Biomedical subjects

François A Lemonnier

Publications and source records attributed to François A Lemonnier.

At least 19 recordsLinked to original sources

PubMed2006

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects.

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.

Adolescent↗

PubMed2006

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells.

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8(+) T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP(86-94) and STEAP(262-270)). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201(+) STEAP(+) human tumor cells. Furthermore, STEAP(86-94) and STEAP(262-270) stimulated specific CD8(+) T cells from HLA-A*0201(+) healthy donors, and these peptide specific CD8(+) T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP(86-94)-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8(+) T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.

Amino Acid Sequence↗

PubMed2006

Persistence of autoreactive myelin oligodendrocyte glycoprotein (MOG)-specific T cell repertoires in MOG-expressing mice.

Experimental autoimmune encephalomyelitis, an experimental murine model for multiple sclerosis, is induced by stimulation of myelin-specific T lymphocytes. Myelin oligodendrocyte glycoprotein (MOG), a minor component of myelin proteins, is a potent autoantigen which contributes extensively to the anti-myelin response. In the present work, immunoscope analyses and sequencing of the oligoclonal expansions revealed anti-MOG Valpha and Vbeta public repertoires in lymphocytes infiltrating the CNS of wild-type (WT) mice. Moreover, a subset of CNS-infiltrating CD4+ T lymphocytes bearing the public Vbeta8.2 segment have an inflammatory phenotype strongly suggesting that it is encephalitogenic. We then observed that, in lymph node cells of MOG-deficient and WT animals, the Valpha and Vbeta public repertoires expressed by MOG-specific T cells are identical in both strains of mice and correspond to those found in the CNS of WT animals. These findings indicate that the MOG immunodominant determinant is unable to induce tolerance by deletion, and public anti-MOG T cell repertoires are selected for, regardless of the presence of MOG in the thymus and peripheral organs.

Animals↗

PubMed2005

Direct recognition by alphabeta cytolytic T cells of Hfe, a MHC class Ib molecule without antigen-presenting function.

Crystallographic analysis of human Hfe has documented an overall structure similar to classical (class Ia) MHC molecules with a peptide binding groove deprived of ligand. Thus, to address the question of whether alphabeta T cells could recognize MHC molecules independently of bound ligands, we studied human and mouse Hfe interactions with T lymphocytes. We provide formal evidence of direct cytolytic recognition of human Hfe by mouse alphabeta T cell receptors (TCR) in HLA-A*0201 transgenic mice and that this interaction results in ZAP-70 phosphorylation. Furthermore, direct recognition of mouse Hfe molecules by cytotoxic T lymphocytes (CTLs) was demonstrated in DBA/2 Hfe knockout mice. These CTLs express predominantly two T cell antigen receptor alpha variable gene segments (AV6.1 and AV6.6). Interestingly, in wild-type mice we identified a subset of CD8+ T cells positively selected by Hfe that expresses the AV6.1/AV6.6 gene segments. T cell antigen receptor recognition of MHC molecules independently of bound ligand has potential general implications in alloreactivity and identifies in the Hfe case a cognitive link supporting the concept that the immune system could be involved in the control of iron metabolism.

Animals↗

PubMed2005

Determination of cellularly processed HLA-A2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE.

PURPOSE: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two "cancer germ line" genes, MAGE-A4 and SAGE. EXPERIMENTAL DESIGN: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8(+) T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8(+) T cells sensitized with autologous CD4(+) PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. RESULTS: After DNA vaccination, murine CD8(+) T cells recognizing MAGE-A4(143-151) or SAGE(715-723) in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8(+) T cells with autologous CD4(+) PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE(715-723)-specific precursors was observed in HLA-A2402-positive healthy individuals. CONCLUSIONS: Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4(143-151) and SAGE(715-723), were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.

Animals↗

PubMed2005

A single amino acid substitution improves the in vivo immunogenicity of the HPV16 oncoprotein E7(11-20) cytotoxic T lymphocyte epitope.

