Vincent Vieillard

CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients.

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA‐E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C+CD56dim NK cells in patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Multi‐color flow cytometry revealed that the expanded NKG2C+CD56dim NK cells displayed a highly differentiated phenotype, expressed high amounts of granzyme B and exhibited polyfunctional responses (CD107a, IFN‐γ, and TNF‐α) to stimulation with antibody‐coated as well as HLA‐E expressing target cells but not when stimulated with IL‐12/IL‐18. More importantly, NKG2C+CD56dim NK cells had a clonal expression pattern of inhibitory killer cell immunoglobulin‐like receptors (KIRs) specific for self‐HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C‐mediated activation suggesting that such biased expression of self‐specific KIRs may preserve self‐tolerance and limit immune‐pathology during viral infection. Together, these findings shed new light on how the human NK‐cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells.

Unconventional Repertoire Profile Is Imprinted during Acute Chikungunya Infection for Natural Killer Cells Polarization toward Cytotoxicity

Chikungunya virus (CHIKV) is a worldwide emerging pathogen. In humans it causes a syndrome characterized by high fever, polyarthritis, and in some cases lethal encephalitis. Growing evidence indicates that the innate immune response plays a role in controlling CHIKV infection. We show here that CHIKV induces major but transient modifications in NK-cell phenotype and function soon after the onset of acute infection. We report a transient clonal expansion of NK cells that coexpress CD94/NKG2C and inhibitory receptors for HLA-C1 alleles and are correlated with the viral load. Functional tests reveal cytolytic capacity driven by NK cells in the absence of exogenous signals and severely impaired IFN-γ production. Collectively these data provide insight into the role of this unique subset of NK cells in controlling CHIKV infection by subset-specific expansion in response to acute infection, followed by a contraction phase after viral clearance.

NK Cell Terminal Differentiation: Correlated Stepwise Decrease of NKG2A and Acquisition of KIRs

Background Terminal differentiation of NK cells is crucial in maintaining broad responsiveness to pathogens and discriminating normal cells from cells in distress. Although it is well established that KIRs, in conjunction with NKG2A, play a major role in the NK cell education that determines whether cells will end up competent or hyporesponsive, the events underlying the differentiation are still debated. Methodology/Principal Findings A combination of complementary approaches to assess the kinetics of the appearance of each subset during development allowed us to obtain new insights into these terminal stages of differentiation, characterising their gene expression profiles at a pan-genomic level, their distinct surface receptor patterns and their prototypic effector functions. The present study supports the hypothesis that CD56dim cells derive from the CD56bright subset and suggests that NK cell responsiveness is determined by persistent inhibitory signals received during their education. We report here the inverse correlation of NKG2A expression with KIR expression and explore whether this correlation bestows functional competence on NK cells. We show that CD56dimNKG2A−KIR+ cells display the most differentiated phenotype associated to their unique ability to respond against HLA-E+ target cells. Importantly, after IL-12 + IL-18 stimulation, reacquisition of NKG2A strongly correlates with IFN-γ production in CD56dimNKG2A− NK cells. Conclusions/Significance Together, these findings call for the reclassification of mature human NK cells into distinct subsets and support a new model, in which the NK cell differentiation and functional fate are based on a stepwise decrease of NKG2A and acquisition of KIRs.

Identification of a cellular ligand for the natural cytotoxicity receptor NKp44

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network.

CD56brightCD16+ NK Cells: A Functional Intermediate Stage of NK Cell Differentiation

Human NK cells comprise two main subsets, CD56bright and CD56dim cells, which differ in function, phenotype, and tissue localization. To further dissect the differentiation from CD56bright to CD56dim cells, we performed ex vivo and in vitro experiments demonstrating that the CD56brightCD16+ cells are an intermediate stage of NK cell maturation. We observed that the maximal frequency of the CD56brightCD16+ subset among NK cells, following unrelated cord blood transplantation, occurs later than this of the CD56brightCD16− subset. We next performed an extensive phenotypic and functional analysis of CD56brightCD16+ cells in healthy donors, which displayed a phenotypic intermediary profile between CD56brightCD16− and CD56dimCD16+ NK cells. We also demonstrated that CD56brightCD16+ NK cells were fully able to kill target cells, both by Ab-dependent cell cytotoxicity (ADCC) and direct lysis, as compared with CD56brightCD16− cells. Importantly, in vitro differentiation experiments revealed that autologous T cells specifically encourage the differentiation from CD56brightCD16− to CD56brightCD16+ cells. Finally, further investigations performed in elderly patients clearly showed that both CD56brightCD16+ and CD56dimCD16+ mature subsets were substantially increased in older individuals, whereas the CD56brightCD16− precursor subset was decreased. Altogether, these data provide evidence that the CD56brightCD16+ NK cell subset is a functional intermediate between the CD56bright and CD56dim cells and is generated in the presence of autologous T CD3+ cells.

