Alpha2beta1 Integrin (VLA-2) Protects Activated Human Effector T Cells From Methotrexate-Induced Apoptosis

β1 integrins are critical for T cell migration, survival and costimulation. The integrin α2β1, which is a receptor for collagen, also named VLA-2, is a major costimulatory pathway of effector T cells and has been implicated in arthritis pathogenesis. Herein, we have examined its ability to promote methotrexate (MTX) resistance by enhancing effector T cells survival. Our results show that attachment of anti-CD3-activated human polarized Th17 cells to collagen but not to fibronectin or laminin led to a significant reduction of MTX-induced apoptosis. The anti-CD3+collagen-rescued cells still produce significant amounts of IL-17 and IFNγ upon their reactivation indicating that their inflammatory nature is preserved. Mechanistically, we found that the prosurvival role of anti-CD3+collagen involves activation of the MTX transporter ABCC1 (ATP Binding Cassette subfamily C Member 1). Finally, the protective effect of collagen/α2β1 integrin on MTX-induced apoptosis also occurs in memory CD4+ T cells isolated from rheumatoid arthritis (RA) patients suggesting its clinical relevance. Together these results show that α2β1 integrin promotes MTX resistance of effector T cells, and suggest that it could contribute to the development of MTX resistance that is seen in RA

Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

International audienceBACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability

Effets des dihydropyridines et autres modulateurs de l'activité calcique chez le lymphocyte T humain Jurkat E6.1

L'augmentation de la concentration cytosolique de calcium ([Ca2+]i) est essentielle pour l'activation des lymphocytes T. Cette augmentation est causée d'une part, par le relâchement du calcium à partir des réserves cytosoliques, et d'autre part, par un influx de calcium qui se fait probablement via des canaux calciques. Les effets des dihydropyridines (DHP) (nifedipine et PN200-110), des bloqueurs de canaux calciques voltage dépendant, ainsi que ceux des inhibiteurs du relâchement du calcium interne (TMB-8, ryanodine) ont été étudiés sur les réponses PHA-dépendantes chez les cellules Jurkat E6-1. Nos résultats montrent que les DHP inhibent la production d'interleukine 2 (IL-2), mais le TPA empêche cette inhibition. De plus les DHP bloquent l'entrée du calcium extracellulaire. Le BAY K 8644, un agoniste de canaux calciques voltage dépendant, interfère avec l'influx de calcium. La ryanodine et le TMB-8 n'ont pas d'effets sur la production d'IL-2 et sur l'influx calcique. Par contre, le relâchement du calcium interne est inhibé par la ryanodine mais non par le TMB-8. La réponse calcique est inhibée par le TPA alors que le TEA, un bloqueur de canaux potassiques, n'a aucun effet. Une dépolarisation de la membrane (KCl, 10 mM) induit une augmentation de [Ca2+]. Nous avons mis en évidence l'existence de sites récepteurs spécifiques pour le PN200-110 sur les cellules Jurkat (Kd = 8.5 + 3.1 nM, 2300 + 500 sites/cellule). Nos résultats suggèrent l'existence de canaux calciques chez les cellules Jurkat, lesquels sont probablement impliqués dans la traduction du signal transmembranaire

Effets des dihydropyridines et autres modulateurs de l'activité calcique chez le lymphocyte T humain Jurkat E6.1

