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1.
Front Immunol ; 11: 471, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32265925

RESUMO

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

2.
Cancer Cell Int ; 19: 67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948927

RESUMO

Background: Low molecular weight protein tyrosine phosphatase (LMW-PTP) is overexpressed in different cancer types and its expression is related to more aggressive disease, reduced survival rate and drug resistance. Morin is a natural polyphenol which negatively modulates, among others, the activity of LMW-PTP, leading to the potentiation of the effects of different antitumoral drugs, representing a potential beneficial treatment against cancer. Methods: LMW-PTP levels were measured by immunoblot analysis both in CLL cells from patients and in chronic lymphocytic leukemia (CLL)-derived Mec-1 cells. Cell viability was assessed in Mec-1 cells treated with morin alone or in combination with either fludarabine or ibrutinib or following siRNA-mediated LMW-PTP knockdown. Furthermore, the expression levels of VLA-4 and CXCR4 were assessed by both qRT-PCR and flow cytometry and both adhesion to fibronectin-coated plates and migration toward CXCL12 were analyzed in Mec-1 cells treated with morin alone or in combination with fludarabine or ibrutinib. Results: We observed that LMW-PTP is highly expressed in Mec-1 cells as well as in leukemic B lymphocytes purified from CLL patients compared to normal B lymphocytes. Morin treatment strongly decreased LMW-PTP expression levels in Mec-1 cells and potentiated the anticancer properties of both fludarabine and ibrutinib by increasing their apoptotic effects on leukemic cells. Moreover, morin negatively regulates adhesion and CXCL12-dependent migration of Mec-1 cells by affecting VLA-4 integrin expression and CXCR4 receptor recycling. Conclusions: Morin treatment in CLL-derived Mec-1 cell line synergizes with conventional anticancer drugs currently used in CLL therapy by affecting leukemic cell viability and trafficking.

3.
Cell Mol Life Sci ; 76(16): 3249-3261, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30830241

RESUMO

In addition to their modulation through de novo expression and degradation, surface levels of chemokine receptors are tuned by their ligand-dependent recycling to the plasma membrane, which ensures that engaged receptors become rapidly available for further rounds of signaling. Dysregulation of this process contributes to the pathogenesis of chronic lymphocytic leukemia (CLL) by enhancing surface expression of chemokine receptors, thereby favoring leukemic cell accumulation in the protective niche of lymphoid organs. In this review, we summarize our current understanding of the process of chemokine receptor recycling, focusing on the impact of its dysregulation in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Receptores de Quimiocinas/metabolismo , Arrestinas/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfonodos/metabolismo , Fosforilação , Transdução de Sinais
4.
Haematologica ; 104(10): 2040-2052, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30819907

RESUMO

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

6.
Oncogene ; 37(11): 1534-1550, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29326436

RESUMO

Neoplastic cell traffic abnormalities are central to the pathogenesis of chronic lymphocytic leukemia (CLL). Enhanced CXC chemokine receptor-4 (CXCR4) and chemokine receptor-7 (CCR7) recycling contributes to the elevated surface levels of these receptors on CLL cells. Here we have addressed the role of p66Shc, a member of the Shc family of protein adaptors the expression of which is defective in CLL cells, in CXCR4/CCR7 recycling. p66Shc reconstitution in CLL cells reduced CXCR4/CCR7 recycling, lowering their surface levels and attenuating B-cell chemotaxis, due to their accumulation in Rab5+ endosomes as serine-phosphoproteins bound to ß-arrestin. This results from the ability of p66Shc to inhibit Ca2+ and PP2B-dependent CXCR4/CCR7 dephosphorylation and ß-arrestin release. We also show that ibrutinib, a Btk inhibitor that promotes leukemic cell mobilization from lymphoid organs, reverses the CXCR4/CCR7 recycling abnormalities in CLL cells by increasing p66Shc expression. These results, identifying p66Shc as a regulator of CXCR4/CCR7 recycling in B cells, underscore the relevance of its deficiency to CLL pathogenesis and provide new clues to the mechanisms underlying the therapeutic effects of ibrutinib.


Assuntos
Endossomos/metabolismo , Leucemia Linfocítica Crônica de Células B , Receptores CCR7/metabolismo , Receptores CXCR4/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , beta-Arrestinas/metabolismo , Adulto , Animais , Estudos de Casos e Controles , Células Cultivadas , Endossomos/efeitos dos fármacos , Endossomos/patologia , Mutação em Linhagem Germinativa , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Knockout , Fosforilação/genética , Proteólise , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico
8.
Methods Mol Biol ; 1584: 143-155, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28255701

RESUMO

Engagement of the T cell antigen receptor (TCR) by specific ligand bound to the major histocompatibility complex is the primary event that leads to the assembly of the immune synapse (IS). Central to this process is TCR clustering at the T cell-APC contact, which is achieved with the contribution of an endosomal pool that is delivered to the IS by polarized recycling. As the TCR recycling process has not been fully elucidated, we developed methods suitable to quantitate recycling to the plasma membrane of TCR/CD3 complexes that have been engaged at the cell surface and track their traffic through the intracellular vesicular compartments toward the IS.


