RESUMEN
We have generated a construct encoding a single-chain H-2D(b) mouse MHC class I molecule in which an influenza virus nucleoprotein (NP) epitope, amino acid sequence ASNENMDAM, is fused to mouse beta(2)-microglobulin and the D(b) H chain via flexible linker sequences. This single-chain trimer (SCT) was efficiently expressed at the cell surface independently of TAP and endogenous beta(2)-microglobulin, and it was recognized directly and efficiently by specific T cells in vitro. A recombinant vaccinia virus encoding the D(b) NP SCT primed a CD8(+) T cell response in C57BL/6 mice 4-fold greater than an equivalent virus expressing the NP epitope as a minigene, as shown by tetramer staining, whether or not the minigene was directed into the endoplasmic reticulum by a signal sequence. This response was functional as shown by in vivo lysis assays with peptide-pulsed target cells, and it was greatly expanded following secondary challenge in vivo with influenza virus. The SCT was also significantly more immunostimulatory for CD8(+) cells than the NP minigene in adoptive transfer experiments using F5 TCR transgenic spleen cells, in which the magnitude of the T cell response was much greater. Our results extend previous DNA vaccination studies using SCTs, which demonstrated that such molecules are capable of generating functional CD8(+) T cell responses. We have shown that class I SCTs are more immunogenic than even preprocessed Ag in the form of an epitope minigene, and they therefore should be considered for use when the generation of optimal CD8(+) T cell responses is required.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Antígenos H-2/inmunología , Nucleoproteínas/inmunología , Orthomyxoviridae/inmunología , Animales , Células CHO , Cricetinae , Cricetulus , Ratones , Ratones Endogámicos C57BL , Microglobulina beta-2/inmunologíaRESUMEN
The survival of dendritic cells (DC) in vivo determines the duration of Ag presentation and is critical in determining the strength and magnitude of the resulting T cell response. We used a mouse model to show that Ag-loaded C57BL/6 DC (MHC class II(+/+) (MHC II(+/+))) that reach the lymph node survived longer than Ag-loaded MHC II(-/-) DC, with the numbers of C57BL/6 DC being approximately 2.5-fold the number of the MHC II(-/-) DC by day 4 and approximately 5-fold by day 7. The differential survival of DC in vivo was not affected by low doses of LPS, but in vitro pretreatment with CD40L or with high doses of LPS increased the numbers of MHC II(-/-) DC to levels approaching those of C57BL/6 DC. Regardless of their numbers and relative survival in lymph nodes, MHC II(-/-) DC were profoundly defective in their ability to induce CTL responses against the gp33 peptide epitope, and were unable to induce expansion and optimal cytotoxic activity of CD8(+) T cells specific for the male Ag UTY. We conclude that CD4(+) T cell help for CD8(+) responses involves mechanisms other than the increased survival of Ag-presenting DC in the lymph node.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Animales , Supervivencia Celular , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunidad Innata/inmunología , Lipopolisacáridos/farmacología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Fenotipo , Glicoproteína IIb de Membrana Plaquetaria/farmacologíaRESUMEN
The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty(246-254) epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7(-/-)) skin grafts by female LMP7(-/-) mice. Second, we ruled out an altered Uty(246-254)-specific T cell repertoire in LMP7(-/-) female mice and demonstrated efficient Uty(246-254) presentation by re-expressing LMP7 in male LMP7(-/-) cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty(246-254)-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7(-/-) female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.
Asunto(s)
Reactividad Cruzada/inmunología , Complejo de la Endopetidasa Proteasomal/fisiología , Animales , Línea Celular Tumoral , Reactividad Cruzada/genética , Epítopos de Linfocito T/genética , Femenino , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Antígenos H-2/genética , Antígeno H-Y/genética , Antígeno de Histocompatibilidad H-2D , Vigilancia Inmunológica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/fisiología , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/genética , Factores Sexuales , Trasplante de Piel/inmunología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
We describe the cloning and characterization of Siglec-H, a novel murine CD33-related siglec-like molecule with 2 immunoglobulin domains. Unlike other CD33-related siglecs, Siglec-H lacks tyrosine-based signaling motifs in its cytoplasmic tail. Although Siglec-H has the typical structural features required for sialic acid binding, no evidence for carbohydrate recognition was obtained. Specific monoclonal and polyclonal antibodies (Abs) were raised to Siglec-H and used to define its cellular expression pattern and functional properties. By flow cytometry, Siglec-H was expressed specifically on plasmacytoid dendritic cell (pDC) precursors in bone marrow, spleen, blood, and lymph nodes. Staining of tissue sections showed that Siglec-H was also expressed in a subset of marginal zone macrophages in the spleen and in medullary macrophages in lymph nodes. Using bone marrow-derived pDC precursors that express Siglec-H, addition of Abs did not influence cytokine production, either in the presence or absence of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG). In comparison, Siglec-H functioned as an endocytic receptor and mediated efficient internalization of anti-Siglec-H Abs. By immunizing mice with ovalbumin-conjugated anti-Siglec-H Ab in the presence of CpG, we demonstrate generation of antigen-specific CD8 T cells in vivo. Targeting Siglec-H may therefore be a useful way of delivering antigens to pDC precursors for cross-presentation.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Endocitosis/inmunología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Clonación Molecular , ADN Complementario/genética , Células Dendríticas/citología , Expresión Génica , Células Madre Hematopoyéticas/citología , Ratones , Ratones Endogámicos , Ratones Noqueados , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/metabolismo , Células Plasmáticas/citología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.
