Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

The Role of NK Cells in EBV Infection and Related Diseases: Current Understanding and Hints for Novel Therapies.

The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus most often transmitted during infancy and infecting the vast majority of human beings. Usually, EBV infection is nearly asymptomatic and results in life-long persistency of the virus in a latent state under the control of the host immune system. Yet EBV can cause an acute infectious mononucleosis (IM), particularly in adolescents, and is associated with several malignancies and severe diseases that pose a serious threat to individuals with specific inborn error of immunity (IEI). While there is a general consensus on the requirement for functional CD8 T cells to control EBV infection, the role of the natural killer (NK) cells of the innate arm of immunity is more enigmatic. Here we provide an overview of the interaction between EBV and NK cells in the immunocompetent host as well as in the context of primary and secondary immunodeficiencies. Moreover, we report in vitro data on the mechanisms that regulate the capacity of NK cells to recognize and kill EBV-infected cell targets and discuss the potential of recently optimized NK cell-based immunotherapies for the treatment of EBV-associated diseases.

Impact of IL-15 and latency reversing agent combinations in the reactivation and NK cell-mediated suppression of the HIV reservoir.

Inhibitors of histone deacetylases (HDACis) are major latency reversing agent (LRA) candidates in 'shock and kill' strategies to eradicate the HIV reservoir in infected patients. The poor achievements of initial HDACi-based trials and subsequent studies have highlighted the need for more efficient approaches such as combinatory and immunostimulating therapies. Here we studied combinations of IL-15 with pan-HDACi (Vorinostat, Romidepsin, Panobinostat) or class I selective-HDACi (Entinostat) with or without a PKC agonist (Prostratin) for their impact on in vitro reactivation and NK cell-mediated suppression of latent HIV. Results showed that pan-HDACis but not Entinostat reduced NK cell viability and function; yet, combined IL-15 reverted the negative effects of pan-HDACis except for Panobinostat. All HDACis were ineffective at reactivating HIV in a CD4+ T cell model of latency, with pan-HDACis suppressing spontaneous and IL-15- or Prostratin-induced HIV release, while IL-15 + Prostratin combination showed maximal activity. Moreover, Panobinostat impaired STAT5 and NF-κB activation by IL-15 and Prostratin, respectively. Finally, by using effectors (NK) and targets (latently infected CD4+ T cells) equally exposed to drug combinations, we found that IL-15-mediated suppression of HIV reactivation by NK cells was inhibited by Panobinostat. Our data raise concerns and encouragements for therapeutic application of IL-15/LRA combinations.

MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia.

The most widely accepted hypothesis for the development of glioblastoma suggests that glioblastoma stem-like cells (GSCs) are crucially involved in tumor initiation and recurrence as well as in the occurrence of chemo- and radio-resistance. Mesenchyme homeobox 2 (MEOX2) is a transcription factor overexpressed in glioblastoma, whose expression is negatively correlated with patient survival. Starting from our observation that MEOX2 expression is strongly enhanced in six GSC lines, we performed shRNA-mediated knock-down experiments in two different GSC lines and found that MEOX2 depletion resulted in the inhibition of cell growth and sphere-forming ability and an increase in apoptotic cell death. By a deep transcriptome analysis, we identified a core group of genes modulated in response to MEOX2 knock-down. Among these genes, the repressed ones are largely enriched in genes involved in the hypoxic response and glycolytic pathway, two strictly related pathways that contribute to the resistance of high-grade gliomas to therapies. An in silico study of the regulatory regions of genes differentially expressed by MEOX2 knock-down revealed that they mainly consisted of GC-rich regions enriched for Sp1 and Klf4 binding motifs, two main regulators of metabolism in glioblastoma. Our results show, for the first time, the involvement of MEOX2 in the regulation of genes of GSC metabolism, which is essential for the survival and growth of these cells.

Case Report: Altered NK Cell Compartment and Reduced CXCR4 Chemotactic Response of B Lymphocytes in an Immunodeficient Patient With HPV-Related Disease.