Assuntos
Complexo CD3/imunologia , Endossomos/imunologia , Sinapses Imunológicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Humanos , Células Jurkat
9.
Oncotarget ; 7(35): 57086-57098, 2016 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-27494881

RESUMO

p66Shc attenuates mitogenic, prosurvival and chemotactic signaling and promotes apoptosis in lymphocytes. Consistently, p66Shc deficiency contributes to the survival and trafficking abnormalities of chronic lymphocytic leukemia (CLL) B cells. The mechanism of p66shc silencing in CLL B cells is methylation-independent, at variance with other cancer cell types. Here we identify STAT4 as a novel transcriptional regulator of p66Shc in B cells. Chromatin immunoprecipitation and reporter gene assays showed that STAT4 binds to and activates the p66shc promoter. Silencing or overexpression of STAT4 resulted in a co-modulation of p66Shc. IL-12-dependent STAT4 activation caused a coordinate increase in STAT4 and p66Shc expression, which correlated with enhanced B cell apoptosis. Treatment with the STAT4 inhibitor lisofylline reverted partly this effect, suggesting that STAT4 phosphorylation is not essential for but enhances p66shc transcription. Additionally, we demonstrate that CLL B lymphocytes have a STAT4 expression defect which partly accounts for their p66Shc deficiency, as supported by reconstitution experiments. Finally, we show that p66Shc participates in a positive feedback loop to promote STAT4 expression. These results provide new insights into the mechanism of p66Shc expression in B cells and its defect in CLL, identifying the STAT4/IL-12 pathway as a potential therapeutic target in this neoplasia.


Assuntos
Linfócitos B/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Fator de Transcrição STAT4/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Humanos , Interleucina-12/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Pentoxifilina/análogos & derivados , Pentoxifilina/química , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Microambiente Tumoral
10.
Sci Transl Med ; 8(321): 321ra7, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26764158

RESUMO

X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Morte Celular/efeitos dos fármacos , Citocinas/biossíntese , Diacilglicerol Quinase/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Ativação Linfocitária , Contagem de Linfócitos , Transtornos Linfoproliferativos/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/deficiência , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/metabolismo , Tiazóis/farmacologia , Proteínas ras/metabolismo
11.
Small GTPases ; 7(1): 16-20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26587735

RESUMO

Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Among them, Rab8 is an essential participant in primary cilium formation. In a report recently published in the Journal of Cell Science, Finetti and colleagues identify Rab8 as a novel player in vesicular traffic in the non-ciliated T lymphocytes, which contributes to the assembly of the specialized signaling platform known as the immune synapse. By interacting with the v-SNARE VAMP-3, Rab8 is indeed responsible for the final docking/fusion step in T cell receptor (TCR) recycling to the immune synapse. A second important take-home message which comes to light from this work is that VAMP-3 also interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened at the plasma membrane. Hence the data presented in this report, in addition to identifying Rab8 as a novel player in vesicular traffic to the immune synapse, reveal how both ciliated and non-ciliated cells take advantage of a conserved pathway to build highly specific cellular structures.


Assuntos
Cílios/metabolismo , Endossomos/metabolismo , Sinapses Imunológicas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte/metabolismo , Camundongos , Células NIH 3T3 , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo
12.
Cancer Res ; 75(19): 4153-63, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26282174

RESUMO

Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome.


Assuntos
Linfócitos B/metabolismo , Quimiotaxia/fisiologia , Endossomos/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Infiltração Leucêmica/fisiopatologia , Linfonodos/patologia , Proteínas de Neoplasias/metabolismo , Receptores CCR7/metabolismo , Receptores CXCR4/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Linfócitos B/patologia , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores CCR7/genética , Receptores CXCR4/genética , Receptores de Retorno de Linfócitos/metabolismo , Receptores de Lisoesfingolipídeo/deficiência , Receptores de Lisoesfingolipídeo/genética
13.
J Cell Sci ; 128(14): 2541-52, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26034069

RESUMO

IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11(+) endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR(+) endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis.


Assuntos
Sinapses Imunológicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Endossomos/genética , Endossomos/metabolismo , Humanos , Sinapses Imunológicas/genética , Células Jurkat , Camundongos , Células NIH 3T3 , Receptores de Antígenos de Linfócitos T/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteína 3 Associada à Membrana da Vesícula/genética , Proteínas rab de Ligação ao GTP/genética
14.
J Cell Sci ; 127(Pt 9): 1924-37, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24554435

RESUMO

T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5(+) endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis.


