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International audience; The antitumor activity of exercise by means of enhanced immune activation is documented, but better identification of the underlying mechanisms is required to develop new therapeutic strategies. Recent work from the Dr Bar-Sagi group reveals that exercise engages IL-15 signaling and pharmacological activation of the IL-15/IL-15R axis mimics the exercise-driven immune cell-mediated cytotoxicity in pancreatic cancer. more...

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein ‘cytokine inducible SH2-containing protein’ (CISH) in NK cells to evaluate the impact on their functions and antitumor properties.MethodsTo further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions.ResultsIn Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions.ConclusionThis study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy. more...

Immunotherapies targeting the “don’t eat me” myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies, and the strong ADCC effector function of Poly Morpho Nuclear Cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cells function while limiting adverse effects to healthy cells able to participate in the anti-tumor immune response.SynopsisDose-limiting toxicities demonstrate the need to investigate anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. We demonstrate that anti-CD47 mAbs stimulate potent killing of T cells by PMN through a novel combination of trogocytosis and ROS regulated by SIRPα.Graphical Abstract more...

International audience

Significance This work provides a report of accumulation of unconventional CD56−CD16+ NK cells in nonvirally induced malignancies. Increased frequency of CD56−CD16+ NK cells is associated with adverse clinical outcome in AML, as well as other maturation defects, and might contribute to a defective control of AML progression. Pseudotime analysis highlights a disruption in the maturation process of conventional NK cells in AML patients, leading to a bifurcation point absent in healthy subjects. This analysis, combined with the reduced frequency of conventional NK cells observed in AML patients, suggests that unconventional CD56−CD16+ NK cells derive from an aberrant maturation of conventional NK cells. Overall, accumulation of CD56−CD16+ NK cells could be an important feature of immune escape from innate immunity., Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56−CD16+ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56−CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56−CD16+ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56−CD16+ NK cells. Overall, our data suggest that the accumulation of CD56−CD16+ NK cells may be the consequence of immune escape from innate immunity during AML progression. more...

Simple Summary Vδ2+ γδ T cells have potent antitumor properties both in vitro and in murine preclinical models of breast cancers. However, in the context of human breast cancer, there is a lack of information for potential phenotypic alterations of this crucial immune cell subset. This is partly due to Vδ2+ γδ T cells scarcity in surgical specimens. To break this deadlock, we assessed Vδ2+ γδ T cell phenotypes using untreated breast cancer patients’ peripheral blood, so-called minimally invasive “liquid biopsy”. We show that circulating Vδ2+ γδ T cell phenotypic alterations are already established at diagnosis and related to tumor progression. Notably, terminally differentiated Vδ2+ γδ T cells expressing canonical markers of replicative senescence and exhaustion were significantly associated with tumor-draining lymph node invasion. Our results shed light on the interest of using liquid biopsy to monitor rare events and support Vδ2+ γδ T cell involvement in breast cancer pathogenesis and progression. Abstract The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αβ T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αβ T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients’ peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients’ peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity. more...

International audience

Natural killer (NK) cells are major anti-leukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the present work, we highlight an accumulation of CD56-CD16+ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly-diagnosed AML (N=48, HEMATOBIO cohort, NCT02320656) and healthy subjects (N=18) by mass cytometry. We evidenced a moderate to drastic accumulation of CD56- CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30, and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56-CD16+ NK cells at diagnosis was associated with an adverse clinical outcome, with decreased overall survival (HR=0.13; P=.0002) and event-free survival (HR=0.33; P=.018), and retained statistical significance in multivariate analysis. Pseudo-time analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56-CD16+ NK cells. Overall, our data suggest that the accumulation of CD56-CD16+ NK cells may be the consequence of immune escape from innate immunity during AML progression.SignificanceThis work provides the first report of accumulation of unconventional CD56-CD16+ NK cells in non-virally induced malignancies. Pseudotime analysis highlights a bifurcation point occurring during the course of NK cell maturation, providing elements regarding the possible origin of CD56-CD16+ NK cells. Increased frequency of CD56-CD16+ NK cells is associated with adverse clinical outcome in AML and might contribute, as well as other maturation defects, to a defective control of AML progression. Overall, accumulation of CD56-CD16+ NK cells could be an important feature of immune escape from innate immunity.Graphical abstractKey pointsA disruption in the maturation process of NK cells leads to accumulation of unconventional CD56- CD16+ NK cells in patients with AMLHigh frequency of CD56-CD16+ NK cells is associated with adverse clinical outcome more...