L'augmentation de la concentration cytosolique de calcium ([Ca2+]i) est essentielle pour l'activation des lymphocytes T. Cette augmentation est causée d'une part, par le relâchement du calcium à partir des réserves cytosoliques, et d'autre part, par un influx de calcium qui se fait probablement via des canaux calciques. Les effets des dihydropyridines (DHP) (nifedipine et PN200-110), des bloqueurs de canaux calciques voltage dépendant, ainsi que ceux des inhibiteurs du relâchement du calcium interne (TMB-8, ryanodine) ont été étudiés sur les réponses PHA-dépendantes chez les cellules Jurkat E6-1. Nos résultats montrent que les DHP inhibent la production d'interleukine 2 (IL-2), mais le TPA empêche cette inhibition. De plus les DHP bloquent l'entrée du calcium extracellulaire. Le BAY K 8644, un agoniste de canaux calciques voltage dépendant, interfère avec l'influx de calcium. La ryanodine et le TMB-8 n'ont pas d'effets sur la production d'IL-2 et sur l'influx calcique. Par contre, le relâchement du calcium interne est inhibé par la ryanodine mais non par le TMB-8. La réponse calcique est inhibée par le TPA alors que le TEA, un bloqueur de canaux potassiques, n'a aucun effet. Une dépolarisation de la membrane (KCl, 10 mM) induit une augmentation de [Ca2+]. Nous avons mis en évidence l'existence de sites récepteurs spécifiques pour le PN200-110 sur les cellules Jurkat (Kd = 8.5 + 3.1 nM, 2300 + 500 sites/cellule). Nos résultats suggèrent l'existence de canaux calciques chez les cellules Jurkat, lesquels sont probablement impliqués dans la traduction du signal transmembranaire

Phospholipase A1 Member A Deficiency Alleviates Mannan-Induced Psoriatic Arthritis in Mice Model

Synovial fluids from rheumatoid and psoriatic arthritis patients have high levels of PLA1A. The current study was to understand PLA1A functions in the pathophysiology of rheumatic diseases. We generated Pla1a−/− mice to assess their phenotype and the impact of PLA1A deficiency on the development of mannan-induced psoriatic arthritis (MIP). Mice were evaluated routinely for the induced symptoms. On the day of sacrifice, blood samples were collected for hematology analysis and prepared for plasma. Livers were collected. Lymph node immune cells were analyzed using flow cytometry. We performed μCT scans of hind paws from naïve and mannan-induced female mice. Cytokines/chemokines were quantified using Luminex in hind paw tissues and plasma of female mice. Pla1a−/− mice showed a slight increase in circulating and lymph node lymphocytes. CD4+ T cells contributed most to this increase in lymph nodes (p = 0.023). In the MIP model, the lymph node ratios of CD3+ to CD19+ and CD4+ to CD8+ were higher in Pla1a−/− mice. Pla1a−/− mice were less susceptible to MIP (p −/− mice also showed reduced IL-17, KC, IP-10, MIP-1β, LIF, and VEGF in hind paw tissues as compared to WT mice (p < 0.05). These findings indicated that PLA1A deficiency protected from the development of the MIP disease. The data suggested that PLA1A could contribute to MIP through increased activation of lymphocytes, possibly those producing IL-17

VLA-4 Induces Chemoresistance of T Cell Acute Lymphoblastic Leukemia Cells via PYK2-Mediated Drug Efflux

Cell adhesion plays a critical role in the development of chemoresistance, which is a major issue in anti-cancer therapies. In this study, we have examined the role of the VLA-4 integrin, a major adhesion molecule of the immune system, in the chemoresistance of T-ALL cells. We found that attachment of Jurkat and HSB-2 T-ALL cells to VCAM-1, a VLA-4 ligand, inhibits doxorubicin-induced apoptosis. However, their adhesion to fibronectin, which is mainly mediated via VLA-5, had no effect. Even the presence of the chemoattractant SDF1α (Stromal cell-derived factor-1α), which enhances the adhesion of T-ALL cells to fibronectin, did not modify the sensitivity of the cells attached on fibronectin towards doxorubicin-induced apoptosis. Mechanistically, we found that VLA-4 promoted T-ALL chemoresistance by inducing doxorubicin efflux. Our results showed that cell adhesion to both fibronectin and VCAM-1-induced Focal adhesion kinase (FAK) phosphorylation in T-ALL cells. However, only cell adhesion to VCAM-1 led to PYK2 phosphorylation. Inhibition studies indicated that FAK is not involved in doxorubicin efflux and chemoresistance, whereas PYK2 inhibition abrogated both VLA-4-induced doxorubicin efflux and chemoresistance. Together, these results indicate that the VLA-4/PYK2 pathway could participate in T-ALL chemoresistance and its targeting could be beneficial to limit/avoid chemoresistance and patient relapse