Chronic lymphocytic leukaemia cells are efficiently killed by an anti‐CD20 monoclonal antibody selected for improved engagement of FcγRIIIA/CD16

Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo‐immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo‐immunotherapy. We generated a chimeric anti‐CD20 monoclonal antibody (mAb), EMAB‐6, which has a low fucose content. Apoptosis and complement activities for EMAB‐6 were similar to those seen for rituximab. By contrast, EMAB‐6 mAb showed improved Fcγ receptor IIIA (FcγRIIIA)/CD16 binding and FcγRIIIA‐dependent effector functions. It induced a higher in vitro antibody‐dependent cellular cytotoxicity against CLL cells and a higher FcγRIIIA‐mediated interleukin‐2 production by FcγRIIIA+ Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB‐6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB‐6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.

Association of KIR2DS1 and KIR2DS3 with fatal outcome in Ebola virus infection.

Zaïre ebolavirus (ZEBOV) infection rapidly outruns the hosts immunity and leads to death within a week. Fatal cases have been associated with an aberrant innate, proinflammatory immune response followed by a suppressed adaptive response leading to the rapid depletion of peripheral NK cells and lymphocytes. A critical role for NK cells has been suggested but not elucidated. In this genetic study, we investigated the association of KIR genotype with disease outcome by comparing genotypes of a Gabonese control population, IgG+ contacts, survivors, and fatalities of ZEBOV infection. We showed that the activating KIR2DS1 and KIR2DS3 genes associate with fatal outcome in Ebola virus infection. In addition, this study brings supplemental evidence in favor of the specificity of the IgG+ contact population. The outcome of fulminating Ebola virus infection could depend in part on the hosts inherited KIR gene repertoire. This supports a key role for KIRs in disease susceptibility to infections.

Control of Acute Dengue Virus Infection by Natural Killer Cells

Dengue fever is the most important arthropod-borne viral disease worldwide, affecting 50–100 million individuals annually. The clinical picture associated with acute dengue virus (DENV) infections ranges from classical febrile illness to life-threatening disease. The innate immunity is the first line of defense in the control of viral replication. This review will examine the particular role of natural killer (NK) cells in DENV infection. Over recent years, our understanding of the interplay between NK cells and viral pathogenesis has improved significantly. NK cells express an array of inhibitory and activating receptors that enable them to detect infected targets while sparing normal cells, and to recruit adaptive immune cells. To date, the exact mechanism by which NK cells may contribute to the control of DENV infection remains elusive. Importantly, DENV has acquired mechanisms to evade NK cell responses, further underlining the relevance of these cells in pathophysiology. Hence, understanding how NK cells affect the outcome of DENV infection could benefit the management of this acute disease.

Expansion of CMV-Mediated NKG2C+ NK Cells Associates with the Development of Specific De Novo Malignancies in Liver-Transplanted Patients

Solid cancers are a major adverse outcome of orthotopic liver transplantation (OLT). Although the use of chronic immunosuppression is known to play a role in T cell impairment, recent insights into the specificities of NK cells led us to reassess the potential modulation of this innate immune cell compartment after transplantation. Our extensive phenotypic and functional study reveals that the development of specific de novo noncutaneous tumors post-OLT is linked to unusual NK cell subsets with maturation defects and to uncommon cytokine production associated with the development of specific cancers. Remarkably, in CMV+ patients, the development de novo head/neck or colorectal tumors is linked to an aberrant expansion of NK cells expressing NKG2C and a high level of intracellular TNF-α, which impact on their polyfunctional capacities. In contrast, NK cells from patients diagnosed with genitourinary tumors possessed a standard immature signature, including high expression of NKG2A and a robust production of IFN-γ. Taken together, our results suggest that under an immunosuppressive environment, the interplay between the modulation of NK repertoire and CMV status may greatly hamper the spectrum of immune surveillance and thus favor outgrowth and the development of specific de novo tumors after OLT.

An HIVgp41 vaccine protects CD4 central memory T cells in SHIV-infected macaques.

BACKGROUNDnThe immunodeficiency defining AIDS results from a progressive decline in the number of CD4(+) T cells. Although no single viral protein is likely to be the sole effector of immune impairment, the gp41 envelope protein is believed to contribute significantly to AIDS pathogenesis. We have shown that 3S, a unique motif of gp41, is highly conserved in HIV-1 strains and specifically induces NKp44L, a ligand of the natural cytotoxicity receptor NKp44, on CD4(+) T cells, rendering them sensitive to NK lysis. We therefore hypothesized that a 3S/gp41 vaccine strategy designed to modulate the NK cell compartment might improve CD4(+) T cell resistance, independently of any effect on viral load.nnnMETHODSnNine macaques were chronically infected with the SHIV163P3; four were then immunized with the 3S/gp41 peptide and five with the carrier alone. Frequency of CD4(+) T cell subsets, proliferation, cell activation and apoptosis were analyzed in the periphery and the lymph nodes, spleen and rectum by flow cytometry.nnnRESULTSnThe anti-3S antibodies prevented NKp44L expression on CD4(+) T cells in vivo and subsequently preserved the CD4(+) central memory T cells in 3S/gp41-vaccinated animals. They also limited the NK cytotoxic activity against autologous CD4(+) T cells, the cell activation, the proliferation, and the apoptosis in peripheral blood and secondary lymphoid tissues remained intact.nnnCONCLUSIONnThese data suggest a new paradigm for AIDS vaccine development, aimed at generating specific responses to protect a specific subset of CD4(+) T cells from attack by activated NK cells.