RESUMO
T cell-specific adapter protein (TSAd) is a SRC-homology-2 (SH2) domain-containing intracellular signaling molecule that is required for T cell antigen receptor (TCR)-induced cytokine synthesis in T cells. How TSAd functions in TCR signal transduction is not clear. Previous work has suggested a nuclear role for this adapter. However, other evidence suggests that TSAd also functions in the cytoplasm. Using T cells from TSAd-deficient mice, we now show that the major role of TSAd in the cytoplasm is in activation of the LCK protein tyrosine kinase at the outset of TCR signal transduction. Consequently, TSAd regulates several downstream signaling events, including intracellular calcium mobilization and activation of the Ras-extracellular signal-regulated kinase signaling pathway. TSAd regulates LCK activity directly through physical interaction with LCK SH3 and SH2 domains. These studies reveal TSAd as a positive regulator of proximal TCR signal transduction and provide important new information on the mechanism of TCR-induced LCK activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
In animal models and clinical trials, adoptive transfer of activated, antigen-specific CD8(+) T cells mediates tumor regression in a cell dose-dependent manner. The cytokine interleukin (IL)-12 promotes CD8(+) T-cell cytotoxicity and, with IL-18, synergistically up-regulates IFN-gamma release. We have shown that culturing CD8(+) T cells ex vivo with IL-12 and IL-18 enhanced antitumor responses in vivo and in vitro using a model of C1498/ovalbumin, a murine acute myeloid leukemia cell line expressing the antigen ovalbumin. Activated ovalbumin-specific CD8(+) T cells cultured with IL-12, IL-18, both, or neither were assayed for antigen-specific cytokine production and cytolytic activity and adoptively transferred to C57BL/6 mice with established tumors. Maximal IFN-gamma release occurred after T-cell culture with IL-12 and IL-18. Tumor-specific in vitro cytotoxicity was enhanced by IL-12, unaffected by addition of IL-18, and abrogated in perforin-deficient T cells irrespective of cytokine exposure. T cells cultured with IL-12 more effectively eliminated tumors, and addition of IL-18 did not further augment responses. IFN-gamma-deficient CD8(+) T cells showed effective antitumor activity that was enhanced by IL-12 with or without IL-18. Perforin-deficient CD8(+) T cells were poor mediators of antitumor activity, though, and showed no improvement after culture with IL-12 and/or IL-18. Thus, ex vivo culture with IL-12 was sufficient to augment antigen-specific in vitro cytotoxicity and antitumor activity in vivo in an IFN-gamma-independent but perforin-dependent manner. Ex vivo culture with IL-12 may improve CD8(+) T-cell immunotherapy of cancer in the absence of donor cell-derived IFN-gamma via perforin-mediated cytolysis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Interleucina-12/imunologia , Interleucina-18/imunologia , Leucemia Mieloide/imunologia , Glicoproteínas de Membrana/imunologia , Doença Aguda , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Sinergismo Farmacológico , Epitopos de Linfócito T/imunologia , Feminino , Interferon gama/deficiência , Interferon gama/imunologia , Interleucina-12/farmacologia , Interleucina-18/farmacologia , Leucemia Mieloide/terapia , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perforina , Proteínas Citotóxicas Formadoras de PorosAssuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tolerância Imunológica/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Doenças Autoimunes/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
BACKGROUND & OBJECTIVE: Chimeric T-cell receptors (chTCR) are recombinant immune receptors with the characteristics of combining exquisite antigen specificity of a monoclonal antibody and activating T lymphocyte function by signal transduction element. Compared to "classic" TCR in mediating T cells immune response to target cells,a significant potential advantage of chTCR is the lack of major histocompatibility complex (MHC) restriction and antigen processing. N29gamma is a chTCR specific for p185HER2. The current study is to investigate the efficacy of N29gamma redirect T cells in response to p185HER2 in a murine model of metastatic breast cancer. METHODS: Splenic T cells from Balb/c mice were purificated by negative selection over sterile brushed nylon wool fiber,activated by immobilized purified anti-mouse CD3 and CD28 antibodies. Then the recombinant retrovirus pRet6N29gamma were transduced by centrifuging (1 300 g for 90 min, at 32 Centigrade). The transduction efficiency was indirectly defermined by green fluorescence protein (GFP) expression of T cells in control group tested by flow cytometry. Mice bearing 3 days or 8 days MT901 or MT901/HER2 were randomized into experiment group or control group to receive IV infusions of polyclonal activated Balb/c splenic T cells transduced with the N29gamma retroviral vector or a control GFP vector(5 x 10(6)-40 x 10(6) of T cells per mouse). Each mouse was injected intraperitoneal with IL-2 of 3 x 10(4) IU every 12 h for 10 times. Mice were sacrificed at 11 days (Day 3 model) or 13 days (Day 8 model) after T cells transfer to enumerate the lung metastases. RESULTS: In Day 3 model, more than 200 lung metastases were present in mice in mock group, received non-transduced T cells group as well as GFP transduced T cells group. Treatment of mice bearing HER2 positive tumor with N29gamma transduced T cells resulted in a significant reduction in the number of lung metastases (3.4+/-3.3/lung). In contrast,N29gamma transduced T cells failed to induce the regression of HER2 negative MT901 pulmonary metastases (>200/lung). In Day 8 model, treatment with N29gamma modified T cells had a cell dose dependent effect on HER2+ lung metastases. Compared with 4 x 10(7) GFP T cells, 107 of N29gamma T cells were not enough to reduce the number of lung metastases (167+/-15.3) (P=0.198). With increasing dosage of N29gamma modified T cells from 2,3 to 4 x 10(7),there was a progressive decrease in the number of pulmonary metastases from 64+/-12.1,34+/-6.3,to 8+/-4.3,respectively (P< 0.01). CONCLUSIONS: Gene engineered T cells expressing chimeric TCR N29gamma induced the HER2-specific regression of lung metastases in breast cancer. Higher doses of gene-modified T cells were necessary to eliminate more advanced lung metastases.