International audience; No abstract available

International audience; Women with low levels of vitamin D have a higher risk of developing breast cancer. Numerous studies associated the presence of a CD8+ T cell infiltration with a good prognosis. As vitamin D may play a key role in the modulation of the immune system, the objective of this work was to evaluate the impact of vitamin D on the breast cancer progression and mammary tumor microenvironment. We show that vitamin D decreases breast cancer tumor growth. Immunomonitoring of the different immune subsets in dissociated tumors revealed an increase in tumor infiltrating CD8+ T cells in the vitamin D-treated group. Interestingly, these CD8+ T cells exhibited a more active T cell (TEM/CM) phenotype. However, in high-fat diet conditions, we observed an opposite effect of vitamin D on breast cancer tumor growth, associated with a reduction of CD8+ T cell infiltration. Our data show that vitamin D is able to modulate breast cancer tumor growth and inflammation in the tumor microenvironment in vivo. Unexpectedly, this effect is reversed in high-fat diet conditions, revealing the importance of diet on tumor growth. We believe that supplementation with vitamin D can in certain conditions represent a new adjuvant in the treatment of breast cancers. more...

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P in human vs. A in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this YAPP sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside., Progetto Ateneo” (Sapienza University of Rome, Italy) and Multiple Sclerosis Italian Foundation (FISM 2016/R/29) more...

International audience; Downstream of Kinase (DOK) proteins represent a multigenic family of adaptors that includes negative regulators of immune cell signaling. Using phylogenetics and intron/exon structure data, we show here that the seven human DOK genes (DOK1 to DOK7) form three highly divergent groups that emerged before the protostome-deuterostome split: DOK1/2/3, DOK4/5/6, and DOK7. For two of these three groups (DOK1/2/3 and DOK4/5/6), further gene duplications occurred in vertebrates and so while chordates only have three DOK genes, vertebrates have seven DOK genes over the three groups. From our expression analysis in humans, we show that each group of DOK genes has a distinct pattern of expression. The DOK1/2/3 group is immune specific, yet each of the three genes in the group has a distinct pattern of expression in immune cells. This immune specificity could thus be ancestral, with the DOK1/2/3 gene also being immune -related in protostomes. The DOK4/5/6 and DOK7 groups represent genes that are much less expressed in immune system than the DOK1/2/3 group. Interestingly, we identify a novel tyrosine based motif that is specific to the vertebrate DOK41516 sequences. The evolution of the DOK genes is thus marked by a pattern of ancient duplications and functional specializations. more...

Background Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation. Methods Whole blood was treated by lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulating factor (GM-CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 – CD45 staining and three phosphorylated proteins such as AKT, ERK-1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis. Results By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK-1/2 phosphorylation where as GM-CSF stimulation induces STAT5 phosphorylation. Conclusions This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society more...

International audience; Crosslinking of regulatory immunoreceptors (RR), such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-α/β and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function-IFN-α production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction. more...

International audience; SIGNIFICANCE: Immune T cells are present in adipose tissues (AT), and the stoichiometry of the different T cell subsets is altered during diet-induced obesity (DIO). T cells contribute to the early steps of AT inflammation during DIO. Recent Advances: Many factors could potentially be responsible for this altered pro-inflammatory versus anti-inflammatory T cell balance.CRITICAL ISSUES: T cells are potentially activated in AT, which vitamin D might contribute to, as will be discussed in this article. In addition, we will review the different possible contributors to T cell activation in AT, such as the CD28 and CD154 T cell costimulatory molecules in AT.FUTURE DIRECTIONS: The potential antigen presentation capacities of adipocytes should be further investigated. Moreover, the properties of these AT resident (or migrating to AT) T cells must be further assessed. Antioxid. Redox Signal. 26, 489-500. more...

