RESUMO
Bacterial cell wall polysaccharides, such as PGN, bind and activate TLR-2, NOD2 and PGRP on monocytes/macrophages and activate inflammation. We found that the peptides containing basic amino acids (cations) at N -terminus and tyrosine at C-terminus interfered with activating ability of PGN. This finding is significant because the ECD of TLR-2 in vivo encounters a large number of proteins or peptides. Some should bind ECD and "pre-form" TLR-2 to respond or not to its activators, although they cannot activate TLR-2 alone. TLR-2 is receptor for a large number of ligands, including lipopeptides and bacterial cell wall glycoproteins. A binding site for lipopeptides has been identified; however, a binding site for soluble or multimeric PGN has not been proposed. To identify the candidate binding sites of peptides and PGN on TLR-2, we modeled docking of peptides and of the PGN monomer (PGN-S-monomer) to extracellular domain (ECD-TLR-2) of the unbound TLR-2. Quantification, in silico, of free energy of binding (DG) identified 2 close sites for peptides and PGN. The PGN-S-monomer binding site is between amino acids TLR-2, 404-430 or more closely TLR-2, 417-428. The peptide-binding site is between amino acids TLR-2, 434-455. Molecular models show PGN-S-monomer inserts its N -acetyl-glucosamine (NAG) deep in the TLR-2 coil, while its terminal lysine interacts with inside (Glu(403)) and outside pocket (Tyr(378)). Peptides insert their two N -terminal arginines or their C-terminal tyrosines in the TLR-2 coil. PGN did not bind the lipopeptide-binding site in the TLR-2. It can bind the C-terminus, 572-586 (DG = 0.026 kcal), of "lipopeptide-bound" TLR-2. An additional, low-affinity PGN-binding site is TLR-2 (227-237). MTP, MDP, and lysine-less PGN bind to TLR-2, 87-113. This is the first report identifying candidate binding sites of monomer PGN and peptides on TLR-2. Experimental verification of our findings is needed to create synthetic adjuvant for vaccines. Such synthetic PGN can direct both adjuvant and cancer antigen to TLR-2.
Assuntos
Sítios de Ligação , Modelos Moleculares , Peptidoglicano/química , Receptor 2 Toll-Like/química , Sequência de Aminoácidos , Biologia Computacional , Glutamina/química , Humanos , Ativação de Macrófagos/imunologia , Dados de Sequência Molecular , Peptidoglicano/imunologia , Peptidoglicano/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Serina/química , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismoRESUMO
ERBB2/neu and Notch signaling are known to be deregulated in many human cancers. However, pathway cross-talk and dependencies are not well understood. In this study, we use an ERBB2-transgenic mouse model of breast cancer (neuT) to show that Notch signaling plays a critical role in tumor maintenance. Inhibition of the Notch pathway with a gamma-secretase inhibitor (GSI) decreased both the Notch and the mammalian target of rapamycin/AKT pathways. Antitumor activity resulting from GSI treatment was associated with decreased cell proliferation as measured by Ki67 and decreased expression of glucose transporter Glut1. Positron emission tomography (PET) imaging showed that the functional consequences of decreased Glut1 translated to reduced glucose uptake and correlated with antitumor effects as measured by micro-computed tomography imaging. The decrease of Glut1 in neuT tumors was also observed in several human breast cancer cell lines following GSI treatment. We provide evidence that approximately 27% of ERBB2-positive human breast cancer specimens display high expression of HES1, phospho-S6RP, and GLUT1. Together, these results suggest that pathways downstream of Notch signaling are, at least in part, responsible for promoting tumor growth in neuT and also active in both neuT and a subset of human breast cancers. These findings suggest that GSI may provide therapeutic benefit to a subset of ERBB2-positive breast cancers and that [(18)F]FDG-PET imaging may be useful in monitoring clinical response.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Glucose/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Oncogênica v-akt/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/metabolismo , Receptores Notch/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Óxidos S-Cíclicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Fluordesoxiglucose F18/farmacocinética , Transportador de Glucose Tipo 1/biossíntese , Humanos , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Oncogênica v-akt/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Tiadiazóis/farmacologiaRESUMO
Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients.
