Shp-2 is dispensable for establishing T cell exhaustion and for PD-1 signaling in vivo

In chronic infection and cancer, T cells acquire a dysfunctional state characterized by the expression of inhibitory receptors. In vitro studies implicated the phosphatase Shp-2 downstream of these receptors, including PD-1. However, whether Shp-2 is responsible in vivo for such dysfunctional responses remains elusive. To address this, we generated T cell- specific Shp-2-deficient mice. These mice did not show differences in controlling chronic viral infections. In this context, Shp-2-deleted CD8+ T lymphocytes expanded moderately better but were less polyfunctional than control cells. Mice with Shp-2-deficient T cells also showed no significant improvement in controlling immunogenic tumors and responded similarly to controls to α-PD-1 treatment. We therefore showed that Shp-2 is dispensable in T cells for globally establishing exhaustion and for PD-1 signaling in vivo. These results reveal the existence of redundant mechanisms downstream of inhibitory receptors and represent the foundation for defining these relevant molecular events

Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion

On the rôle of the tyrosine phosphatase Shp-2 and the transcription factor Myc in innate and adaptive cytotoxic responses

NK cells are innate lymphocytes that mediate protection against tumors and viral infections. Their effector fonctions are highly controlled by inhibitory and activating receptors. The phosphatase SH2 domain-containing tyrosine phosphatase 1 (Shp-1) has been shown to relay signais important for NK cell responsiveness downstream of inhibitory receptors. The closest homolog of Shp-1, called Shp-2, was proposed to exert a similar fonction in NI< cells as it could associate with inhibitory receptors. Nevertheless, its contribution to NK cell responsiveness has not been investigated in vivo. By generating a mouse model in which Shp-2 was selectively deleted in NK cells, we observed - surprisingly - that the effector fonctions of Shp-2-deficient NI< cells were not altered. In contrast, we found that Shp-2-deficient NK cells presented a compromised response to interleukin (LL)-15, an essential cytokine for NK cell development, survival, and activation. While knockout cells survived better in conditions of low doses of IL-15 due to increased expression of the anti-apoptotic protein Bcl-2, their maintenance and prolifération were strongly reduced at high doses of IL-15 due to impaired mammalian target of rapamycin (mTOR) and abrogated extracellular signal-regulated kinase (ERK) signalings. These results emphasize a crucial rôle of Shp-2 in the IL-15 signaling in order to enhance NK cell responses under inflammatory conditions. In NK cells, in addition to mTOR and ERK, IL-15 engages the janus kinase (Jak)/signal transducers and activators of transcription 5 (STAT5) pathway. Interestingly, these three signaling cascades have been reported to control the expression of the proto-oncogene Myelocytomatosis (Myc) in différent cell types and IL-15 was shown to upregulate Myc levels in NK cells. Moreover, Myc has been associated to NK cell cancer progression, suggesting an important rôle for this transcription factor in these lymphocytes. Because its fonction is not characterized in NK cells, we generated mice with a conditional deletion of Myc in the NK cell lineage. Our data revealed that in the absence of Myc NK cells presented reduced prolifération and numbers, and this affected their anti-tumoral immunity. Mechanistically, we found that Myc protein levels were upregulated upon IL- 15 stimulation through engagement of mTOR and ERK signalings. Moreover, we showed that Myc controls a transcriptional program associated to protein translation within NK cells, and this might explain the ability of these cells to proliferate. Altogether, this thesis work describes the essential rôle of Shp-2 and Myc in regulating NIC cell responses through IL-15 signaling. A better understanding of IL-15 signaling in NK cells and of their biology more broadly might help develop therapeutic stratégies for the treatment of immune diseases and cancer. -- Les cellules NK sont des lymphocytes innés qui assurent une protection contre les tumeurs et les infections virales. Leurs fonctions effectrices sont hautement contrôlées par des récepteurs