Dok1 and Dok2 proteins play a crucial role in myeloid cell proliferation as demonstrated by Dok1 and Dok2 gene inactivation, which induces a myeloproliferative disease in aging mice. In this study, we show that Dok1/Dok2 deficiency affects myeloproliferation even at a young age. An increase in the cellularity of multipotent progenitors is observed in young Dok1/Dok2-deficient mice. This is associated with an increase in the cells undergoing cell cycle, which is restricted to myeloid committed progenitors. Furthermore, cellular stress triggered by 5-fluorouracil (5-FU) treatment potentiates the effects of the loss of Dok proteins on multipotent progenitor cell cycle. In addition, Dok1/Dok2 deficiency induces resistance to 5-FU–induced hematopoietic stem cell exhaustion. Taken together, these results demonstrate that Dok1 and Dok2 proteins are involved in the control of hematopoietic stem cell cycle regulation. more...

Key players on the immunosurveillance program, T cells are regulated by their surface receptors such as T-cell receptor (TCR), and costimulatory molecules, optimizing T-cell activation. Some of these costimulatory molecules, such as the cytotoxic T-lymphocyte antigen 4 (CTLA-4), induce inhibitory effects on T cells. By "inhibiting the inhibitor" CTLA-4-blocking monoclonal antibodies represent a novel class of weapons against cancers. To better understand the promising future and the limits of this immunotherapy, this review dissects the molecular inhibitory mechanisms induced by CTLA-4 in T cells. more...

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85α subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50α, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50α accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50α plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50α at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50α is known to carry a stronger lipid kinase activity compared with p85α. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50α is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules. more...

International audience; CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) . more...

Q fever is an infectious disease caused byCoxiella burnetii, an obligate intracellular bacterium that replicates in macrophages. As cell-mediated immune response to microbial pathogens requires signals mediated by T-cell receptors and costimulatory molecules such as CD28, we wondered if CD28 is involved in protection againstC. burnetiiinfection. CD28-deficient (CD28−/−) mice were inoculated withC. burnetiiby intraperitoneal and intravenous routes. With both wild-type and CD28−/−mice,C. burnetiiorganisms were detected exclusively in spleen and liver. The antibody response againstC. burnetiiwas impaired in CD28−/−animals, but, surprisingly, the lack of CD28 decreasedC. burnetiiburden in the infected tissues, whatever the manner of inoculation of bacteria. The CD28 deficiency had no effect on either granuloma formation, which reflects cell-mediated immunity againstC. burnetii, or the production of gamma interferon and tumor necrosis factor, two cytokines known to be involved in granuloma formation. On the other hand, the production of interleukin-10 (IL-10) by peritoneal macrophages was highly impaired in CD28−/−mice. The results suggest that CD28 initiates a signal that favorsC. burnetiireplication through the modulation of the IL-10 pathway. more...

Tec is the prototypical member of the Tec tyrosine kinases family, which plays an important role in T cell signaling. We show in this study that Tec translocates to the immunological synapse when a T cell contacts a dendritic cell. Surprisingly, the presence of the pleckstrin homology (PH) domain of Tec is not required for this accumulation, and despite a strong activation of 3′-phosphorylated phosphoinositide lipids synthesis during the synapse formation, the Tec PH domain is not redistributed to the T cell plasma membrane. In contrast, we demonstrate that an active Src homology 3 domain is absolutely required, underlining the essential role played by this part of the molecule in the recruitment and/or stabilization of Tec at the immunological synapse. Our results nevertheless suggest that the PH domain controls the kinase activity of the molecule in vivo. We finally demonstrate that the two domains are necessary to trigger transcriptional events following Ag presentation. These data support a model in which the plasma membrane recruitment of the PH-containing protein Tec is not dependent on the production of 3′-phosphorylated phosphoinositide lipids by the PI3K, but rather on an intact Src homology 3 domain. more...

