Biomedical subjects
Publications and source records attributed to Eric Tartour.
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PURPOSE: The human telomerase reverse transcriptase (hTERT) is considered as a potential target for cancer immunotherapy because it is preferentially expressed in tumor cells. To increase the applicability of hTERT-based immunotherapy, we set out to identify CTL epitopes in hTERT restricted by HLA-B*0702 molecule, a common MHC class I allele. EXPERIMENTAL DESIGN: HLA-B*0702-restricted peptides from hTERT were selected by using a method of epitope prediction and tested for their immunogenicity in human (in vitro) and HLA-B*0702 transgenic mice (in vivo). RESULTS: All the six hTERT peptides that were predicted to bind to HLA-B*0702 molecule were found to induce primary human CTL responses in vitro. The peptide-specific CD8+ CTL lines were tested against various hTERT+ tumor cells. Although differences were observed according to the tumor origin, only three CTL lines specific for p277, p342, and p351 peptides exhibited cytotoxicity against tumor cells in a HLA-B*0702-restricted manner. In addition, this cytotoxicity was inhibited by the addition of peptide-loaded cold target cells and indicated that these epitopes are naturally processed and presented on the tumor cells. Further, in vivo studies using humanized HLA-B*0702 transgenic mice showed that all the candidate peptides were able to induce CTL responses after peptide immunization. Furthermore, vaccination with a plasmid DNA encoding full-length hTERT elicited peptide-specific CTL responses, indicating that these epitopes are efficiently processed in vivo. CONCLUSIONS: Together with previously reported hTERT epitopes, the identification of new CTL epitopes presented by HLA-B*0702 increases the applicability of hTERT-based immunotherapy to treating cancer.
PURPOSE: CD4(+) T cells play a central role in initiating and maintaining anticancer immune responses. However, regulatory CD4(+)CD25(+) T cells which express Foxp3 have also been shown to inhibit antitumor effector T cells. In view of these heterogeneous CD4(+) T-cell populations, this study was designed to determine the prognostic value of various tumor-infiltrating CD4(+) T-cell populations in head and neck squamous cell carcinoma. EXPERIMENTAL DESIGN: Eighty-four newly diagnosed untreated patients with histologically proven primary head and neck squamous cell carcinoma were included in this study. Double or triple immunofluorescence staining was done to assess and quantify the activated CD4(+)CD69(+) T cells, regulatory CD4(+)Foxp3(+) T cells, and mixed CD4(+)CD25(+) T cells comprising both activated and regulatory T cells. RESULTS: On univariate analysis, high levels of tumor-infiltrating CD4(+)CD69(+) T cells were correlated with both better locoregional control (P = 0.01) and longer survival (P = 0.01). Infiltration by regulatory Foxp3(+)CD4(+) T cells was positively associated with a better locoregional control of the tumor. Multivariate analysis showed that the only significant prognostic factors related to locoregional control were T stage (P = 0.02) and CD4(+)Foxp3(+) T-cell infiltration of the tumor (P = 0.02). In the Cox multivariate analysis, only two variables influenced overall survival probability: T stage (P = 0.036) and CD4(+)CD69(+) T-cell infiltration (P = 0.017). CONCLUSION: This study shows that tumor-infiltrating activated CD4(+)CD69(+) T cells are associated with a good prognosis in head and neck squamous cell carcinoma. In addition, regulatory Foxp3(+)CD4(+) T cells are positively correlated with locoregional control may be through down-regulation of harmful inflammatory reaction, which could favor tumor progression.
