RESUMEN
Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. Here we describe a recombinant poxvirus vaccine that codes for ten HLA-A2-restricted epitopes derived from five melanoma Ags conjoined in an artificial polyepitope or polytope construct. Target cells infected with the melanoma polytope vaccinia were recognized by three different epitope-specific CTL lines derived from HLA-A2 melanoma patients, and CTL responses to seven of the epitopes were generated in at least one of six HLA-A2-transgenic mice immunized with the construct. CTL lines derived from vaccinated transgenic mice were also able to kill melanoma cells in vitro. Multiple epitopes within the polytope construct were therefore shown to be individually immunogenic, illustrating the feasibility of the polytope approach for melanoma immunotherapy. Tumor escape from CTL surveillance, through down regulation of individual tumor Ags and MHC alleles, might be overcome by polytope vaccines, which simultaneously target multiple cancer Ags.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/inmunología , Melanoma/inmunología , Melanoma/terapia , Vacunación/métodos , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/uso terapéutico , Secuencia de Bases , Vacunas contra el Cáncer/genética , Pruebas Inmunológicas de Citotoxicidad , Epítopos de Linfocito T/genética , Antígeno HLA-A2/genética , Humanos , Activación de Linfocitos , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/uso terapéutico , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas , Virus Vaccinia/genética , Virus Vaccinia/inmunologíaRESUMEN
The immunodominant, CD8(+) cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein-Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide-RSKFRQIV-located in a serine/threonine kinase and a bacterial peptide-RRKYKQII-located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted alphabeta TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8(+) CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Antígeno HLA-B8/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Imitación Molecular , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Proteínas Bacterianas/inmunología , Secuencia de Bases , Citotoxicidad Inmunológica , ADN Helicasas/inmunología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/inmunología , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Herpesvirus Humano 4/inmunología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Staphylococcus aureus/inmunología , Linfocitos T Citotóxicos/clasificación , Transactivadores/química , Transactivadores/inmunología , Proteínas Virales/inmunologíaRESUMEN
Five healthy human leukocyte antigen-B8 (HLA-B8)-positive virus carriers were studied to investigate the CD8+ cytotoxic T lymphocyte (CTL) response to an HLA-B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early trans-activator protein, BZLF1. Of the 5 virus carriers, 4 were infected with type A and 1 with type B EBV. Using limiting-dilution analysis of peripheral blood mononuclear cells, a high RAKFKQLLQ-specific CTL precursor frequency was demonstrated after specific peptide or autologous lymphoblastoid cell line stimulation in both type A and type B EBV carriers. The RAKFKQLLQ-specific CTL precursor frequencies in all 5 persons were at least as dominant as those observed with two other EBV-associated, HLA-B8-restricted latent epitopes, FLRGRAYGL and QAKWRLQTL. These findings show that healthy virus carriers maintain a high frequency of BZLF1-specific memory T cells, potentially to control virus spread from lytically infected cells.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/inmunología , Linfocitos T Citotóxicos/inmunología , Transactivadores/inmunología , Antígenos Virales/análisis , Antígenos Virales/inmunología , Antígenos CD8/inmunología , Enfermedad Crónica , Pruebas Inmunológicas de Citotoxicidad , Epítopos/análisis , Epítopos/inmunología , Antígeno HLA-B8/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Memoria Inmunológica , Leucocitos Mononucleares/inmunología , Proteínas Virales/análisis , Proteínas Virales/inmunologíaRESUMEN
We investigated the CD8+ cytotoxic T lymphocyte (CTL) repertoire to an HLA B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early protein, BZLF1. Repertoire selection was monitored by determining the TCR beta chain sequences of RAKFKQLLQ-specific CTL established from primary infected and healthy virus carriers. PCR analysis of spontaneous EBV-transformed lymphoblastoid cell lines (LCL) from three individuals with primary infection showed that two were infected with type A and one with type B EBV. Polyclonal and clonal CTL that were generated by stimulating peripheral blood mononuclear cells with an HLA B8+ homozygous LCL lysed T cell blasts pulsed with the peptide, RAKFKQLLQ; lysis of certain HLA B8+ LCL targets was associated with the abundance of BZLF1 transcripts. TCR beta analysis showed that while there was loop length restriction in the putative peptide contact site of all responding beta chains, diverse and unique (non-recurrent) TCR beta clonotypes were selected in individuals during primary infection and continued to emerge after long-term virus exposure. TCR-contact site heterogeneity was excluded as the selective force in diversity generation since the epitope-encoded sequences were found to be identical within endogenous virus isolates. In this first study of TCR repertoire selection for an EBV lytic antigen, a BZLF1-reactive component of diverse clonotypes was identified in primary type A or type B EBV infection which was sustained in the EBV-specific memory response throughout life-long infection. This diversity selection is likely to play a critical role in maintaining a balanced viral load throughout EBV persistence.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al ADN/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 4/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Citotóxicos/inmunología , Transactivadores/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Portador Sano , Proteínas de Unión al ADN/genética , Epítopos/inmunología , Infecciones por Herpesviridae/sangre , Infecciones por Herpesviridae/virología , Herpesvirus Humano 4/genética , Humanos , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Transactivadores/genética , Proteínas Virales/genéticaRESUMEN
The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.