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1.
J Immunol ; 186(11): 6157-64, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21518970

RESUMEN

TGF-ß is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-ß on pDC, and to our knowledge, no study has assessed the capacity of TGF-ß-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-ß-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-ß/Smad signal, because TGF-ß treatment induced Smad2 phosphorylation in pDC and blockade of TGF-ß signaling with the SD208 TGF-ßRI kinase inhibitor abrogated Th17 commitment induced by TGF-ß-treated pDC. Moreover, TGF-ß mRNA synthesis and active TGF-ß release were induced in TGF-ß-treated pDC and anti-TGF-ß Ab blocked Th17 commitment. Unexpectedly, TGF-ß treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-ß-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-ß-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-ß-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-ß-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Th17/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Immunoblotting , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteína Smad2/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo
2.
Blood ; 115(12): 2420-9, 2010 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-20089966

RESUMEN

Human CD20 is a B-cell lineage-specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and is a target for rituximab (RTX) immunotherapy. A CD20 reverse transcriptase-polymerase chain reaction (PCR) on B-cell lines cDNA yielded a short PCR product (DeltaCD20) corresponding to a spliced mRNA transcript linking the exon 3 and exon 7 ends. We established here that this novel, alternatively spliced CD20 transcript is expressed and detectable at various levels in leukemic B cells, lymphoma B cells, in vivo tonsil- or in vitro CD40L-activated B cells, and Epstein-Barr virus (EBV)-transformed B cells, but not in resting CD19(+)- or CD20(+)-sorted B cells from peripheral blood or bone marrow of healthy donors. The truncated CD20 sequence is within the reading frame, codes a protein of 130 amino acids ( approximately 15-17 kDa) lacking large parts of the 4 transmembrane segments, suggesting that DeltaCD20 is a nonanchored membrane protein. We demonstrated the translation into a DeltaCD20 protein which is associated with the membrane CD20 protein and showed its involvement in RTX resistance. Study of patient samples before and after RTX resistance or escape confirms our in vitro findings.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Leucemia de Células B/tratamiento farmacológico , Proteínas de la Membrana/genética , Empalme Alternativo/fisiología , Anticuerpos Monoclonales de Origen Murino , Linfocitos B/citología , Linfocitos B/fisiología , Biomarcadores de Tumor/metabolismo , Western Blotting , Compartimento Celular , Línea Celular Transformada , Línea Celular Tumoral , Humanos , Leucemia de Células B/genética , Leucemia de Células B/patología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Neoplasia Residual/genética , Neoplasia Residual/patología , Tonsila Palatina/citología , ARN Mensajero/genética , Rituximab
3.
Cancer Res ; 71(5): 1615-26, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21257710

RESUMEN

The role of natural killer group 2, member D receptor (NKG2D)-expressing natural killer (NK) cells in tumor immunosurveillance is now well established. Nevertheless, tumor progression occurs despite tumor immunosurveillance, leading to cancer persistence in immunocompetent hosts. STAT3 plays a pivotal role both in oncogenic functions and in immunosuppression. In this study, we investigated the role of STAT3 in suppressing NK cell-mediated immunosurveillance. Using a colorectal cancer cell line (HT29) that can poorly activate NK, we neutralized STAT3 with pharmacologic inhibitors or siRNA and found that this led to an increase in NK degranulation and IFN-γ production in a TGF-ß1-independent manner. Exposure to NKG2D-neutralizing antibodies partially restored STAT3 activity, suggesting that it prevented NKG2D-mediated NK cell activation. On this basis, we investigated the expression of NKG2D ligands after STAT3 activation in HT29, mesenchymal stem cells, and activated lymphocytes. The NK cell recognition receptor MHC class I chain-related protein A (MICA) was upregulated following STAT3 neutralization, and a direct interaction between STAT3 and the MICA promoter was identified. Because cross-talk between DNA damage repair and NKG2D ligand expression has been shown, we assessed the influence of STAT3 on MICA expression under conditions of genotoxic stress. We found that STAT3 negatively regulated MICA expression after irradiation or heat shock, including in lymphocytes activated by CD3/CD28 ligation. Together, our findings reveal a novel role for STAT3 in NK cell immunosurveillance by modulating the MICA expression in cancer cells.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Vigilancia Inmunológica/inmunología , Células Asesinas Naturales/inmunología , Factor de Transcripción STAT3/inmunología , Western Blotting , Inmunoprecipitación de Cromatina , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Células HT29 , Humanos , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/inmunología , Mutagénesis Sitio-Dirigida , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Reacción en Cadena de la Polimerasa , Factor de Transcripción STAT3/metabolismo
4.
PLoS One ; 6(7): e20444, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21747928

RESUMEN

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-ß1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-ß1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFß-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFß-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-ß1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.