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates cell survival and proliferation mediated by phosphoinositol 3 kinases. We have explored the role of the phosphoinositol(3,4,5)P3-phosphatase PTEN in T cell development by analyzing mice with a T cell–specific deletion of PTEN. Ptenflox/floxLck-Cre mice developed thymic lymphomas, but before the onset of tumors, they showed normal thymic cellularity. To reveal a regulatory role of PTEN in proliferation of developing T cells we have crossed PTEN-deficient mice with mice deficient for interleukin (IL)-7 receptor and pre–T cell receptor (TCR) signaling. Analysis of mice deficient for Pten and CD3γ; Pten and γc; or Pten, γc, and Rag2 revealed that deletion of PTEN can substitute for both IL-7 and pre-TCR signals. These double- and triple-deficient mice all develop normal levels of CD4CD8 double negative and double positive thymocytes. These data indicate that PTEN is an important regulator of proliferation of developing T cells in the thymus. more...

The Dok adaptor family of proteins binding to RasGAP, consisting of Dok-1 and Dok-2, are critical regulators in cell proliferation. These molecules are partners and/or substrates of different protein tyrosine kinases considered as oncoproteins. Here, we show that Dok-1 and Dok-2 are the major tyrosine-phosphorylated proteins associated to Tec, a protein tyrosine kinase expressed in T cells. Furthermore, we evaluate the effect of Dok-1 or Dok-2 on Tec-mediated signalling pathways in T cells. Here, we provide evidence that Dok-1 and Dok-2 proteins are involved in a negative feedback regulation of Tec via a downregulation of its tyrosine phosphorylation and downstream signalling pathways including the Ras pathway. Either Dok-1 or Dok-2 therefore represents a mean of potent retrograde control for protein tyrosine kinase signalling, and then possibly of tumor development. more...

IFN-α is an important cytokine for the generation of a protective T cell-mediated immune response to viruses. In this study, we asked whether IFN-α can regulate the functional properties of dendritic cells (DCs). We show that monocytes cultured in the presence of GM-CSF and IFN-α can differentiate into DCs (IFN-α-derived DCs (IFN-DCs)). When compared with DCs generated in the presence of GM-CSF and IL-4 (IL-4-derived DCs), IFN-DCs exhibited a typical DC morphology and expressed, in addition to DC markers CD1a and blood DC Ag 4, a similar level of costimulatory and class II MHC molecules, but a significantly higher level of MHC class I molecules. After maturation with CD40 ligand, IFN-DCs up-regulated costimulatory, class I and II MHC molecules and expressed mature DC markers such as CD83 and DC-lysosome-associated membrane protein. IFN-DCs were endowed with potent functional activities. IFN-DCs secreted large amounts of the inflammatory cytokines IL-6, IL-10, TNF-α, IL-1β, and IL-18, and promoted a Th1 response that was independent of IL-12p70 and IL-18, but substantially inhibited by IFN-α neutralization. Furthermore, immature IFN-DCs induced a potent autologous Ag-specific immune response, as evaluated by IFN-γ secretion and expansion of CD8+ T cells specific for CMV. Also, IFN-DCs expressed a large number of Toll-like receptors (TLRs), including acquisition of TLR7, which is classically found on the natural type I IFN-producing plasmacytoid DCs. Like plasmacytoid DCs, IFN-DCs could secrete IFN-α following viral stimulation or TLR7-specific stimulation. Taken together, these results illustrate the critical role of IFN-α at the early steps of immune response to pathogens or in autoimmune diseases. more...

International audience; The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knockout) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail. more...

Background/Objectives Lymphocytes play a critical role in visceral adipose tissue inflammation. The CD28 costimulatory molecule is required for lymphocyte activation and for the development of a functional regulatory T cells (Treg) compartment; however its role during obesity is unknown. Methods During diet-induced obesity, we investigated the effects of selective interference with CD28 signaling using knock-out mice (Cd28KO) and a CTLA4-Ig fusion protein inhibiting CD28-B7 interactions. Results Cd28 deficiency decreased pathogenic T-cells and Treg content within adipose tissue without changing macrophages number. Cd28KO epididymal but not subcutaneous fat was characterized by enlarged adipocytes, reduced levels of inflammatory cytokines and increased Glut4, adiponectin and lipogenic enzymes mRNA levels. This was associated with reduced inflammation, fat accumulation and enhanced glucose metabolism in liver. Weight gain and fasting glucose tolerance were not affected. CTLA4-Ig injections reduced the number T-cells in epididymal adipose tissue but not inflammatory cytokines levels and failed to improve liver fat accumulation. Conclusions Deletion of CD28 creates a new pro/anti-inflammatory balance in epididymal adipose tissue and liver and exerts a protective effect against hepatic steatosis. more...

