RESUMEN
Primary T-cell acute lymphoblastic leukemia (T-ALL) cells require stromal-derived signals to survive. Although many studies have identified cell-intrinsic alterations in signaling pathways that promote T-ALL growth, the identity of endogenous stromal cells and their associated signals in the tumor microenvironment that support T-ALL remains unknown. By examining the thymic tumor microenvironments in multiple murine T-ALL models and primary patient samples, we discovered the emergence of prominent epithelial-free regions, enriched for proliferating tumor cells and dendritic cells (DCs). Systematic evaluation of the functional capacity of tumor-associated stromal cells revealed that myeloid cells, primarily DCs, are necessary and sufficient to support T-ALL survival ex vivo. DCs support T-ALL growth both in primary thymic tumors and at secondary tumor sites. To identify a molecular mechanism by which DCs support T-ALL growth, we first performed gene expression profiling, which revealed up-regulation of platelet-derived growth factor receptor beta (Pdgfrb) and insulin-like growth factor I receptor (Igf1r) on T-ALL cells, with concomitant expression of their ligands by tumor-associated DCs. Both Pdgfrb and Igf1r were activated in ex vivo T-ALL cells, and coculture with tumor-associated, but not normal thymic DCs, sustained IGF1R activation. Furthermore, IGF1R signaling was necessary for DC-mediated T-ALL survival. Collectively, these studies provide the first evidence that endogenous tumor-associated DCs supply signals driving T-ALL growth, and implicate tumor-associated DCs and their mitogenic signals as auspicious therapeutic targets.
Asunto(s)
Células Dendríticas/inmunología , Proteínas de Neoplasias/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Receptores de Somatomedina/inmunología , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Supervivencia Celular , Células Dendríticas/patología , Femenino , Humanos , Masculino , Ratones , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor IGF Tipo 1 , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/inmunología , Receptores de Somatomedina/genética , Transducción de Señal/genética , Microambiente Tumoral/genéticaRESUMEN
The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Organogénesis , Proteínas de Dominio T Box/fisiología , Timo/embriología , Alelos , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Endodermo , Femenino , Factores de Transcripción Forkhead/fisiología , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Nucleares/fisiología , Células Madre/citología , Proteínas de Dominio T Box/genética , Factores de Tiempo , Factores de Transcripción/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets. RESULTS: TrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38-), and late memory (CD24-CD38-) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation. CONCLUSIONS: We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.