The non-toxic B-subunit of Shiga toxin (STxB) interacts with the glycolipid Gb3, which is preferentially expressed on dendritic cells (DC) and B cells. After administration of STxB chemically coupled to OVA (STxB-OVA) in mice, we showed that the immunodominant OVA(257-264) peptide restricted by K(b) molecules is specifically presented by CD11c+ CD8alpha- DC, some of them displaying a mature phenotype. Using mice carrying a transgene encoding a diphtheria toxin receptor (DTR) under the control of the murine CD11c promoter, which allows inducible ablation of DC, we showed that DC are required for efficient priming of CTL after STxB-OVA vaccination. Immunization of mice with STxB-OVA induced OVA-specific CD8+ T cells detected ex vivo; these cells were long lasting, since they could be detected even 91 days after the last immunization and were composed of both central and memory T cells. Vaccination of mice with STxB-OVA and STxB coupled to E7, a protein derived from HPV16, inhibited tumor growth in prophylactic and therapeutic experiments. This effect was mainly mediated by CD8+ T cells. STxB therefore appears to be a powerful carrier directly targeting DC in vivo, resulting in a strong and durable CTL response associated with tumor protection.
In preclinical tumor model and in human cancer, tumor antigen specific T lymphocytes play a key role in the control of tumor development. Nevertheless in numerous cases, the infiltrating tumor T cells do not seem to influence the clinical progression of the tumor. A better phenotypic and functional characterization of T cells in close contact with tumor associated with a comprehensive analysis of tumor evasion mechanism to the host response should lead to an optimization of cancer immunotherapy protocols.
The conventional enzyme-linked immunospot (ELISPOT) technique detects only one secreted cytokine, which constitutes a major pitfall for the accurate characterization of the various T-cell subpopulations. We have therefore developed a fluorospot assay, which is a modification of the ELISPOT and is based on the use of multiple fluorescent-labeled anticytokines detection antibodies. A special automated ELISPOT reader consisting of a light microscope with incident fluorescence illumination and an integrating digital color camera has been adapted for this technique. This technique has been applied for the analysis of subpopulations of T-cells and polarized antigen-specific T-cells.
Interleukin-17 (IL-17) is a proinflammatory cytokine mainly produced by activated CD4+ CD45RO T cells. In mice, we have demonstrated that, depending on the model, IL-17 may act as a tumor growth-promoting or -inhibiting factor. In order to address the relevance of these models in human tumors, we look for the natural expression and activity of IL-17 in mycosis fungoides (MF) and Sezary syndrome (SS). These cutaneous T-cell lymphomas were selected because they are usually CD3+ CD4+ CD45RO+, a phenotype similar to nontransformed T cells producing IL-17. We show that in vitro activated malignant T cells derived from MF or SS patients express IL-17 mRNA and secrete this cytokine. However, IL-17 does not act in vitro as a growth factor for MF or SS cell lines. In addition, 5 out of 10 MF/SS biopsies expressed IL-17 mRNA, while this cytokine was not detected in normal skin. IL-17 was not observed in the biopsies derived from 2 patients initially identified as MF, whereas an upregulation of this cytokine was clearly demonstrated during progression of the disease in these patients. An association between IL-17 expression and polymorphonuclear neutrophil infiltration was also recorded in this group of MF/SS patients. A more detailed analysis of 2 patients with a pustular form of MF and SS revealed that IL-17 may participate in the recruitment of polymorphonuclear neutrophils via a paracrine mechanism involving keratinocyte-released IL-8. This study is the first report demonstrating that some human tumor cells could express IL-17, a cytokine that represents an early event in the development of the inflammatory reaction within the tumor microenvironment, a process that may influence tumor phenotype and growth.
Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.