The study of inborn errors of immunity (IEI) provides unique opportunities to elucidate the microbiome and pathogenic mechanisms related to severe viral infection. Several immunological and genetic anomalies may contribute to the susceptibility to develop Human Papillomavirus (HPV) pathogenesis. They include different acquired immunodeficiencies, EVER1-2 or CIB1 mutations underlying epidermodysplasia verruciformis (EV) syndrome and multiple IEI. Whereas EV syndrome patients are specifically unable to control infections with beta HPV, individuals with IEI show broader infectious and immune phenotypes. The WHIM (warts, hypogammaglobulinemia, infection, and myelokathexis) syndrome caused by gain-of-CXCR4-function mutation manifests by HPV-induced extensive cutaneous warts but also anogenital lesions that eventually progress to dysplasia. Here we report alterations of B and NK cells in a female patient suffering from cutaneous and mucosal HPV-induced lesions due to an as-yet unidentified genetic defect. Despite no detected mutations in CXCR4, B but not NK cells displayed a defective CXCR4-dependent chemotactic response toward CXCL12. In addition, NK cells showed an abnormal distribution with an expanded CD56bright cell subset and defective cytotoxicity of CD56dim cells. Our observations extend the clinical and immunological spectrum of IEI associated with selective susceptibility toward HPV pathogenesis, thus providing new insight on the immune control of HPV infection and potential host susceptibility factors.

Case Report: EBV Chronic Infection and Lymphoproliferation in Four APDS Patients: The Challenge of Proper Characterization, Therapy, and Follow-Up.

Activated PI3K-kinase Delta Syndrome (APDS) is an autosomal-dominant primary immunodeficiency (PID) caused by the constitutive activation of the PI3Kδ kinase. The consequent hyperactivation of the PI3K-Akt-mTOR pathway leads to an impaired T- and B-cells differentiation and function, causing progressive lymphopenia, hypogammaglobulinemia and hyper IgM. Patients with APDS show recurrent sinopulmonary and chronic herpes virus infections, immune dysregulation manifestations, including cytopenia, arthritis, inflammatory enteropathy, and a predisposition to persistent non-neoplastic splenomegaly/lymphoproliferation and lymphoma. The recurrence of the lymphoproliferative disorder and the difficulties in the proper definition of malignancy on histological examination represents the main challenge in the clinical management of APDS patients, since a prompt and correct diagnosis is needed to avoid major complications. Targeted therapies with PI3Kδ-Akt-mTOR pathway pharmacologic inhibitors (i.e., Rapamycin, Theophylline, PI3K inhibitors) represent a good therapeutic strategy. They can also be used as bridge therapies when HSCT is required in order to control refractory symptoms. Indeed, treated patients showed a good tolerance, improved immunologic phenotype and reduced incidence/severity of immune dysregulation manifestations. Here, we describe our experience in the management of four patients, one male affected with APDS1 (P1) and the other three, a male and two females, with APDS2 (P2, P3, P4) presenting with chronic EBV replication, recurrent episodes of immune dysregulation manifestations and lymphomas. These cases highlighted the importance of a tailored and close follow-up, including serial endoscopic and lymph nodes biopsies control to detect a prompt and correct diagnosis and offer the best therapeutic strategy.

Combinations of Histone Deacetylase Inhibitors with Distinct Latency Reversing Agents Variably Affect HIV Reactivation and Susceptibility to NK Cell-Mediated Killing of T Cells That Exit Viral Latency.

The 'shock-and-kill' strategy to purge the latent HIV reservoir relies on latency-reversing agents (LRAs) to reactivate the provirus and subsequent immune-mediated killing of HIV-expressing cells. Yet, clinical trials employing histone deacetylase inhibitors (HDACis; Vorinostat, Romidepsin, Panobinostat) as LRAs failed to reduce the HIV reservoir size, stressing the need for more effective latency reversal strategies, such as 2-LRA combinations, and enhancement of the immune responses. Interestingly, several LRAs are employed to treat cancer because they up-modulate ligands for the NKG2D NK-cell activating receptor on tumor cells. Therefore, using in vitro T cell models of HIV latency and NK cells, we investigated the capacity of HDACis, either alone or combined with a distinct LRA, to potentiate the NKG2D/NKG2D ligands axis. While Bortezomib proteasome inhibitor was toxic for both T and NK cells, the GS-9620 TLR-7 agonist antagonized HIV reactivation and NKG2D ligand expression by HDACis. Conversely, co-administration of the Prostratin PKC agonist attenuated HDACi toxicity and, when combined with Romidepsin, stimulated HIV reactivation and further up-modulated NKG2D ligands on HIV+ T cells and NKG2D on NK cells, ultimately boosting NKG2D-mediated viral suppression by NK cells. These findings disclose limitations of LRA candidates and provide evidence that NK cell suppression of reactivated HIV may be modulated by specific 2-LRA combinations.

