RESUMO
Various T cell adhesion molecules and their cognate receptors on target cells promote T cell receptor (TCR)-mediated cell killing. In this report, we demonstrate that the interaction of epithelial cell marker E-cadherin with integrin alpha(E)(CD103)beta(7), often expressed by tumor-infiltrating lymphocytes (TILs), plays a major role in effective tumor cell lysis. Indeed, we found that although tumor-specific CD103(+) TIL-derived cytotoxic T lymphocyte (CTL) clones are able to kill E-cadherin(+)/intercellular adhesion molecule 1(-) autologous tumor cells, CD103(-) peripheral blood lymphocyte (PBL)-derived counterparts are inefficient. This cell killing is abrogated after treatment of the TIL clones with a blocking anti-CD103 monoclonal antibody or after targeting E-cadherin in the tumor using ribonucleic acid interference. Confocal microscopy analysis also demonstrated that alpha(E)beta(7) is recruited at the immunological synapse and that its interaction with E-cadherin is required for cytolytic granule polarization and subsequent exocytosis. Moreover, we report that the CD103(-) profile, frequently observed in PBL-derived CTL clones and associated with poor cytotoxicity against the cognate tumor, is up-regulated upon TCR engagement and transforming growth factor beta1 treatment, resulting in strong potentiation of antitumor lytic function. Thus, CD8(+)/CD103(+) tumor-reactive T lymphocytes infiltrating epithelial tumors most likely play a major role in antitumor cytotoxic response through alpha(E)beta(7)-E-cadherin interactions.
Assuntos
Caderinas/fisiologia , Polaridade Celular/imunologia , Grânulos Citoplasmáticos/imunologia , Exocitose/imunologia , Integrinas/fisiologia , Neoplasias Pulmonares/imunologia , Linfócitos T Citotóxicos/imunologia , Caderinas/metabolismo , Carcinoma de Células Grandes/imunologia , Carcinoma de Células Grandes/prevenção & controle , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , Humanos , Integrinas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Linfócitos T Citotóxicos/metabolismoRESUMO
Natural cytotoxicity receptors and NKG2D correspond to major activating receptors involved in triggering of tumor cell lysis by human NK cells. In this report, we investigated the expression of NKG2D ligands (NKG2DLs), MHC class I-related chain (MIC) A, MICB and UL16-binding proteins 1, 2 and 3, on a panel of human non-small-cell lung carcinoma cell lines, and we analyzed their role in tumor cell susceptibility to NK cell lysis. Although adenocarcinoma (ADC) cells expressed heterogeneous levels of NKG2DLs, they were often resistant to NK cell-mediated killing. Resistance of a selected cell line, ADC-Coco, to allogeneic polyclonal NK cells and autologous NK cell clones correlated with shedding of NKG2DLs resulting from a matrix metalloproteinase (MMP) production. Treatment of ADC-Coco cells with a MMP inhibitor (MMPI) combined with IL-15 stimulation of autologous NK cell clones lead to a potentiation of NK cell-mediated cytotoxicity. This lysis is mainly NKG2D mediated, since it is abrogated by anti-NKG2D-neutralizing mAb. These results suggest that MMPIs, in combination with IL-15, may be useful for overcoming tumor cell escape from the innate immune response.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Anticorpos Bloqueadores/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Citotoxicidade Imunológica/efeitos dos fármacos , Proteínas Ligadas por GPI , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-15/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Quinurenina 3-Mono-Oxigenase/farmacologia , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
The precise role of alpha-actinin-4 encoding gene (ACTN4) is not very well understood. It has been reported to elicit tumor suppressor activity and to regulate cellular motility. To further assess the function of human ACTN4, we studied a lung carcinoma cell line expressing a mutated alpha-actinin-4, which is recognized as a tumor antigen by autologous CD8(+) cytotoxic T lymphocytes (CTL). Confocal immunofluorescence microscopy indicated that, while wild-type (WT) alpha-actinin-4 stains into actin cytoskeleton and cell surface ruffles, the mutated protein is only dispersed in the cytoplasm of the lung carcinoma cells. This loss of association with the cell surface did not appear to correlate with a decrease in in vitro alpha-actinin-4 crosslinking to filamentous (F)-actin. Interestingly, experiments using cell lines stably expressing ACTN4 demonstrated that as opposed to WT gene, mutant ACTN4 was unable to inhibit tumor cell growth in vitro and in vivo. Moreover, the expression of mutant alpha-actinin-4 resulted in the loss of tumor cell capacity to migrate. The identification of an inactivating mutation in ACTN4 emphasizes its role as a tumor suppressor gene and underlines the involvement of cytoskeleton alteration in tumor development and metastasis.