Immunodeficiency related to HIV may increase the incidence of EBV-associated lymphomas, by altering EBV-specific immune control and consequently favoring EBV reactivation. The aim of the present study was to assess the relationship between the decrease of EBV-specific cellular immunity and the increase of EBV reactivation in a prospective cohort of 72 unselected HIV-infected individuals. EBV-specific immunity was evaluated by a highly sensitive IFN-gamma ELISPOT assay using 22 peptides mimicking latent and lytic antigens, and circulating mononuclear (PBMC) EBV DNA load was quantified by real-time quantitative PCR. The mean circulating cell-associated EBV DNA load was higher in HIV-infected patients (639 copies/10(6) PBMC) than in healthy controls (21, n = 14) ( P = 0.005) and was higher in patients with CD4(+) T-cell count below 350/microL than that in patients harboring higher CD4(+) T-cell count (1112 vs. 389, P = 0.003). The mean intensity of EBV-specific cellular responses was lower in HIV-infected patients than in controls ( P = 0.001), even in patients with CD4(+) T-cell count above 350/-microL ( P = 0.007). The number of EBV peptides recognized was lower in HIV-infected patients than in controls (frequency: 0.44 vs. 0.67; P = 0.02), indicating reduced polyclonality in HIV-infected patients. The polyclonality was 1.5-fold lower in HIV-infected patients with CD4(+) T-cell count below 350/-microL ( P =0.007). For EBV load >1000 copies/10(6) PBMC, the levels of cell-associated EBV DNA and those of EBV-specific cellular immunity, either in intensity or in polyclonality, or both, were inversely correlated. These findings demonstrate early impairment of the EBV-specific cellular immune control with progressive increase of EBV reactivation in the course of HIV infection. These observations likely provide a basis for appreciating the risk to develop non-Hodgkin's lymphomas in HIV-infected individuals.
BACKGROUND: The MUC1 protein is a highly glycosylated mucin normally found at the apical surface of mucin-secreting epithelial cells in many types of tissues. MUC1 is expressed, but heavily underglycosylated, in different human tumors. TG4010 is a viral suspension of a recombinant vaccinia vector (MVA) containing DNA sequences coding for the human MUC1 antigen and interleukin-2 (IL-2). This product was developed for use as a vaccine in cancer patients whose tumors express the MUC1 antigen. The objective of the present study was to determine the safety of the product and to define the dose of TG4010 to be used in further clinical trials. MATERIALS AND METHODS: Thirteen patients with different solid tumors were treated by repeated intramuscular injection with increasing doses of TG4010 in two separate phase I studies, one in Europe (Basel-CR) and one in the United States (UCLA-RF): a total of 6 patients were treated at a dose of 5 x 10(6) pfu, 3 patients at 5 x 10(7) pfu, and 4 patients at 10(8) pfu. Safety, efficacy, and different immunological tests were the endpoints of the study. RESULTS: Tolerance of TG4010 was excellent, and side effects mainly consisted of injection site pain and influenza-like symptoms. There was no apparent detrimental effect of repeated injections of the vaccinia virus. Four of thirteen evaluable patients showed stabilization of their disease for 6 to 9 months. One lung cancer patient who was initially progressing after the first injections later showed a marked decrease in the size of his metastases that lasted for 14 months. Some T cell proliferative immune responses were seen in five patients. CONCLUSIONS: The administration of TG4010 was generally well tolerated in patients with metastatic tumors, and transient disease stabilization was observed in several patients, warranting further clinical studies with the product.
Various subpopulations of T lymphocytes-i.e. Type 1, Type 2, Tr1 T cells-play a major role in the homeostasis of the immune system and in the pathogenesis of many inflammatory and auto-immune diseases. At present, in the absence of specific surface markers, these T cells can only be reliably distinguished on the basis of their cytokine production profile. The Elispot assay detects cytokine-producing cells, but in most cases can detect only one secreted cytokine, which represents a major limitation of this technique. We have developed a Fluorospot assay to detect single cells that simultaneously produce multiple cytokines. The Fluorospot assay permits the detection of regulatory T cells with an immunosuppressive activity, identified by their coexpression of IL-10 and IFNgamma. Polarized type 1 and type 2 specific tetanus toxoid T cells are also directly detected using a dual color Fluorospot. This technique will therefore be useful for detailed analysis of T lymphocytes in various disease states in which an imbalance of T cell subpopulations is suspected, but will also provide a better characterization of polarized specific immune responses.
