RESUMEN
Many cancers are termed immunoevasive due to expression of immunomodulatory ligands. Programmed death ligand-1 (PD-L1) and cluster of differentiation 80/86 (CD80/86) interact with their receptors, programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4), respectively, on tumor-infiltrating leukocytes eliciting immunosuppression. Immunotherapies aimed at blocking these interactions are revolutionizing cancer treatments, albeit in an inadequately described patient subset. To address the issue of patient stratification for immune checkpoint intervention, we quantitatively imaged PD-1/PD-L1 interactions in tumor samples from patients, employing an assay that readily detects these intercellular protein-protein interactions in the less than or equal to 10 nm range. These analyses across multiple patient cohorts demonstrated the intercancer, interpatient, and intratumoral heterogeneity of interacting immune checkpoints. The PD-1/PD-L1 interaction was not correlated with clinical PD-L1 expression scores in malignant melanoma. Crucially, among anti-PD-1-treated patients with metastatic non-small cell lung cancer, those with lower PD-1/PD-L1 interaction had significantly worsened survival. It is surmised that within tumors selecting for an elevated level of PD-1/PD-L1 interaction, there is a greater dependence on this pathway for immune evasion and hence, they exhibit more impressive patient response to intervention. SIGNIFICANCE: Quantitation of immune checkpoint interaction by direct imaging demonstrates that immunotherapy-treated patients with metastatic NSCLC with a low extent of PD-1/PD-L1 interaction show significantly worse outcome.
Asunto(s)
Antígeno B7-H1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Neoplasias Pulmonares/inmunología , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Adulto , Anciano , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Femenino , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/mortalidad , Persona de Mediana Edad , Terapia Molecular Dirigida , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Reproducibilidad de los Resultados , Resultado del TratamientoRESUMEN
The adaptor protein Gab-2 coordinates the assembly of the IL-3 signalsome comprising Gab-2, Grb2, Shc, SHP-2 and PI3K. To investigate the role of the pleckstrin homology domain of Gab-2 in this process, epitope-tagged wild type Gab-2 (WTGab-2), Gab-2 lacking its PH domain (DeltaPHGab-2) and the Gab-2 PH domain alone (PHGab-2) were inducibly expressed in IL-3-dependent BaF/3 cells. Expression of DeltaPHGab-2 reduced IL-3-dependent proliferation and long-term activation of ERK1 and 2 and PKB by IL-3. While we demonstrate that the Gab-2 PH domain can bind PI(3,4,5)P3, it is dispensable for Gab-2 membrane localisation, tyrosine phosphorylation and signalsome formation. Rather, the proline-rich motifs of Gab-2 appear to contribute to the constitutive membrane localisation we observe, independently of tyrosine phosphorylation or the PH domain. Taken together, these findings suggest that once Gab-2 is tyrosine phosphorylated its PH domain is required for the optimal stabilisation of the signalsome, enabling full activation of downstream signals.
Asunto(s)
Interleucina-3/farmacología , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatos de Inositol/metabolismo , Ratones , Mutación/genética , Fosfatidilinositoles/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Grb-2-associated binder (Gab)2 is a scaffolding adaptor protein that has been reported to promote growth factor and cytokine receptor signal transduction, but inhibit TCR-mediated signaling events. In this study, we show that ligation of CD28 by its natural ligand B7-1/CD80, induces tyrosine phosphorylation of Gab2 and its coassociation with Src homology phosphatase (SHP)-2 and class IA PI3K in Jurkat cells. Overexpression of wild-type Gab2 revealed a negative role in regulation of CD3/CD28 induction of the transcription factors NF-kappaB and AP-1. To characterize this inhibitory function further, we used Gab2 mutants unable to bind either PI3K or SHP-2 and a PH domain deletion mutant. Although PI3K has previously been implicated as necessary for Gab2-mediated inhibition of TCR signaling, Gab2 mutants defective in their ability to bind PI3K or SHP-2 retained their inhibitory function, whereas deletion of the PH domain ablated the inhibitory effect of Gab2. Together, these data demonstrate that CD28 stimulation of T cells is sufficient to induce an inhibitory multimeric signaling complex involving Gab2, SHP-2, and PI3K. Furthermore, the inhibitory capacity of Gab2 is strictly dependent upon the integrity of its PH domain, suggesting phosphoinositide-mediated membrane recruitment is important to Gab2 function in T cells.
Asunto(s)
Antígenos CD28/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Fosfoproteínas/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/metabolismo , Antígenos CD28/metabolismo , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Linfocitos T/inmunología , Factor de Transcripción AP-1/inmunología , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 -14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281-29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764-23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911-914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the beta-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in suboptimal IL-3 and upon IL-3 withdrawal. These findings indicate that SHP-2 plays an important role in mediating the anti-apoptotic effect of IL-3 and raises the possibility that PECAM-1 participates in the modulation of cytokine-induced signals.