Asunto(s)
Epítopos Inmunodominantes , Poxviridae/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/inmunología , Virus Vaccinia/inmunología , Antígenos HLA-A , Antígeno HLA-A2 , Humanos , Cinética , Melanoma/inmunología , Melanoma/terapia , Especificidad del Receptor de Antígeno de Linfocitos T , Vacunas Sintéticas/normas , Vacunas Sintéticas/uso terapéuticoRESUMEN
A characteristic of the secondary response of CD8(+) T cells that distinguishes it from the primary response is the generation of greater numbers of effector cells. Because effector CD8(+) T cells are derived from a pool of less differentiated, replicating cells in secondary lymphoid organs, and because IL-2 mediates effector differentiation, the enhanced secondary response may reflect the enlargement of this generative pool by the transient repression of IL-2-mediated differentiation. We have examined for this function the transcriptional repressor BCL6b, a homologue of BCL6 that represses IL-2-induced B cell differentiation. BCL6b is expressed in a small subset of antigen-experienced CD8(+) T cells. Ectopic expression of BCL6b in CD8(+) T cells diminishes their growth in response to IL-2 in vitro. Female mice in which the BCL6b gene has been interrupted have normal primary responses of CD8(+) T cells to infection with vaccinia expressing the H-Y epitope, Uty, but Uty-specific, BCL6b(-/-), memory CD8(+) T cells have diminished recall proliferative responses to this epitope in vitro. BCL6b(-/-) mice also have normal primary CD8(+) T cell responses to influenza infection, but nucleoprotein peptide-specific, BCL6b(-/-), memory CD8(+) T cells have a cell autonomous defect in the number of effector cells generated in response to reinfection. Therefore, BCL6b is required for the enhanced magnitude of the secondary response of memory CD8(+) T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Memoria Inmunológica , Proteínas Represoras/metabolismo , Elementos Silenciadores Transcripcionales/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Cartilla de ADN , Epítopos/metabolismo , Femenino , Componentes del Gen , Marcación de Gen , Vectores Genéticos , Antígeno H-Y/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Ratones , Ratones Noqueados , Virus de la Leucemia Murina de Moloney , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vaccinia/inmunologíaRESUMEN
Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a "prime-boost" DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A(26-35) analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naive patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029-0.19% CD8(+) T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.
Asunto(s)
Antígenos de Neoplasias/inmunología , Melanoma/inmunología , Melanoma/terapia , Recurrencia Local de Neoplasia/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapia , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/inmunología , Adulto , Anciano , Formación de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Epítopos , Femenino , Ingeniería Genética , Humanos , Inmunoterapia/métodos , Masculino , Melanoma/patología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Plásmidos/genética , Neoplasias Cutáneas/patología , Vacunas de ADN , Virus Vaccinia/genéticaRESUMEN
Plasmacytoid dendritic cells (PDCs) are a unique leukocyte population capable of secreting high levels of type I interferon (IFN) in response to viruses and bacterial stimuli. In vitro experiments have shown that upon maturation, human and murine PDCs develop into potent immunostimulatory cells; however, their ability to prime an immune response in vivo remains to be addressed. We report that CpG-matured murine PDCs are capable of eliciting in naive mice antigen-specific CTLs against endogenous antigens as well as exogenous peptides, but not against an exogenous antigen. Type I IFN is not required for priming, as injection of CpG-matured PDCs into type I IFN receptor-deficient mice elicits functional CTL responses. Mature PDCs prime CTLs that secrete IFN-gamma and protect mice from a tumor challenge. In contrast, immature PDCs are unable to prime antigen-specific CTLs. However, mice injected with immature PDCs are fully responsive to secondary antigenic challenges, suggesting that PDCs have not induced long-lasting tolerance via anergic or regulatory T cells. Our results underline the heterogeneity and plasticity of different antigen-presenting cells, and reveal an important role of mature PDCs in priming CD8 responses to endogenous antigens, in addition to their previously reported ability to modulate antiviral responses via type I IFN.
Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Fosfatos de Dinucleósidos/inmunología , Isoantígenos/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Secuencia de Aminoácidos , Animales , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Fluorometría/métodos , Células Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos/administración & dosificación , Línea Celular , Citometría de Flujo/métodos , Colorantes Fluorescentes , Humanos , Inmunización , Técnicas In Vitro , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1(157-165)-specific CD8(+) cells, detected ex vivo with an A2/H-2K(b) chimeric class I tetramer. These NY-ESO-1(157-165)-specific CD8(+) cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1(157-165)-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.