inhibiteurs et activateurs. La tyrosine phosphatase Shp-1 a été montré comme relayant des signaux importants pour la réactivité des cellules NI< en dessous des récepteurs inhibiteurs. L'homologue le plus proche de Shp-1, appelé Shp-2, a été proposé pour exercer une fonction similaire dans les cellules NK car il pouvait s'associer à des récepteurs inhibiteurs. Néanmoins, sa contribution à la réactivité des cellules NK n'a pas été étudiée in vivo. En générant un modèle de souris où Shp-2 a été sélectivement supprimée dans les cellules NK, nous avons observé - étonnamment - que les fonctions effectrices des cellules NK déficientes en Shp-2 n'étaient pas altérées. En revanche, nous avons constaté que ces cellules présentaient une réponse compromise à l'interleukine (IL)-15, une cytokine essentielle au développement, à la survie et l'activation des cellules NK. Alors que les cellules knockout survivaient mieux dans des conditions de faibles doses d'IL-15 à cause d'une expression augmentée de la protéine Bcl-2, leur maintien et leur prolifération étaient fortement réduits à des doses élevées d'IL-15, en raison de l'altération des protéines mTOR et ERK. Ces résultats soulignent le rôle crucial de Shp-2 dans la signalisation de l'IL-15 afin d'augmenter les réponses des cellules NK en conditions inflammatoires. Dans les cellules NK, en plus de mTOR et ERK, l'IL-15 engage aussi la voie Jak/STAT5. Ces trois cascades de signalisation ont été montré comme contrôlant l'expression du proto-oncogène Myc dans différents types cellulaires et l'IL-15 a été révélé comme augmentant les niveaux de Myc dans les cellules NK. De plus, Myc a été associé à des cancers impliquant les cellules NK, ce qui suggère un rôle important pour ce proto-oncogène dans ces lymphocytes. Puisque sa fonction n'est pas caractérisée dans les cellules NK, nous avons généré des souris avec une délétion conditionnelle de Myc dans ces lymphocytes. Nos données ont révélé qu'en l'absence de Myc, les cellules NK présentaient une prolifération et des nombres réduits, ce qui a affecté leur immunité anti-tumorale. D'un point de vue mécanique, nous avons trouvé que les niveaux protéiques de Myc étaient augmentés par l'IL-15 grâce à l'engagement des voies mTOR et ERK. Enfin nous avons montré que Myc contrôle un programme transcriptionnel associé à la traduction protéique au sein des cellules NK, et ceci pourrait expliquer la capacité de ces cellules à proliférer. Dans l'ensemble, ce travail de thèse décrit le rôle essentiel de Shp-2 et Myc dans la régulation des réponses des cellules NK par la signalisation IL-15. Une meilleure compréhension de la voie de l'IL-15 et plus largement de la biologie des cellules NK aiderait à développer des stratégies thérapeutiques pour le traitement des maladies immunitaires et du cancer

Unexpected cross-reactivity of anti-cathepsin B antibodies leads to uncertainties regarding the mechanism of action of anti-CD20 monoclonal antibody GA101

International audienceGA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode

The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence

Myc controls NK cell development, IL-15-driven expansion, and translational machinery

MYC is a pleiotropic transcription factor involved in cancer, cell proliferation, and metabolism. Its regulation and function in NK cells, which are innate cytotoxic lymphocytes important to control viral infections and cancer, remain poorly defined. Here, we show that mice deficient for Myc in NK cells presented a severe reduction in these lymphocytes. Myc was required for NK cell development and expansion in response to the key cytokine IL-15, which induced Myc through transcriptional and posttranslational mechanisms. Mechanistically, Myc ablation in vivo largely impacted NK cells’ ribosomagenesis, reducing their translation and expansion capacities. Similar results were obtained by inhibiting MYC in human NK cells. Impairing translation by pharmacological intervention phenocopied the consequences of deleting or blocking MYC in vitro. Notably, mice lacking Myc in NK cells exhibited defective anticancer immunity, which reflected their decreased numbers of mature NK cells exerting suboptimal cytotoxic functions. These results indicate that MYC is a central node in NK cells, connecting IL-15 to translational fitness, expansion, and anticancer immunity