Assuntos
Antagonistas de Androgênios/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Transporte/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Sirolimo/farmacologia , Antagonistas de Androgênios/administração & dosagem , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Imageamento Tridimensional/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR , Ultrassonografia/métodosRESUMO
Mobilization of tumor-reactive CD8+ T cells remains the major challenge of cancer immunotherapy. Knowing how and when the T cell response expands and differentiates after antigen stimulation would make a significant contribution to the development of tumor vaccines. In the current study, we used CFSE-based cell sorting and cDNA microarray to identify the gene expression profile of adjacent generations of T cells after PHA stimulation. Early-divided generations of T cells responded to stimulation by activating cell cycle and surviving gene pathways, while late generations of T cells had more dramatic changes in transcription of cytokine genes. Reconstruction of biochemical pathways, activated in both early and late generations of T cells, also confirmed the impact of division in focal-adhesion kinases. Because most tumors are infiltrated by lymphocytes, our studies indicate a novel approach to identify 'systemic biological responses' of T cells, which could determine the design, and optimization of effective tumor vaccines.
Assuntos
DNA Complementar , Mitógenos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Citometria de Fluxo , Fluoresceína , Corantes Fluorescentes , Perfilação da Expressão Gênica , Humanos , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Fito-Hemaglutininas/farmacologiaRESUMO
Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-alpha/beta) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-alpha. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-alpha/beta response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-gamma in PBMC. The levels of IFN-gamma were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Influenza A/imunologia , Ativação Linfocitária , Linfócitos T Citotóxicos/imunologia , Proteínas não Estruturais Virais/fisiologia , Regulação Viral da Expressão Gênica , Humanos , Linfócitos T Citotóxicos/virologia , Células Tumorais Cultivadas , Proteínas não Estruturais Virais/químicaRESUMO
MicroRNA (miRNA) are a class of non-coding RNAs found both in normal tissues and cancer cells. The natural miRNA, which target cancer genes for transcriptional repression are still unknown. Approaches for synthetic miRNA design targeting cancer genes have not yet been established. We designed miRNAs targeting the 3' UTR (nt 4350-4372) of HER-2 proto-oncogene. One miRNA (miR-14U) was designed by introducing a mutation in the nt 14 counting from the 5' end of anti-sense RNA. Two others (miR4350-10GGA and miR4350-11AAGCU) were designed by introducing either loop-forming nucleotides in position 10 or a part of the complementary sequence of the Brd-box consensus sequence, in position 11. miR4350-10GGA was more effective than the anti-sense strand in decreasing the numbers of ovarian tumor SKOV3 cells, which over-expressed HER-2 protein. Its inhibitory effects were lower than that of corresponding double-stranded (ds) RNA4350-4372. Inhibition of HER-2 expression mediated by miRNAs was higher in cells expressing higher levels of HER-2 protein than in cells expressing lower levels of HER-2 protein. This is the first demonstration of inhibition of expression of a constitutively over-expressed tumor protein by designed synthetic miRNA and its cor-responding dsRNA targeting 3' untranslated regions in mRNA. Our results also show that measuring the effects of miRNA and dsRNA in pooled populations of tumor cells, which express various levels of target oncogenes, may reflect the survival responses of siRNA resistant tumors rather than the growth inhibitory responses of siRNA-sensitive tumors.
Assuntos
MicroRNAs , Neoplasias Ovarianas/patologia , RNA de Cadeia Dupla , Receptor ErbB-2/biossíntese , Regiões 3' não Traduzidas , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Terapia Genética/métodos , Humanos , Proto-Oncogene Mas , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific effector and memory cells. The later are particularly relevant for prevention of disease relapse. Efficient induction of memory cells is hindered by the lack of information about the relationship between TCR stimulation and the cytokines required for Ag-specific memory CD8+ cells and proliferation and survival. Since viruses are known to induce memory T cells, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the HER-2 CTL E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of PBMC from healthy donors and of tumor-associated lymphocytes (TAL) from ovarian cancer patients with dendritic cells (DC) infected with KIF-NS (KIF-NS-DC), induced higher numbers of immediate memory effector CD8+ CD44hi CD122hi cells, expressing TCR specific for E75 (E75-TCR) than stimulation with peptide E75. Survival of CD44hi CD122hi cells was dependent on the levels of TCR; cells expressing lower levels of E75-TCR (MFI: 10(2)-10(3)) survived better in IL-2 while cells expressing high levels of TCR (MFI: 10(3)-10(4)) survived better in IL-15. This is the first report demonstrating induction of human Ag-specific memory CD8+ cells against a human tumor-antigen using a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for preventive immunity in human cancer vaccine development.