Assuntos
Actinina/genética , Actinina/fisiologia , Neoplasias Pulmonares/metabolismo , Proteínas dos Microfilamentos , Mutação , Actinas/química , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Divisão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Clonagem Molecular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Microscopia Confocal , Dados de Sequência Molecular , Metástase Neoplásica , Transplante de Neoplasias , Mutação Puntual , Ligação Proteica , Homologia de Sequência de Aminoácidos , Linfócitos T Citotóxicos/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
T lymphocytes infiltrating a human lung carcinoma stimulated in vitro with autologous tumor cell line showed a TCRVbeta13.6(+) T-cell expansion. This subset was isolated using TCRVbeta-specific antibody and several T-cell clones were generated. All these clones expressed a unique Vbeta13.6-Jbeta2.7 TCR with the same junctional region strongly suggesting that they derived from the same cell. They were CD8(+)/CD28(-) and expressed the MHC class I binding killer cell Ig-like receptor (KIR)3DL2/p140, but not KIR3DL1/p70, KIR2DL1/p58.1 and KIR2DL2/3/p58.2. Sequence analysis indicated that KIR3DL2/p140 cDNA was identical to the previously reported 3DL2*002 allele except for two nucleic acid substitutions. Functional studies showed that KIR3DL2/p140(+) CTL secrete a significant level of IFNgamma and mediate an HLA-A2-restricted cytotoxicity against the autologous and some allogeneic tumor cells but not towards the autologous EBV-B cells. Strikingly, both the lytic and the cytokine secretion activities induced upon specific cell interactions were unaffected by anti-KIR3DL2/p140 antibody. In addition, crosslinking KIR3DL2/p140 molecules on CTL did not result into the modification of cytotoxicity and cytokine production triggered by anti-CD3 antibody. These results strongly suggest that, as opposed to distinct KIR expressed by CTL, the in vitro KIR3DL2/p140 engagement does not result into inhibitory (nor activatory) effects on tumor-specific CTL.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores Imunológicos/imunologia , Linfócitos T Citotóxicos/fisiologia , Antígenos CD/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Clonais , Citotoxicidade Imunológica , Imunofluorescência , Humanos , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/patologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores Imunológicos/genética , Receptores KIR , Receptores KIR2DL1 , Receptores KIR2DL2 , Receptores KIR2DL3 , Receptores KIR3DL1 , Receptores KIR3DL2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Células Tumorais CultivadasRESUMO
We have isolated from tumor-infiltrating lymphocytes (TIL) and PBL of a lung carcinoma patient several tumor-specific T cell clones displaying similar peptide-MHC tetramer staining and expressing a unique TCR. Although these clones elicited identical functional avidity and similar cytolytic potential, only T cell clones derived from TIL efficiently lysed autologous tumor cells. Interestingly, all of these clones expressed the same T cell surface markers except for the TCR inhibitory molecule CD5, which was expressed at much lower levels in TIL than in PBL. Video-imaging recordings demonstrated that, although both T cell clones could form stable conjugates with tumor cells, the Ca(2+) response occurred in TIL clones only. Significantly, analysis of a panel of circulating clones indicated that antitumor cytolytic activity was inversely proportional to CD5 expression levels. Importantly, CD5 levels in TIL appeared to parallel the signaling intensity of the TCR/peptide-MHC interaction. Thus, in situ regulation of CD5 expression may be a strategy used by CTL to adapt their sensitivity to intratumoral peptide-MHC levels.
Assuntos
Comunicação Celular/imunologia , Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Fragmentos de Peptídeos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adaptação Fisiológica/imunologia , Antígenos CD5/biossíntese , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Linhagem Celular Tumoral , Células Clonais , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/fisiologia , Humanos , Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Linfócitos do Interstício Tumoral/patologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Transdução de Sinais/imunologia , Coloração e Rotulagem , Subpopulações de Linfócitos T/patologiaRESUMO
NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking beta2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I- tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.