Assuntos
Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Células Jurkat , Microscopia de Fluorescência , Ligação Proteica/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
15.
Front Immunol ; 4: 213, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23908653

RESUMO

Glycerophosphoinositols (GPIs) are bioactive, diffusible phosphoinositide metabolites of phospholipase A2 that act both intracellularly and in a paracrine fashion following their uptake by specific transporters. The most representative compound, glycerophosphoinositol (GroPIns), is a ubiquitous component of eukaryotic cells that participates in central processes, including cell proliferation and survival. Moreover, glycerophosphoinositol 4-phosphate (GroPIns4P) controls actin dynamics in several cell systems by regulating Rho GTPases. Recently, immune cells have emerged as targets of the biological activities of the GPIs. We have shown that exogenous GroPIns4P enhances CXCL12-induced T-cell chemotaxis through activation of the kinase Lck in a cAMP/PKA-dependent manner. While highlighting the potential of GroPIns4P as an immunomodulator, this finding raises questions on the role of endogenously produced GroPIns4P as well as of other GPIs in the regulation of the adaptive immune responses under homeostatic and pathological settings. Here we will summarize our current understanding of the biological activities of the GPIs, with a focus on lymphocytes, highlighting open questions and potential developments in this promising new area.

16.
Blood ; 120(22): 4391-9, 2012 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-23033271

RESUMO

Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.


Assuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Lisoesfingolipídeo/genética , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Adulto , Animais , Feminino , Regulação da Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Camundongos , Camundongos Knockout , Oxidantes/metabolismo , Prognóstico , Receptores de Lisoesfingolipídeo/fisiologia , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais Cultivadas
17.
Apoptosis ; 17(2): 174-86, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21983898

RESUMO

p66Shc, an adaptor molecule which enhances reactive oxygen species (ROS) production by mitochondria, promotes T-cell apoptosis by inducing mitochondrial dysfunction and impairing Ca(2+) homeostasis. We have addressed the potential role of Lck, a kinase which has been implicated in T-cell apoptosis induced by a number of stimuli, in the proapoptotic activity of p66Shc. Lck deficiency in Jurkat T cells overexpressing p66Shc leads to impaired apoptotic responses to supraphysiological increases in [Ca(2+)](c). This defect could be rescued by reconstitution of Lck expression, indicating that Lck is required for p66Shc-dependent apoptosis. Furthermore, p66Shc phosphorylation on serine 36 (S36), an event on which the proapoptotic function of p66Shc depends, requires Lck. p66Shc-dependent mitochondrial dysfunction, altered Ca(2+) homeostasis and S36 phosphorylation require moreover the activity of CaMKII, a Ca(2+)/calmodulin-dependent kinase known to be implicated in the proapoptotic activity of Lck in T cells. The results suggest that increases in [Ca(2+)](c) lead to CaMKII activation and subsequent Lck-dependent p66Shc phosphorylation on S36. This event causes both mitochondrial dysfunction and impaired Ca(2+) homeostasis, which synergize in promoting Jurkat T-cell apoptosis.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Calcimicina/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Regulação da Expressão Gênica , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Estresse Oxidativo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Serina , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
18.
J Immunol ; 187(11): 5941-51, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22048771

RESUMO

Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.


Assuntos
Diacilglicerol Quinase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Western Blotting , Diglicerídeos/metabolismo , Imunofluorescência , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Jurkat , Transporte Proteico/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção
19.
J Exp Med ; 208(6): 1317-30, 2011 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-21576384

RESUMO

The Bordetella pertussis adenylate cyclase toxin (CyaA) assists infection by potently suppressing the host immune response. Although CyaA effectively targets T lymphocytes, its putative receptor on these cells is unknown. Here, we show that CyaA binds to T cells via the ß2 integrin LFA-1 in its active conformation. CyaA clusters with LFA-1 at the immune synapse (IS), from which it induces the premature disengagement of LFA-1 concomitant with the dissipation of talin, which tethers the integrin to the underlying actin cytoskeleton. The CyaA-induced redistribution of LFA-1 was cAMP- and protein kinase A (PKA)-dependent. These results not only identify LFA-1 as a CyaA receptor on T cells but unveil a novel mechanism of immunosuppression whereby the toxin parasitizes its interaction with LFA-1 to inhibit signaling at the IS through the local production of cAMP. The data also provide novel insights into the role of cAMP/PKA signaling in controlling the dynamics of the IS.


Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos T/microbiologia , Linfócitos B/citologia , Antígeno CD11a/biossíntese , Complexo CD3/imunologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/citologia , Citometria de Fluxo/métodos , Humanos , Sistema Imunitário , Células Jurkat , Microscopia de Fluorescência/métodos , Linfócitos T/imunologia
20.
Immunol Lett ; 115(2): 75-82, 2008 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-18054087

RESUMO

The signaling pathways induced in T lymphocytes by CXCR4-CXCL12 interaction, which lead to the cytoskeletal macro-rearrangements observable in migrating cells, are as yet largely uncharacterized. The aim of this review is to briefly summarize the current knowledge of the signaling machinery which controls the process of chemotaxis in CXCL12-stimulated T lymphocytes.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores CXCR4/metabolismo , Linfócitos T/fisiologia , Animais , Quimiotaxia de Leucócito/fisiologia , Citoesqueleto/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
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