Natural Killer Cells Promote Early CD8 T Cell Responses against Cytomegalovirus

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-α/β production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-α administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity

Développement d'anticorps bispécifiques anti-CEA / anti-FcyRIII pour l'immunothérapie des cancers

Le laboratoire a développé une nouvelle génération d anticorps bispécifiques, basée sur l utilisation de domaines variables d anticorps de lamas simple-chaîne lourde (VHH). Les domaines humains Ck et CH1 ont été utilisés comme motif de dimérisation de 2 VHH, liant l antigène carcinoembryonnaire (CEA) et le récepteur activateur FcgRIIIA (ou CD16A) exprimé par les cellules Natural Killer. Différents formats d anticorps bispécifiques ont été produits en système bactérien et caractérisés in vitro et in vivo. Après incubation à 37C en sérum humain, les formats bsFab sont stables au moins 7 jours, et les bsFab2 31h. Une biodistribution en souris nude montre une rétention des bsAb dans les tumeurs CEA+. Un suivi de la compétition avec les IgG humaines sériques pour la liaison au CD16A humain a montré que les bsAb ne sont pas en compétition avec les IgG humaines. De plus, les bsAb sont capables d induire une cytotoxicité de Natural Killer de donneurs à très faibles concentrations in vitro.The lab has designed a new kind of bispecific antibodies, based on the use of llama domain antibodies (VHH). Human Ck and CH1 domains have been used as a natural dimerization motif to bring together two domain antibodies binding to carcinoembryonic antigen (CEA) and activating receptor FcgRIIIA (CD16A) expressed by NK cells. Differents bispecific antibodies formats (bsFab and bsFab2) with variable binding profiles on the target cells have been produced in bacterial system and caracterized in vitro and in vivo. BsFab are stable at least for 7 days when incubated in human serum at 37C, and bsFab2 are stables for 31h. Biodistribution in nude mice showed that bsAb are retained in CEA+ tumor. A competition study of CD16A binding with human IgG showed that bsAb are not in competition with human IgG, contrary to Fc-bearing molecules. Moreover, bsAb are able to induce, via CD16A binding, donnor s Natural Killer cytotoxicity in vitro et very low concentrations.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Llama single-domain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross-reacting antigen.

International audienceSingle-domain antibodies (sdAbs), which occur naturally in camelids, are endowed with many characteristics that make them attractive candidates as building blocks to create new antibody-related therapeutic molecules. In this study, we isolated from an immunized llama several high-affinity sdAbs directed against human carcinoembryonic antigen (CEA), a heavily glycosylated tumor-associated molecule expressed in a variety of cancers. These llama sdAbs bind a different epitope from those defined by current murine mAbs, as shown by binding competition experiments using immunofluorescence and surface plasmon resonance. Flow cytometry analysis shows that they bind strongly to CEA-positive tumor cells but show no cross-reaction toward nonspecific cross-reacting antigen, a highly CEA-related molecule expressed on human granulocytes. When injected into mice xenografted with a human CEA-positive tumor, up to 2% of the injected dose of one of these sdAbs was found in the tumor, despite rapid clearance of this 15 kDa protein, demonstrating its high potential as a targeting moiety. The single-domain nature of these new anti-CEA IgG fragments should facilitate the design of new molecules for immunotherapy or diagnosis of CEA-positive tumors

All-trans retinoic acid (ATRA) induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity

International audienceNK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model

Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity against multiple tumors

International audienceMonoclonal antibodies (mAbs) have significantly improved the treatment of certain cancers. However, in general mAbs alone have limited therapeutic activity. One of their main mechanisms of action is to induce antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated by natural killer (NK) cells. Unfortunately, most cancer patients have severe immune dysfunctions affecting NK activity. This can be circumvented by the injection of allogeneic, expanded NK cells, which is safe. Nevertheless, despite their strong cytolytic potential against different tumors, clinical results have been poor. Methods: We combined allogeneic NK cells and mAbs to improve cancer treatment. We generated expanded NK cells (e-NK) with strong in vitro and in vivo ADCC responses against different tumors and using different therapeutic mAbs, namely rituximab, obinutuzumab, daratumumab, cetuximab and trastuzumab. Results: Remarkably, e-NK cells can be stored frozen and, after thawing, armed with mAbs. They mediate ADCC through degranulation-dependent and -independent mechanisms. Furthermore, they overcome certain anti-apoptotic mechanisms found in leukemic cells. Conclusion: We have established a new protocol for activation/expansion of NK cells with high ADCC activity. The use of mAbs in combination with e-NK cells could potentially improve cancer treatment

MicroRNAs: new candidates for the regulation of the human cumulus–oocyte complex

International audienceWhat is the expression pattern of microRNAs (miRNAs) in human cumulus-oocyte complexes (COCs)?Not applicable

Significance of heat-shock protein (HSP) 90 expression in acute myeloid leukemia cells

The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of a variety of client proteins with receptor and signal transduction functions. The objective of this study was to assess its expression in primary acute myeloid leukemia (AML) cells and to evaluate its biological and clinical significance. The in vitro effects of 17-AAG, a selective inhibitor of HSP90, was also evaluated. Cells from 65 patients with newly diagnosed AML were studied. The expression of HSP90 correlated with that of CD34, p170, and bcl-2 proteins but not with white cell counts, FAB or WHO subtype, or cytogenetics. HSP90 levels were also higher in samples exhibiting an autonomous growth in liquid culture or forming spontaneous colonies. A concomitant constitutive activation of the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/AKT pathways was observed in a majority of samples and was significantly correlated with HSP90 expression. All patients received induction chemotherapy. The percentages of HSP90-, CD34-, bcl-2-, and p170-positive cells were higher in patients who did not attain complete remission. Survival was also shorter in patients with high levels of HSP90. In vitro exposure of leukemic cells to 17-allylamino-demethoxy geldanamycin (17-AAG) resulted in inhibition of growth in liquid and clonogeneic cultures and in apoptosis, at concentrations which in most cases were not toxic for normal CD34-positive or progenitor cells. The concentration inhibiting 50% growth at 72 h in liquid culture correlated with HSP90 expression. Our study suggests that HSP90 is overexpressed in poor-prognosis AML cells and plays a role in cell survival and resistance to chemotherapy. Targeted therapy with 17-AAG represents a promising antileukemic strategy in adult AML