Asunto(s)
Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/genética , Citotoxicidad Inmunológica/genética , VIH-1/genética , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/genética , Linfocitos T Citotóxicos/inmunología , Transducción Genética/métodos , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Presentación de Antígeno/genética , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Vectores Genéticos , VIH-1/inmunología , Humanos , Inyecciones Intravenosas , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones TransgénicosRESUMEN
Immunotherapy could be combined with conventional chemotherapeutic modalities aimed at reducing tumor burden. Such combination therapy may be most useful when "metronomic" doses of antineoplastic drugs are used, thereby potentially avoiding some of the immunosuppressive effects of these drugs. Recent studies have shown that some conventional antineoplastic drugs can be exploited for antiangiogenic capacities, a strategy that requires drugs to be administered at regular intervals. We therefore investigated whether such metronomic therapy with the alkylating agent cyclophosphamide (CTX) could be effectively combined with immunotherapy eliciting tumor-reactive CTLs. An immunization protocol using injection of recombinant DNA followed by injection of recombinant modified vaccinia virus Ankara strain was used to initiate a specific CTL response in mice capable of providing resistance to challenge with the murine melanoma B16.F10. Combining this immunotherapeutic regime with metronomic delivery of CTX resulted in antitumor activity that was dramatically enhanced over either treatment administered alone and was also significantly greater than combining immunotherapy with CTX administered by a maximum tolerated dose regime. Whereas both metronomic and maximum tolerated dose delivery of CTX did cause deletion of proliferating tumor-specific CTLs in the blood, this deletion occurred with slower kinetics with the metronomic schedule. Further analysis showed that metronomic CTX treatment did not delete cells with low expression of CD43, a "memory" phenotype, and that these cells maintained potent restimulatory capacity. The combination of immunotherapy and metronomic CTX therapy may be well suited to clinical management of cancer.
Asunto(s)
Antineoplásicos Alquilantes/administración & dosificación , Ciclofosfamida/administración & dosificación , Melanoma Experimental/terapia , Vacunación/métodos , Animales , Terapia Combinada , Esquema de Medicación , Epítopos de Linfocito T/biosíntesis , Epítopos de Linfocito T/inmunología , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Antígenos H-2/genética , Antígenos H-2/inmunología , Antígeno HLA-A1/genética , Antígeno HLA-A1/inmunología , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno de Histocompatibilidad H-2D , Recuento de Leucocitos , Masculino , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Vacunas de ADN/inmunología , Vacunas de ADN/farmacología , Virus Vaccinia/genéticaRESUMEN
Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.
Asunto(s)
Antígenos CD8/análisis , Citotoxicidad Inmunológica , Epítopos de Linfocito T/análisis , Proteínas de la Membrana , Coloración y Etiquetado , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Animales , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/metabolismo , Sitios de Unión/genética , Sitios de Unión/inmunología , Antígenos CD8/genética , Antígenos CD8/metabolismo , Línea Celular , Línea Celular Tumoral , Células Clonales , Citotoxicidad Inmunológica/genética , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Inmunización Secundaria , Células Jurkat , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Plásmidos/administración & dosificación , Proteínas/análisis , Proteínas/genética , Proteínas/metabolismo , Linfocitos T Citotóxicos/química , Vaccinia/genética , Vaccinia/inmunología , Microglobulina beta-2/análisis , Microglobulina beta-2/metabolismoRESUMEN
Plasmacytoid dendritic cells (PDC) are a small population of leukocytes specialized in the production of type I IFN. It has been shown that PDC have a potent T cell stimulatory capacity in allogeneic mixed lymphocyte reaction, However, their role in initiating primary immune responses remains elusive. We report that blood PDC efficiently prime naive CD8(+) lymphocytes specific for the melan-A(26-35) epitope to become IFN-gamma producing cells in vitro. In addition, we found that CD40L-stimulated PDC induce expression on primed melan-A-specific T cells of cutaneous lymphocyte antigen and L-selectin (CD62L), homing receptors that allow the migration of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri-tumoral area of most primary cutaneous melanomas in vivo and that type I IFN-containing supernatants derived from PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro. Our results suggest a new immunomodulatory role for tissue infiltrating PDC, which may prime tumor-specific T cell responses and affect tumor growth via soluble factors.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Antígenos de Diferenciación de Linfocitos T , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Células Dendríticas/clasificación , Antígenos de Histocompatibilidad/biosíntesis , Humanos , Interferón Tipo I/farmacología , Interleucina-12/farmacología , Activación de Linfocitos , Antígeno MART-1 , Melanoma/patología , Glicoproteínas de Membrana/biosíntesis , Proteínas de Neoplasias/inmunología , Células Tumorales Cultivadas , Receptor fas/biosíntesisRESUMEN
Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2K(b) H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.