The RNA editing enzyme ADAR2 restricts L1 mobility.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosines to inosines in double-stranded RNAs (RNA editing A-to-I). ADAR1 and ADAR2 were previously reported as HIV-1 proviral factors. The aim of this study was to investigate the composition of the ADAR2 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 10 non-ribosomal ADAR2-interacting factors. A significant fraction of these proteins was previously found associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons. Considering that we previously demonstrated that ADAR1 is an inhibitor of LINE-1 retrotransposon activity, we investigated whether also ADAR2 played a similar function. To reach this goal, we performed specific cell culture retrotransposition assays in cells overexpressing or ablated for ADAR2. These experiments unveil a novel function of ADAR2 as suppressor of L1 retrotransposition. Furthermore, we showed that ADAR2 binds the basal L1 RNP complex.Overall, these data support the role of ADAR2 as regulator of L1 life cycle.

Early ART initiation during infancy preserves natural killer cells in young European adolescents living with HIV (CARMA cohort).

INTRODUCTION: HIV infection causes pathological changes in the natural killer (NK) cell compartment that can be only partially restored by antiretroviral therapy (ART). We investigated NK cells phenotype and function in children with perinatally acquired HIV (PHIV) and long-term viral control (five years) due to effective ART in a multicentre cross-sectional European study (CARMA, EPIICAL consortium). The impact of age at ART start and viral reservoir was also evaluated. METHODS: Peripheral blood mononuclear cells (PBMCs) from 40 PHIV who started ART within two years of life (early treated patients (ET), ≤6 months; late treated patients (LT), > 6 months), with at least five years of HIV-1 suppression (<40 HIV copies/mL), were collected between November 2017 and August 2018. NK phenotype and function were analysed by flow cytometry and transcriptional profile of PBMCs by RNA-Seq. HIV-1 DNA was measured by real-time polymerase chain reaction (Data were analysed by Spearman correlation plots and multivariable Poisson regression model (adjusted for baseline %CD4 and RNA HIV viral load and for age at ART start as an interaction term, either ET or LT) to explore the association between NK cell parameters and HIV reservoir modulated by age at ART start. RESULTS: A significantly higher frequency of CD56neg NK cells was found in LT compared with ET. We further found in LT a positive correlation of CD56neg NK cells with HIV-1 DNA. LT also displayed increased expression of the NKG2D and NKp46 activating receptors and perforin compared with ET. Moreover, CD107a+ and IFN-γ+ frequencies in non-stimulated NK were associated with HIV-1 DNA in LT patients. Finally, RNA-Seq analysis showed in LT an up-regulation of genes related to NK-activating pathways and susceptibility to apoptosis compared with ET. CONCLUSIONS: We show that early initiation of ART during infancy preserves the NK compartment and is associated with lower HIV-1 reservoir. Such condition persists over adolescence due to long-term viral control achieved through effective ART.

Altered NK-cell compartment and dysfunctional NKG2D/NKG2D-ligand axis in patients with ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a multisystem disorder caused by biallelic pathogenic variants in the gene encoding A-T mutated (ATM) kinase, a master regulator of the DNA damage response (DDR) pathway. Most A-T patients show cellular and/or humoral immunodeficiency that has been associated with cancer risk and reduced survival, but NK cells have not been thoroughly studied. Here we investigated NK cells of A-T patients with a special focus on the NKG2D receptor that triggers cytotoxicity upon engagement by its ligands (NKG2DLs) commonly induced via the DDR pathway on infected, transformed, and variously stressed cells. Using flow cytometry, we examined the phenotype and function of NK cells in 6 A-T patients as compared with healthy individuals. NKG2D expression was evaluated also by western blotting and RT-qPCR; plasma soluble NKG2DLs (sMICA, sMICB, sULBP1, ULBP2) were measured by ELISA. Results showed that A-T NK cells were skewed towards the CD56neg anergic phenotype and displayed decreased expression of NKG2D and perforin. NKG2D was reduced at the protein but not at the mRNA level and resulted in impaired NKG2D-mediated cytotoxicity in 4/6 A-T patients. Moreover, in A-T plasma we found 24-fold and 2-fold increase of sMICA and sULBP1, respectively, both inversely correlated with NKG2D expression. Overall, NK cells are disturbed in A-T patients showing reduced NKG2D expression, possibly caused by persistent engagement of its ligands, that may contribute to susceptibility to cancer and infections and represent novel targets for therapeutic interventions.

Expanding Phenotype of Schimke Immuno-Osseous Dysplasia: Congenital Anomalies of the Kidneys and of the Urinary Tract and Alteration of NK Cells.

