The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes ...natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56bright, NK cells. Most NK cells in healthy human donors are CD45RA+CD45RO-. The CD45RA-RO+ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RAdimRO- (CD45RAdim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RAdim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RAdim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.
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-alpha/beta 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-alpha 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.
Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ ...receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.
Monoclonal 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.
We combined allogeneic NK cells and mAbs to improve cancer treatment. We generated expanded NK cells (e-NK) with strong
and
ADCC responses against different tumors and using different therapeutic mAbs, namely rituximab, obinutuzumab, daratumumab, cetuximab and trastuzumab.
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.
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.
Natural killer (NK) cells, a cytotoxic lymphocyte lineage, are able to kill tumor cells in vitro and in mouse models. However, whether these cells display an anti-tumor activity in cancer patients ...has not been demonstrated. Here we have addressed this issue in patients with several hematological cancers. We found a population of highly activated CD56dimCD16+ NK cells that have recently degranulated, evidence of killing activity, and it is absent in healthy donors. A high percentage of these cells expressed natural killer cell p46-related protein (NKp46), natural-killer group 2, member D (NKG2D) and killer inhibitory receptors (KIRs) and a low percentage expressed NKG2A and CD94. They are also characterized by a high metabolic activity and active proliferation. Notably, we found that activated NK cells from hematological cancer patients have non-NK tumor cell antigens on their surface, evidence of trogocytosis during tumor cell killing. Finally, we found that these activated NK cells are distinguished by their CD45RA+RO+ phenotype, as opposed to non-activated cells in patients or in healthy donors displaying a CD45RA+RO− phenotype similar to naïve T cells. In summary, we show that CD45RA+RO+ cells, which resemble a unique NK population, have recognized tumor cells and degranulate in patients with hematological neoplasias.
•Expression of both CD45 isoforms RA and RO identifies anti-leukemia NK cells.•Anti-leukemia NK cells proliferate, degranulate and perform trogocytosis in vivo.•The presence of CD45RARO population identifies hematological cancer patients.
NK cells are thought to have an intrinsic anti-tumor activity. However, the presence of anti-leukemia NK cells in patients is unknown. We present a relatively simple protocol to identify and characterize them. This is based on differential protein expression and on the fact that they gain tumor cell proteins by the process of trogocytosis. These phenotypic differences should be taken into account in analysis to identify different NK cell subpopulations. Hence, NK cells are actively recognizing tumor cells in leukemia patients; but this seems to be insufficient to eradicate disease. Future efforts should enhance the antitumor activity of this population.
Single-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.
STUDY QUESTION
What is the expression pattern of microRNAs (miRNAs) in human cumulus–oocyte complexes (COCs)?
SUMMARY ANSWER
Several miRNAs are enriched in cumulus cells (CCs) or oocytes, and are ...predicted to target genes involved in biological functions of the COC.
WHAT IS KNOWN ALREADY
The transcriptional profiles of human MII oocytes and the surrounding CCs are known. However, very limited data are available about post-transcriptional regulators, such as miRNAs. This is the first study focussing on the identification and quantification of small RNAs, including miRNAs, in human oocytes and CCs using a deep-sequencing approach.
STUDY DESIGN, SIZE, DURATION
MII oocytes and CCs were collected from women who underwent IVF.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Using the Illumina/deep-sequencing technology, we analyzed the small RNAome of pooled MII oocytes (n = 24) and CC samples (n = 20). The mRNA targets of CC and MII oocyte miRNAs were identified using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in oocytes (10 pools of 19 ± 3 oocytes/each) and 10 individual CC samples. TaqMan miRNA assays were used to confirm the sequencing results in independent pools of MII oocytes (3 pools of 8 ± 3 oocytes/each) and CC samples (3 pools of 7 ± 3 CCs/each). The functional role of one miRNA, MIR23a, was assessed in primary cultures of human CCs.
MAIN RESULTS AND THE ROLE OF CHANCE
Deep sequencing of small RNAs yielded more than 1 million raw reads. By mapping reads with a single location to the human genome, known miRNAs that were abundant in MII oocytes (MIR184, MIR100 and MIR10A) or CCs (MIR29a, MIR30d, MIR21, MIR93, MIR320a, MIR125a and the LET7 family) were identified. Predicted target genes of the oocyte miRNAs were associated with the regulation of transcription and cell cycle, whereas genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including PTGS2, CTGF and BMPR1B that are important for cumulus–oocyte communication. Functional analysis using primary CC cultures revealed that BCL2 and CYP19A1 mRNA levels were decreased upon MIR23a overexpression.
LIMITATIONS, REASONS FOR CAUTION
Only known miRNAs were investigated in the present study on COCs. Moreover, the source of the material is MII oocytes that failed to fertilize.
WIDER IMPLICATIONS OF THE FINDINGS
The present findings suggest that miRNA could play a role in the regulation of the oocyte and CC crosstalk.
STUDY FUNDING/COMPETING INTEREST(S)
This work was partially supported by a grant from Ferring Pharmaceuticals. The authors of the study have no conflict of interest to report.
TRIAL REGISTRATION NUMBER
Not applicable.
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-allylaminodemethoxy 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.
Abstract We analysed, by flow cytometry, the “adhesive” phenotype of acute myelogenous leukemia (AML) cells from 36 patients treated in our institution. In univariate analysis, the main prognostic ...factor for CR achievement was lower CXCR4 expression ( p = 0.03). Overall survival (OS) was negatively influenced by higher CXC chemokine receptor 4 (CXCR4) ( p = 0.01), very late antigen-4 (VLA-4) ( p = 0.01), and focal adhesion kinase (FAK) expression ( p = 0.04). Combination of these markers allowed to distinguish two prognostic groups: patients overexpressing 2 or 3 factors had a significantly shorter OS ( p = 0.015). CXCR4, VLA-4 and FAK are new phenotypic markers which could be helpful to establish risk-stratified therapeutic strategies.
Perception of treatment effect (TE) in cosmetics is multifaceted and influenced by multiple parameters that need to be considered simultaneously when evaluating TE. Here we provide a global approach ...to predicting TE perception using Random Forest (RF) classifier. Data from three randomized double-blind clinical studies with a total of 50 subjects were used. Different products were applied to each facial cheek of subjects at each visit, and post-application photographs were taken. Nine primary endpoints relating to skin pores were assessed by a specific image analysis algorithm. Twenty judges evaluated the relative pore visibility in all possible pairs of cheek photographs. RF was used to construct a prediction model for TE perception based on the primary endpoints and judge's evaluation. Intra-study product ranking was done using the Bradley-Terry model on mean judges' predicted preference. RF demonstrated overall good accuracy in predicting TE perception. Applying RF technique not only addresses issues of multiplicity, nonlinearity and interactions between multiple criteria but also focuses decision-making on one discrete parameter thereby simplifying interpretability and allowing for more consumer-centered claim substantiation in clinical trials.
Abbreviations: RF: Random Forest classifier; FDA: The US Food and Drug Agency; ID: Identifier; MCID: Minimal clinical important difference; Param: Parameter; PGIC: Patients' Global Impression of Change; TE: Treatment effect; TRT: Treatment
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