International audience; Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK-cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK-cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK-cell-activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK-cell maturation defect and leads to increased IFN-γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK-cell-activating receptors in mouse and human. more...

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) critically regulates the T-cell immune response, and the a chain CD25/IL-2Ra is required for the formation of the high-affinity receptor. Tissuespecific, inducible expression of the IL-2Ra gene is regulated by at least three positive regulatory regions (PRRI, PRRII, and PRRIII), but none responded to CD28 engagement in gene reporter assays although CD28 costimulation strongly amplifies IL-2Ra gene transcription. By DNase I hypersensitivity analysis, we have identified a novel TCR-CD3- and CD28-responsive enhancer (CD28rE) located 8.5 kb 5* of the IL-2Ra gene. PRRIV/CD28rE contains a functional CRE/TRE element required for CD28 signaling. The T-cell-specific, CD28-responsive expression of the IL-2Ra gene appears controlled through PRRIV/CD28rE by cooperation of CREB/ATF and AP-1 family transcription factors. Complete T-cell activation requires antigen-mediated signaling through the TCR-CD3 complex and costimulatory signals that can be provided by CD28 and its counterreceptor, B7. The B7-CD28 signaling pathways synergize with mitogenic signal from the TCR-CD3 complex to promote prolonged T-cell proliferation and increase interleukin-2 (IL-2) secretion (27, 33, 36). Although stimulation via CD28 alone usually cannot induce effector functions, its signaling pathways involve sitespecific tyrosine phosphorylation of several effector proteins that are crucial for these functions (52). Triggering these cosignaling downstream pathways ultimately leads to the transcriptional activation and mRNA stabilization of genes encoding a number of cytokines critically involved in the regulation of Th1-Th2 differentiation (8). Among them, IL-2 plays a major role in the regulation of the magnitude and duration of T-cell activation in the immune responses (41). IL-2 promotes T-cell proliferation by interacting with its high-affinity receptor composed of three transmembrane polypeptides, the a, b, and gc chains. The a chain (CD25) is induced during T-cell activation, and its association with b and gc chains constitutes the high-affinity receptor. Most of the biologic effects of IL-2 are mediated through the high-affinity complex as evidenced by the almost identical phenotype of mice with targeted inactivation of the genes for IL-2 (53) and IL-2Ra (66). Furthermore, the importance of the IL-2Ra gene in the immune system is dramatically illustrated by the profound immunodeficiency observed in patient lacking CD25/IL-2Ra expression (12, 51). Expression of IL-2Ra gene is tightly controlled at the transcriptional level (34, 61) and by mRNA stabilization (28). Previous studies have identified three positive regulatory regions, PRRI, PRRII, and PRRIII, within the 59 noncoding region of the human IL-2Ra gene. Each regulatory region appears to play a well-defined and complementary role. PRRI, located between nucleotides 2276 and 2244, contributes to the inducibility of the IL-2Ra gene, whereas PRRII, located between nucleotides 2137 and 264, is involved in basal promoter activity as well as in T-cell-specific expression (25). PRRIII, located between nucleotides 23786 and 23701, is a specific IL-2-responsive enhancer playing a crucial role in the response to the IL-2/IL-2R autoregulatory loop which amplifies IL-2Ra gene transcription in the late stage of T-cell activation (26, 32, 57). The transcriptional activity of PRRI can be regulated by the viral protein Tax (transcription activator protein of human T-cell leukemia virus type 1), and the transcription factors NF-kB and a serum response factor-related protein (2, 30, 35, 47). Elf-1, a member of the Ets family of proteins specific for lymphoid tissue, and the nonhistone chromatin-associated protein, HMG-I(Y), regulate PRRII (25). The transcriptional activity of PRRIII/IL-2rE is controlled by the functional interaction of Stat5, Ets-1, and Ets-2 in response to IL-2 stimulation (50). more...