Assuntos
Epitopos de Linfócito T/imunologia , Genes erbB-2/imunologia , Interleucina-15/imunologia , Interleucina-2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Epitopos de Linfócito T/genética , Feminino , Vetores Genéticos/genética , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/imunologia , Memória Imunológica/imunologia , Vírus da Influenza A/genética , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Neoplasias Ovarianas/imunologia , Peptídeos/imunologia , Peptídeos/farmacologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Interleucina-2/biossíntese , Receptores de Interleucina-2/imunologia , Regulação para Cima , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologiaRESUMO
CD8(+) cells expressing high numbers of TCR per cell (TCR(hi)) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CD8(+) cells expressing high levels of E75-TCR (TCR(hi)) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-gamma(+) Perf(-) and to TCR(hi) IFN-gamma(+) Perf(+) cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCR(hi) Perf(hi) cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCR(hi) Perf(+) CTL required an agonist of optimal CH(2) side chain length, which in this study was equal to two CH(2) groups appended to E75 at the Gly(4) position. Side chains one CH(2) shorter or longer than optimal were either less stimulatory or induced death of TCR(hi) Perf(+) cells. Differentiation of TCR(hi) CD8(+) cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Antígenos de Neoplasias/química , Apoptose/imunologia , Linfócitos T CD8-Positivos/citologia , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Diferenciação Celular/imunologia , Ativação Enzimática , Epitopos de Linfócito T/química , Feminino , Humanos , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Modelos Moleculares , Oligopeptídeos/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptor ErbB-2/imunologia , Relação Estrutura-Atividade , Linfócitos T Citotóxicos/imunologiaRESUMO
HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)(+) melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11-30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11-30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11-30 VH chain, VH (3-11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76-84). Lymphocytes from HLA-A2(+)-immunized patients proliferated to VH (3-11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76-84). Significant increase in IFN-gamma production but not in interleukin 10 production in response to VH (3-11) and to variant HMW-MAA peptide (76-84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2(+) patients with the two peptides induced CTL, which lysed HMW-MAA(+)/HLA-A2(+) A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Melanoma/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Divisão Celular , Mapeamento de Epitopos , Epitopos , Humanos , Imunização , Interferon gama/metabolismo , Ativação Linfocitária , Dados de Sequência Molecular , Neoplasias Cutâneas/imunologiaRESUMO
Breast cancer metastasizes from the primary site to the axillary lymph nodes (LN). It is unknown whether tumor metastasis abolishes or enhances the ability of LN cells to develop a specific response to the Ag expressed by the tumor, and whether an immune response to the same Ag is present in the tumor-free LN. We stimulated lymphocytes from a metastasis negative (Met-) and a metastasis positive (Met+) LN, invaded by a HER-2+ tumor, from the same patient, with HER-2 peptides E75 (369-377) and G89 (776-778). E75 define a CTL epitope presented by HLA-A2, while G89 define a CD4+ cell recognized epitope. Met- LN responded to E75+G89 with higher expansion of E75 TCR+ CD45RO+ CCR7- (CCR7-) and E75-TCR+ CD45RO+ CCR7+ (CCR7+) cells than Met+ LN. Stimulation with E75+G89 induced a significant increase in CCR7+ cells in Met- LN compared with Met+ LN. The levels of IFN-alpha and IL-15 were higher in Met- LN cultures stimulated with E75+G89 than in Met+ LN cultures. This increase did not correlate with the levels of induction of IFN-gamma, IL-4, and IL-10. The finding of higher expansion of Ag specific CCR7+ cells and of differentiation to CCR7- cells, which define the TCM and TEM subsets respectively, in Met- LN, by G89 is novel for tumor systems. This may have implications for preventative vaccination strategies for breast and ovarian cancer.