A number of studies in animal models and humans have shown that both humoral andcell-mediated immune responses play an important role in the control of viral infection and tumor development. In most cases, vaccination with non-vectorized peptides or proteins induces low antibody responses and fails to elicit specific cytotoxic T lymphocytes (CTL). In order to make vaccination more efficient, we chemically coupled the non-toxic B subunit of Shiga toxin (STxB) to a full-size antigenic model protein, ovalbumin (OVA), yielding STxB-OVA. We found that STxB-OVA delivers OVA-derived peptides into both the MHC class I- and II-restricted presentation pathways in mouse dendritic cells. Accordingly, the study of STxB trafficking in these cells revealed that, after internalization, a fraction of STxB followed the retrograde transport pathway to the endoplasmic reticulum, while another fraction was targeted to late endosomes/lysosomes. Vaccination of mice with STxB-OVA primed a specific anti-OVA CTL response without the use of adjuvants. Splenocytes and, particularly, CD4(+) T cells from mice immunized with STxB-OVA also produced higher amounts of the T(h)1 cytokine IFN-gamma and IgG2a-type antibodies than mice immunized with non-vectorized ovalbumin. In conclusion, this study identifies a unique non-live vaccine delivery system for polyepitopic antigens that elicits antigen-specific CTL, a humoral immune response and a T(h)1-type polarization without the use of adjuvant.
Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated CD4(+) memory T cells. We previously showed that IL-17 increased the growth rate of human cervical tumors transplanted into athymic nude mice. To address the possible role of T cells in the biologic activity of IL-17 for tumor control, we grafted 2 murine hematopoietic immunogenic tumors (P815 and J558L) transfected with a complementary DNA encoding murine IL-17 into syngeneic immunocompetent mice. We found that growth of the 2 IL-17-producing tumors was significantly inhibited compared with that of mock-transfected tumors. In contrast to the antitumor activity of IL-17 observed in immunocompetent mice, we observed no difference in the in vivo growth of IL-17-transfected or mock-transfected P815 cells (P815-IL-17 and P815-Neo, respectively) transplanted into nude mice. We then showed that IL-17 increased generation of specific cytolytic T lymphocytes (CTLs) directed against the immunodominant antigens from P815 called A, B, C, D, and E, since all mice injected with P815-IL-17 developed a P815-specific CTL response, whereas only 6 of 16 mice immunized with P815-Neo had a specific CTL response against the antigens. The induction of CTLs was associated with establishment of a tumor-protective immunity. These experiments suggest that T lymphocytes are involved in the antitumor activity of IL-17. Therefore, IL-17, like other cytokines, appears to be a pleiotropic cytokine with possible protumor or antitumor effects on tumor development, which often depends on the immunogenicity of tumor models.
A number of bacterial toxins have evolved diverse strategies for crossing membrane barriers in order to reach their substrates in the mammalian cytosol. Recent studies show that this property can be exploited for the delivery of fused antigens into the major histocompatibility complex class I-restricted presentation pathway, with the goal of eliciting a specific immune response. Here we discuss the peculiarities of the trafficking pathways of a variety of toxins, and how these may allow the toxins to be used as delivery vehicles for therapeutic and diagnostic purposes.
Experiments in mice and recent human clinical studies have clearly shown the contribution of CD8+ T lymphocyte in the control of tumor development. CD8+ T lymphocytes are a constitutive component of the immune response during the development of cancer. In murine models, the efficiency of various cancer vaccines mainly depends on their ability to induce CD8+ T lymphocytes. Clinical responses in immunotherapy treated cancer patients have been associated with the presence of antitumor specific CD8+ T lymphocytes. In spontaneous regressive melanomas, intratumor antigen specific CD8+ cytotoxic T cells were expanded suggesting their involvement in the tumor shrinkage. Administration of antitumor specific cytotoxic T clones in mice resulted in antitumor responses which directly demonstrated the therapeutic efficiency of these cells. However, in most cases during cancer progression, the presence of antitumor CD8 T lymphocyte is not associated with clinical responses. Intrinsic functional abnormalities of these cells or a defect of CD8+ T cell migration to the tumor may in part explain their failure to inhibit tumor development. On the other hand, tumors also develop immune escape mechanisms (down modulation of tumor antigens, secretion of immunosuppressive factors, expression of anti-apoptotic molecules by the tumors, or pro-apoptotic factors inducing T cell death) to resist to the CD8+ T cell attack. To circumvent these tumor escape mechanisms, efficient cancer vaccines will have to recruit CD8+ T cells associated with other immune effectors.