper cell responses in C57BL/6 mice. The corresponding region of the human survivin molecule represented by peptide SVN53-67 is 100% homologous to the murine protein, but SVN53-67 is weakly immunogenic in man. We evaluated several amino acid substitutions in putative human MHC I anchor positions in SVN53-67 to identify potential peptide mimics that could provide an enhanced antitumor immune response against human glioma and primary central nervous system lymphoma (PCNSL) cells in culture. We evaluated survivin peptides with predicted binding to human HLA-A*0201 antigen using peptide-loaded dendritic cells from PBMC of patients with these malignancies. One alteration (M57) led to binding to HLA-A*0201 with significantly higher affinity. We compared the ability of autologous dendritic cells loaded with SVN53-67 peptide and SVN53-67/M57 in CTL assays against allomatched and autologous, survivin-expressing, human malignant glioma and PCNSL cells. Both SVN53-67 and SVN53-67/M57 produced CTL-mediated killing of malignant target cells; however, SVN53-67/M57 was significantly more effective than SVN53-67. Thus, SVN53-67/M57 may act as a peptide mimic to induce an enhanced antitumor CTL response in tumor patients. The use of SVN53-67/M57 as a cancer vaccine might have application for cancer vaccine therapy.     

Exploratory Study on Th1 Epitope-Induced Protective Immunity against Coxiella burnetii Infection

Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs) of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-A^b based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4⁺ T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4⁺ T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4⁺ T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.     

Defining MHC class II T helper epitopes for WT1 tumor antigen

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4⁺ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1_124–138 and WT1_247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.     

烟曲霉抗原Asp f16HLA-A*0201限制性的CD8+CTL抗原表位生物信息学预测与实验室鉴定

目的 预测与鉴定烟曲霉抗原Asp f16的HLA-A *0201限制性CD8+细胞毒性T细胞(CTL)抗原表位.方法 以国人常见的HLA-A*0201位点为靶点,依据生物信息学软件扫描烟曲霉特异性抗原Asp f16的全部427个氨基酸序列.使用HLA-A *0201转基因小鼠制备骨髓来源的树突状细胞(DC)和CTL.流式细胞仪技术检测DC表面MHC Ⅱ类抗原,CD80,CD86和CD11c的表达来验证其是否成熟.ELISPOT试验检测烟曲霉抗原多肽特异性CTL产生的细胞因子IFN-γ.四聚体(Tetramer)试验证实烟曲霉特异性CTL与抗原肽,HLA-A*0201分子复合体的亲和性.结果 根据与MHC I类分子结合的半衰期评分,选择了3个HLA-A*0201限制性抗原表位.流式细胞仪分析示成熟DC高表达HLA Ⅱ类抗原,CD80,CD86和CD11c.Tetramer试验证实烟曲霉特异性T细胞受体与抗原肽,HLA-A*0201分子复合体的高亲和性.ELISPOT实验结果 表明烟曲霉抗原肽体外可以活化CD8+CTL,被负载了抗原肽的DC刺激活化后可以产生IFN-γ.结论 本研究成功鉴定烟曲霉抗原Asp f16的HLA-A*0201限制性CD8+CTL表位,可作为疫苗设计的候选表位,为进一步研发新型抗烟曲霉疫苗提供参考.