Asunto(s)
Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neuropilina-2/genética , Factor de Crecimiento Transformador beta1/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Neuropilina-2/deficiencia , Fosforilación/efectos de los fármacos , Fosforilación/genética , ARN Interferente Pequeño/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína Smad2/metabolismo , Proteína smad3/metabolismo
5.
J Clin Invest ; 120(2): 457-71, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20093776

RESUMEN

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.


Asunto(s)
Proteínas del Choque Térmico HSP72/fisiología , Amilorida/farmacología , Amilorida/uso terapéutico , Animales , Línea Celular , Línea Celular Tumoral , Ciclofosfamida/uso terapéutico , Exosomas/efectos de los fármacos , Exosomas/inmunología , Exosomas/fisiología , Humanos , Terapia de Inmunosupresión , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología
6.
Hum Gene Ther ; 19(7): 699-709, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18557699

RESUMEN

Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.


Asunto(s)
Antígenos CD34/metabolismo , Antivirales/farmacología , Farmacorresistencia Viral , Ganciclovir/farmacología , Proteínas Recombinantes de Fusión/genética , Timidina Quinasa/genética , Transgenes , Antígenos CD34/genética , Western Blotting , Línea Celular , Células Cultivadas , Humanos , Lentivirus/genética , Proteínas Recombinantes de Fusión/metabolismo , Simplexvirus/enzimología , Simplexvirus/genética , Linfocitos T/inmunología , Linfocitos T/virología , Timidina Quinasa/metabolismo , Transducción Genética
7.
J Immunol ; 179(11): 7593-604, 2007 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-18025205

RESUMEN

Immunological tolerance is maintained by specialized subsets of T cells including CD4(+)CD25(+)FOXP3(+) regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4(+)CD25(-) T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4(+)CD25(-) T lymphocytes leads to STAT3 Tyr(705) phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4(+)CD25(-) T cell conversion into Treg through costimulation with TCR/CD28 and TGF-beta1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-gamma. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4(+)CD25(+) T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4(+)CD25(+) T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4(+) lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.


Asunto(s)
Factores de Transcripción Forkhead/biosíntesis , Enfermedad Injerto contra Huésped/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/inmunología , Factor de Transcripción STAT3/fisiología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD28/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Fenotipo , Fosforilación , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/trasplante
8.
Blood ; 105(3): 1256-64, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15388576

RESUMEN

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.


Asunto(s)
Antígenos CD/sangre , Antígenos de Diferenciación Mielomonocítica/sangre , Células Dendríticas/inmunología , Células Plasmáticas/inmunología , Enfermedad Aguda , Anciano , Biomarcadores de Tumor/inmunología , Complejo CD3/sangre , Antígenos CD4/sangre , Antígenos CD55/sangre , Citometría de Flujo , Humanos , Inmunofenotipificación , Leucemia de Células Plasmáticas/inmunología , Masculino , Lectina 3 Similar a Ig de Unión al Ácido Siálico
9.
Blood ; 102(4): 1241-8, 2003 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12730101

RESUMEN

To modulate alloreactivity after hematopoietic stem cell transplantation, "suicide" gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell-depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirus-mediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)-specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the "recent activation" phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA-CD27+CCR7- effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27-CCR7- terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27- cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.


Asunto(s)
Apoptosis/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Citomegalovirus/inmunología , Técnicas de Transferencia de Gen , Herpesvirus Humano 4/inmunología , Retroviridae/genética , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Complejo CD3/farmacología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Transformación Celular Viral , Células Cultivadas , Humanos , Inmunofenotipificación , Lectinas Tipo C , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/metabolismo
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