One of the few and most extensively studied human papilloma virus (HPV) type 16 oncoprotein E7-derived cytotoxic T lymphocyte (CTL) epitopes is YMLDLQPETT, presented to CTL by HLA-A2.1. We previously identified an altered peptide ligand (APL) of this epitope with increased binding affinity for HLA-A2.1, YMLDLQPETV. Herein, the in vivo immunogenicity of this APL was investigated in HLA-A2.1 transgenic HHD mice. Both in vitro and direct ex vivo analysis, performed using newly generated HHD tetramers, showed a significant increase in the number of specific CD8+ T cells upon vaccination with the APL as compared to its unmodified counterpart. Improved immunogenicity of the APL was also observed in functional analyses, including antigen-specific lytic activity and cytokine production of primed CD8+ T cells. Consequently, the YMLDLQPETV peptide may prove useful when included in vaccination strategies against HPV16-induced cervical carcinoma.

Amino Acid Substitution↗

PubMed2005

Enhanced multiepitope-based vaccines elicit CD8+ cytotoxic T cells against both immunodominant and cryptic epitopes.

A frequent issue in vaccinology is to elicit balanced T cell responses against both immunodominant and cryptic T cell epitopes, from one or several antigens presented at the same time to the immune system. Using HLA-A2.1.1 restricted epitopes from the Melan A/MART-1 or gp 100 melanoma-associated antigens as a model, we engineered a series of constructs in the ALVAC canarypox vector system: T cell epitopes were expressed either as linear polyepitopes (with or without spacers), or as minigenes encoding a single epitope. The latter were found to allow the best processing and presentation of most T cell epitopes, following infection by ALVAC recombinants of the HLA A2+ bladder carcinoma cell line and stimulation of epitope-specific human TIL lines. These various constructs were also used to immunize HLA-A2.1.1 HHD transgenic mice to compare their capacity to elicit T cells responses. Polyepitopes but also minigenes encoding wild-type epitopes could not elicit in a reliable manner balanced CTL responses against all target epitopes from gp100. We could rescue T cells responses against poorly immunogenic epitopes after introducing appropriate point mutations to enhance their interaction with MHC Class I molecules. Epitope enhancement within either polyepitope, multiepitopes (i.e. minigenes expressed under the control of separate promoters) or full length immunogens should be systematically considered when designing vaccines containing both cryptic and immunodominant target epitopes.

Animals↗

PubMed2005

Identification of naturally processed HLA-A2--restricted proinsulin epitopes by reverse immunology.

Type 1 diabetes is thought to result from the destruction of beta-cells by autoantigen-specific T-cells. Observations in the NOD mouse model suggest that CD8+ cytotoxic T-cells play an essential role in both the initial triggering of insulitis and its destructive phase. However, little is known about the epitopes derived from human beta-cell autoantigens and presented by HLA class I molecules. We used a novel reverse immunology approach to identify HLA-A2-restricted, naturally processed epitopes derived from proinsulin, an autoantigen likely to play an important role in the pathogenesis of type 1 diabetes. Recombinant human proinsulin was digested with purified proteasome complexes to establish an inventory of potential COOH-terminals of HLA class I-presented epitopes. Cleavage data were then combined with epitope predictions based on the SYFPEITHI and BIMAS algorithms to select 10 candidate epitopes; 7 of these, including 3 with a sequence identical to murine proinsulin, were immunogenic in HLA-A2 transgenic mice. Moreover, six of six tested peptides were processed and presented by proinsulin-expressing cells. These results demonstrate the power of reverse immunology approaches. Moreover, the novel epitopes may be of significant interest in monitoring autoreactive T-cells in type 1 diabetes.

ATP-Binding Cassette Transporters↗

PubMed2004

Comparison of HLA-DR1-restricted T cell response induced in HLA-DR1 transgenic mice deficient for murine MHC class II and HLA-DR1 transgenic mice expressing endogenous murine MHC class II molecules.