Schimke immuno-osseous dysplasia (SIOD) is a rare multisystemic disorder with a variable clinical expressivity caused by biallelic variants in SMARCAL1. A phenotype-genotype correlation has been attempted and variable expressivity of biallelic SMARCAL1 variants may be associated with environmental and genetic disturbances of gene expression. We describe two siblings born from consanguineous parents with a diagnosis of SIOD revealed by whole exome sequencing (WES). Results: A homozygous missense variant in the SMARCAL1 gene (c.1682G>A; p.Arg561His) was identified in both patients. Despite carrying the same variant, the two patients showed substantial renal and immunological phenotypic differences. We describe features not previously associated with SIOD-both patients had congenital anomalies of the kidneys and of the urinary tract and one of them succumbed to a classical type congenital mesoblastic nephroma. We performed an extensive characterization of the immunophenotype showing combined immunodeficiency characterized by a profound lymphopenia, lack of thymic output, defective IL-7Rα expression, and disturbed B plasma cells differentiation and immunoglobulin production in addition to an altered NK-cell phenotype and function. Conclusions: Overall, our results contribute to extending the phenotypic spectrum of features associated with SMARCAL1 mutations and to better characterizing the underlying immunologic disorder with critical implications for therapeutic and management strategies.

Partial T cell defects and expanded CD56bright NK cells in an SCID patient carrying hypomorphic mutation in the IL2RG gene.

X-linked severe combined immunodeficiency (X-SCID) caused by full mutation of the IL2RG gene leads to T- B+ NK- phenotype and is usually associated with severe opportunistic infections, diarrhea, and failure to thrive. When IL2RG hypomorphic mutation occurs, diagnosis could be delayed and challenging since only moderate reduction of T and NK cells may be present. Here, we explored phenotypic insights and the impact of the p.R222C hypomorphic mutation (IL2RGR222C ) in distinct cell subsets in an 8-month-old patient with atypical X-SCID. We found reduced CD4+ T cell counts, a decreased frequency of naïve CD4+ and CD8+ T cells, and an expansion of B cells. Ex vivo STAT5 phosphorylation was impaired in CD4+ CD45RO+ T cells, yet compensated by supraphysiological doses of IL-2. Sanger sequencing on purified cell subsets showed a partial reversion of the mutation in total CD3+ cells, specifically in recent thymic emigrants (RTE), effector memory (EM), and CD45RA+ terminally differentiated EM (EMRA) CD4+ T cells. Of note, patient's NK cells had a normal frequency compared to age-matched healthy subjects, but displayed an expansion of CD56bright cells with higher perforin content and cytotoxic potential, associated with accumulation of NK-cell stimulatory cytokines (IL-2, IL-7, IL-15). Overall, this report highlights an alteration in the NK-cell compartment that, together with the high disease-phenotype variability, should be considered in the suspicion of X-SCID with hypomorphic IL2RG mutation.

Vδ2 T-Cells Kill ZIKV-Infected Cells by NKG2D-Mediated Cytotoxicity.

An expansion of effector/activated Vδ2 T-cells was recently described in acute Zika virus (ZIKV)-infected patients, but their role in the protective immune response was not clarified. The aim of this study was to define the antiviral activity of Vδ2 T-cells against ZIKV-infected cells. The Vδ2 T-cells expansion and their cytotoxic activity against ZIKV-infected cells were tested in vitro and analyzed by RT-PCR and flow cytometry. We found that ZIKV infection was able to induce Vδ2 T-cells expansion and sensitized A549 cells to Vδ2-mediated killing. Indeed, expanded Vδ2 T-cells killed ZIKV-infected cells through degranulation and perforin release. Moreover, ZIKV infection was able to increase the expression on A549 cells of NKG2D ligands (NKG2DLs), namely MICA, MICB, and ULBP2, at both the mRNA and protein levels, suggesting the possible involvement of these molecules in the recognition by NKG2D-expressing Vδ2 T-cells. Indeed, the killing of ZIKV-infected cells by expanded Vδ2 T-cells was mediated by NKG2D/NKG2DL interaction as NKG2D neutralization abrogated Vδ2 cytotoxicity. Our data showed a strong antiviral activity of Vδ2 T-cells against ZIKV-infected cells, suggesting their involvement in the protective immune response. Other studies are necessary to investigate whether the lack of Vδ2 T-cells expansion in vivo may be associated with disease complications.

Potential of the NKG2D/NKG2DL Axis in NK Cell-Mediated Clearance of the HIV-1 Reservoir.