Mutational analysis of the DOK2 haploinsufficient tumor suppressor gene in chronic myelomonocytic leukemia (CMML)

International audience; Background:The phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers,including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not beenyet described.Methods:To clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somaticmutations in exon 9 and 20 using direct sequencing. The same samples were analyzed using western blotting andimmunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases ofbenign lymphadenitis were used as controls.Results:AKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis casesexpressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benignlymphadenitis samples expressed low level of pAKT. PTEN expression was observed in 30/38 (79%) FL and 2/2benign lymphadenitis cases, whereas 8/38 (21%) FL cases showed loss of PTEN expression. 3 cases with positivepAKT did not express PTEN. PIK3CA mutations were not detected in any sample.Conclusions:These data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases,due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations. more...

A signaling module consisting of the adaptor ADAP and the Carma1–Bcl-10–MALT1 (CBM)–TAK1 signalosome has been shown to selectively induce cytokine secretion but not cytotoxicity in natural killer cells. more...

The present study compares the mitogen-activated protein (MAP) kinase responses in T cells activated with the CD28 ligands B7-1 (CD80) and B7-2 B70 (CD86). Ligands B7-1 and B7-2 do not activate the Raf-1 ERK2 cascade, but share the ability to activate related Jun kinases. These natural ligands for CD28 had no stimulatory effect alone on Jun kinase activation, but the data show that B7-1 and B7-2 could both co-operate with intracellular Ca 2+ increase and protein kinase C (PKC) activation to stimulate Jun kinases. The present study shows that the interaction of CD28 with its ligands B7-1 and B7-2 can induce identical signal transduction through the MAP kinase cascades. more...

The elimination of hepatitis C virus (HCV) in > 50% of chronically infected patients by treatment with IFN-α suggests that plasmacytoid dendritic cells (pDCs), major producers of IFN-α, play an important role in the control of HCV infection. However, despite large amounts of Toll-like receptor 7-mediated IFN-α, produced by pDCs exposed to HCV-infected hepatocytes, HCV still replicates in infected liver. Here we show that HCV envelope glycoprotein E2 is a novel ligand of pDC C-type lectin immunoreceptors (CLRs), blood DC antigen 2 (BDCA-2) and DC-immunoreceptor (DCIR). HCV particles inhibit, via binding of E2 glycoprotein to CLRs, production of IFN-α and IFN-λ in pDCs exposed to HCV-infected hepatocytes, and induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of monoclonal antibodies preserves the capacity of pDCs to produce type I and III IFNs in the presence of HCV particles. Thus, negative interference of CLR signaling triggered by cell-free HCV particles with Toll-like receptor signaling triggered by cell-associated HCV results in the inhibition of the principal pDC function, production of IFN. more...

12 pages; All the cells are engaged on a cell communication by sending and receiving signals. Cells having the correct receptors on their surfaces will encode the plasma membrane signal recognition to intracellular signals corresponding to cellular signaling events. This process corresponds to a huge intracellular network of protein-protein or lipid-protein interactions. To avoid to be lost in this kind of maze, some general signaling pathways are most of the time present in many cell systems such as MAP kinases or PI-3 kinases pathways. These signaling pathways induce some protein phosphorylation events that can be identified by specific antibodies directed against these phosphosites (anti-phospho antibodies). Thus, these crossroads can be determined by using antibodies. A widely used method to identify these signaling events is the Western blotting or protein immunoblotting from cell extracts. However, several research groups are now developing new analytical techniques based also on the use of anti-phospho antibodies, but allowing them to detect signaling events at the single cell level. These experimental approaches are using the principles of the flow cytometry (FCM). Phosphoflow technology will provide rapid, quantitative and also multi-parameter analyses on single cells. more...