Transgenic mice expressing human HLA class II molecules provide a useful model for identifying HLA-restricted CD4+ epitopes. However, the influence of endogenous murine H-2-restricted T cell responses on HLA-restricted responses is not known. In the present study, we show that HLA-DR1 transgenic mice deficient for H-2 class II expression (HLA-DR1+/+/IAbeta0/0) exhibit an equivalent expression level of the transgene HLA-DR1 and a similar diversity in the TCR repertoire, but a slightly different number of CD4+ peripheral T cells, when compared to HLA-DR1 transgenic mice in which H-2 class II molecules were retained (HLA-DR1+/+/IAbeta+/+). More importantly, a strong antigen-specific HLA-DR1-restricted response was observed in nearly all HLA-DR1+/+/IAbeta0/0 mice immunized with HBV envelope protein (HBs) or capsid protein (HBc), whereas weak HBs- or HBc-specific HLA-DR1-restricted responses were detected in half of the immunized HLA-DR1+/+/IAbeta+/+ mice. Conversely, strong HBs- or HBc-specific H-2-restricted T cell responses were detected in HLA-DR1+/+/IAbeta+/+ mice but not in HLA-DR1+/+/IAbeta0/0 mice. Our results indicate that the coexpression of endogenous H-2 class II molecules reduces the intensity of HLA-DR1-restricted antigen-specific responses in transgenic mice, by favoring murine over human MHC recognition and education. Thus, HLA-DR1+/+/IAbeta0/0 mice represent a better model for identifying and characterizing HLA-DR1-restricted epitopes relevant for human disease.

Animals↗

PubMed2004

Infection-induced expansion of a MHC Class Ib-dependent intestinal intraepithelial gammadelta T cell subset.

Salmonella species invade the host via the intestinal epithelium. Hence, intestinal intraepithelial lymphocytes (iIELs) are potentially the first element of the immune system to encounter Salmonella during infection. In this study, we demonstrate, in a mouse model, the expansion of a CD8alphabeta(+)CD94(-)TCRgammadelta(+) T cell subset within the iIEL population in response to oral infection with virulent or avirulent Salmonella. This population can be detected 3 days following infection, represents up to 15% of the TCRgammadelta(+) iIELs, and is dependent on the MHC class Ib molecule T23 (Qa-1). Qa-1 is expressed by intestinal epithelial cells and thus accessible for iIEL recognition. Such cells may play a role in the early immune response to Salmonella.

ATP-Binding Cassette Transporters↗

PubMed2004

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules.

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-alpha, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DC(GM/IL-4)) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DC(GM/IL-4) capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Identification of cryptic MHC I-restricted epitopes encoded by HIV-1 alternative reading frames.

Human immunodeficiency virus (HIV) 1 major histocompatibility complex (MHC) I-restricted epitopes are widely believed to be derived from viral proteins encoded by primary open reading frames. However, the HIV-1 genome contains alternative reading frames (ARFs) potentially encoding small polypeptides. We have identified a panel of epitopes encoded by ARFs within the gag, pol, and env genes. The corresponding epitopic peptides were immunogenic in mice humanized for MHC-I molecules. In addition, cytotoxic T lymphocytes recognizing these epitopes were found in HIV-infected patients. These results reveal the existence of atypical mechanisms of HIV-1 epitope generation. They indicate that the repertoire of epitopes recognized by the cellular anti-HIV-1 immune response is broader than initially thought. This should be taken into account when designing vaccine strategies aimed at activating these responses.

Adult↗

PubMed2004

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes.

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress-inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA-A*0201. These peptides were able to trigger a CTL response in vivo in HLA-A*0201-transgenic HHD mice and in vitro in HLA-A*0201+ healthy donors. p391- and p393-specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA-A*0201-restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA-A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70-derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.

Animals↗

PubMed2004

A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice.

HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice were created and their immunological potential evaluated in response to hepatitis B DNA vaccine. Every single immunized mouse developed hepatitis B virus-specific antibodies, HLA-DR1-restricted helper, and HLA-A2.1-restricted cytolytic T cell responses directed at the same immunodominant epitopes as those identified in naturally infected or vaccinated humans. These mice were specifically protected against a hepatitis B-recombinant vaccinia virus infection with a 10,000-fold or more reduction of the virus load at day 4 post-challenge. These mice represent a unique in vivo experimental model for human immune function studies without any interference with mouse MHC response which dwarfed the prediction of human responses. Furthermore, they enable the complete monitoring of immune adaptative responses for preclinical screening of candidate vaccines.

Amino Acid Sequence↗

PubMed2004

Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Amino Acid Sequence↗

PubMed2004

Adjuvant activities of novel cytokines, interleukin-23 (IL-23) and IL-27, for induction of hepatitis C virus-specific cytotoxic T lymphocytes in HLA-A*0201 transgenic mice.

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-gamma)-producing, HCV-specific CD8+ cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-gamma-producing, HCV-specific CD8+ cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.

Adenoviridae↗

PubMed2004

High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy.

Most of the human tumor-associated antigens (TAAs) characterized thus far are derived from nonmutated "self"-proteins. Numerous strategies have been developed to break tolerance to TAAs, combining various forms of antigens with different vectors and adjuvants. However, no study has yet determined how to select epitopes within a given TAA to induce the highest antitumor effector response. We addressed this question by evaluating in HLA-A*0201-transgenic HHD mice the antitumor vaccination efficacy of high- and low-affinity epitopes from the naturally expressed murine telomerase reverse transcriptase (mTERT). Immunity against low-affinity epitopes was induced with heteroclitical variants. We show here that the CTL repertoire against high-affinity epitopes is partially tolerized, while that against low-affinity epitopes is composed of frequent CTLs with high avidity. The high-affinity p797 and p545 mTERT epitopes are not able to protect mice from a lethal challenge with the mTERT-expressing EL4-HHD tumor. In contrast, mice developing CTL responses against the p572 and p988 low-affinity epitopes exhibit potent antitumor immunity and no sign of autoimmune reactivity against TERT-expressing normal tissues. Our results strongly argue for new TAA epitope selection and modification strategies in antitumor immunotherapy applications in humans.

Animals↗

PubMed2003

EphA2 as target of anticancer immunotherapy: identification of HLA-A*0201-restricted epitopes.

EphA2 (Eck) is a tyrosine kinase receptor that is overexpressed in several human cancers such as breast, colon, lung, prostate, gastric carcinoma, and metastatic melanoma but not in nonmalignant counterparts. To validate EphA2 as a tumor antigen recognized by CD8+ T lymphocytes, we used reverse immunology approach to identify HLA-A*0201-restricted epitopes. Peptides bearing the HLA-A*0201-specific anchor motifs were analyzed for their capacity to bind and stabilize the HLA-A*0201 molecules. Two peptides, EphA2(58) and EphA2(550), with a high affinity for HLA-A*0201 were selected. Both peptides were immunogenic in the HLA-A*0201-transgenic HHD mice. Interestingly, peptide-specific murine CTLs cell lines responded to COS-7 cells coexpressing HLA-A*0201 and EphA2 and to EphA2-positive human tumor cells of various origin (renal cell, lung, and colon carcinoma and sarcoma). This demonstrates that EphA2(58) and EphA2(550) are naturally processed from endogenous EphA2. In addition, EphA2(58) and EphA2(550) stimulated specific CD8(+) T cells from healthy donor peripheral blood mononuclear cells. These T cells recognized EphA2-positive human tumor cells in an HLA-A*0201-restricted manner. Interestingly, EphA2-specific CD8+ T cells were detected in the peripheral blood mononuclear cells of prostate cancer patients. These results show for the first time that EphA2 is a tumor rejection antigen and lead us to propose EphA2(58) and EphA2(550) peptides for a broad-spectrum-tumor immunotherapy.

Animals↗