Assuntos
Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Linfonodos/citologia , Metástase Linfática/patologia , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/citologia , Carcinoma Ductal/imunologia , Carcinoma Ductal/metabolismo , Carcinoma Ductal/secundário , Epitopos de Linfócito T/imunologia , Feminino , Antígeno HLA-A2/metabolismo , Humanos , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-15/metabolismo , Interleucina-4/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Linfonodos/imunologia , Ativação Linfocitária , Invasividade Neoplásica/patologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CCR7 , Receptores de Quimiocinas/metabolismo , Linfócitos T Citotóxicos/citologia , Células Tumorais CultivadasRESUMO
Many clinical studies have been undertaken to assess the therapeutic potential of vaccination and have included a large variety of cancer immunogens. Most of these studies involved patients with metastatic cancer, which is characterized by the most aggressive malignant cells, the longest-lasting disease, and the failure of all standard cytotoxic treatments. The presence of tumor over long periods and the toxicity of previous treatments tend to negatively affect immune responsiveness to tumor antigens presented by the vaccine. In this review, we analyze the ability of past and current vaccine therapies to induce clinical responses in breast cancer. To date, clinical responses have been observed by using vaccines targeting HER-2/neu protein, human telomerase reverse transcriptase, carcinoembryonic antigen, and carbohydrate antigen given after stem cell rescue. The review concludes with a discussion of possible future directions for vaccine development and applications.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Vacinas Anticâncer , Animais , Antígenos de Neoplasias/química , Carboidratos/química , Antígeno Carcinoembrionário/biossíntese , Proteínas de Ligação a DNA , Humanos , Modelos Químicos , Metástase Neoplásica , Receptor ErbB-2/metabolismo , Telomerase/metabolismo , Fatores de TempoRESUMO
The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-gamma) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75(+) CD45RO(+) cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-alpha in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.
Assuntos
Antígenos de Neoplasias/imunologia , Epitopos/imunologia , Vírus da Influenza A/imunologia , Ativação Linfocitária , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Neoplasias da Mama/imunologia , Bovinos , Linhagem Celular , Epitopos/química , Feminino , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neoplasias Ovarianas/imunologia , Proto-Oncogene Mas , Linfócitos T Citotóxicos/virologiaRESUMO
To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser(5) (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-gamma production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75(+) TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.
Assuntos
Substituição de Aminoácidos/imunologia , Antígenos de Neoplasias/imunologia , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Genes erbB-2/imunologia , Antígeno HLA-A2/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/farmacologia , Alanina/genética , Apresentação de Antígeno , Apoptose/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Adesão Celular/imunologia , Linhagem Celular , Sobrevivência Celular/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/farmacologia , Glicina/genética , Antígeno HLA-A2/química , Humanos , Interferon gama/biossíntese , Ativação Linfocitária , Lisina/genética , Modelos Moleculares , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Serina/genética , Linfócitos T Citotóxicos/citologia , Células Tumorais CultivadasRESUMO
The transmembrane (TM) receptor encoded by the HER-2 proto-oncogene (HER-2) is amplified in several types of human carcinomas and premalignant states and provides an important target for cancer therapy. While overexpression of HER-2 should lead to increased CTL epitope formation due to the attendant increase in higher protein turnover, breast tumors are poor stimulators of CTL. In this report, we show that treatment of SKBR3.A2 tumor cells with HER-2 receptor agonists (EGF and NDF) enhanced tumor ability to activate CTL from tumor associated lymphocytes (TAL) and from T cells from peripheral blood in vitro. The enhanced ability of tumor cells to stimulate CTL was paralleled by tyrosine phosphorylation of HER-2, and its oligo-ubiquitination compared with control untreated, or TPA-treated tumor cells. Our results demonstrate that HER-2 ligands used at concentrations which induce tyrosine phosphorylation but not downregulation of the receptor can be used to enhance the ability of tumor cells to activate CTL. This may have implications for overcoming Ag ignorance and tolerance in human cancers.
Assuntos
Receptor ErbB-2/metabolismo , Linfócitos T Citotóxicos/metabolismo , Apresentação de Antígeno , Western Blotting , Citotoxicidade Imunológica , Epitopos , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Immunoblotting , Leucócitos Mononucleares/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Ovário/citologia , Fosforilação , Testes de Precipitina , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Fatores de Tempo , Células Tumorais Cultivadas , Tirosina/metabolismo , Ubiquitina/metabolismoRESUMO
The CTL response to Ag expands after priming and subsequently contracts reducing the number of effectors. CD4+ cells are described as regulators of CTL immunity. To elucidate whether CD4+ cells are involved in survival of effector CTL and the survival signals, we used CTL and Th peptides form the HER-2 protooncogene recognized in association with HL-A2 and HLA-DR4, respectively. We analyzed the effect of cells stimulated with G89 (777-789) in survival and expression of lytic function of CTL specific for the epitope E75 (369-377). G89 primed cells (G89-PR) and G89 enhanced expansion and Ag-specific cytolyis of CTL at priming with E75, but inhibited survival of E75-specific CTL at restimulation. These effects were not simply a reflection of the increases in IFN-gamma and IL-10, but the ratio IFN-gamma/lL-10 modified by G89 differentially regulated the survival of stimulated cells. This suggests that the use of helper antigens in cancer vaccines should be evaluated in the context of their CTL survival inducing effect.