Viral persistency in latently infected CD4+ T cells despite antiretroviral therapy (ART) represents a major drawback in the fight against HIV-1. Efforts to purge latent HIV-1 have been attempted using latency reversing agents (LRAs) that activate expression of the quiescent virus. However, initial trials have shown that immune responses of ART-treated patients are ineffective at clearing LRA-reactivated HIV-1 reservoirs, suggesting that an adjuvant immunotherapy is needed. Here we overview multiple lines of evidence indicating that natural killer (NK) cells have the potential to induce anti-HIV-1 responses relevant for virus eradication. In particular, we focus on the role of the NKG2D activating receptor that crucially enables NK cell-mediated killing of HIV-1-infected cells. We describe recent data indicating that LRAs can synergize with HIV-1 at upregulating ligands for NKG2D (NKG2DLs), hence sensitizing T cells that exit from viral latency for recognition and lysis by NK cells; in addition, we report in vivo and ex vivo data showing the potential benefits and drawbacks that LRAs may have on NKG2D expression and, more in general, on the cytotoxicity of NK cells. Finally, we discuss how the NKG2D/NKG2DLs axis can be exploited for the development of effective HIV-1 eradication strategies combining LRA-induced virus reactivation with recently optimized NK cell-based immunotherapies.

DNAM-1 Activating Receptor and Its Ligands: How Do Viruses Affect the NK Cell-Mediated Immune Surveillance during the Various Phases of Infection?

Natural Killer (NK) cells play a critical role in host defense against viral infections. The mechanisms of recognition and killing of virus-infected cells mediated by NK cells are still only partially defined. Several viruses induce, on the surface of target cells, the expression of molecules that are specifically recognized by NK cell-activating receptors. The main NK cell-activating receptors involved in the recognition and killing of virus-infected cells are NKG2D and DNAM-1. In particular, ligands for DNAM-1 are nectin/nectin-like molecules involved also in mechanisms allowing viral infection. Viruses adopt several immune evasion strategies, including those affecting NK cell-mediated immune surveillance, causing persistent viral infection and the development of virus-associated diseases. The virus's immune evasion efficacy depends on molecules differently expressed during the various phases of infection. In this review, we overview the molecular strategies adopted by viruses, specifically cytomegalovirus (CMV), human immunodeficiency virus (HIV-1), herpes virus (HSV), Epstein-Barr virus (EBV) and hepatitis C virus (HCV), aiming to evade NK cell-mediated surveillance, with a special focus on the modulation of DNAM-1 activating receptor and its ligands in various phases of the viral life cycle. The increasing understanding of mechanisms involved in the modulation of activating ligands, together with those mediating the viral immune evasion strategies, would provide critical tools leading to design novel NK cell-based immunotherapies aiming at viral infection control, thus improving cure strategies of virus-associated diseases.

Hexamethylene bisacetamide impairs NK cell-mediated clearance of acute T lymphoblastic leukemia cells and HIV-1-infected T cells that exit viral latency.

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here, we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model, HMBA did not reactivate HIV-1, yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However, HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells, hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside, HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall, our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells, providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug.

Dual regulation of L-selectin (CD62L) by HIV-1: Enhanced expression by Vpr in contrast with cell-surface down-modulation by Nef and Vpu.

The HIV-1 accessory protein Vpr displays various activities that can favor viral replication such as G2 cell cycle arrest. Vpr also modulates host gene expression, although this property is poorly characterized. Here, we investigated the effect of Vpr on L-selectin (CD62L), which crucially controls leukocytes circulation and generation of immune responses against pathogens. We report that Vpr up-regulates CD62L mRNA level when individually expressed in Jurkat T cells as well as during HIV-1 infection of primary CD4+ T cells. Vpr mutant analysis and use of inhibitors suggest that the effect of Vpr on CD62L occurs independently of G2 arrest but requires activation of the ATR kinase. Yet, induction of CD62L expression by Vpr is contrasted by down-regulation of CD62L protein by Nef that, together with Vpu, induces a net reduction of cell-surface CD62L on HIV-1-infected cells, which may impact viral spread and evasion of immune responses.

In Vitro Exposure to Prostratin but Not Bryostatin-1 Improves Natural Killer Cell Functions Including Killing of CD4+ T Cells Harboring Reactivated Human Immunodeficiency Virus.

In the attempt of purging the HIV-1 reservoir through the "shock-and-kill" strategy, it is important to select latency-reversing agents (LRAs) devoid of deleterious effects on the antiviral function of immune effector cells. Here, we investigated two LRAs with PKC agonist activity, prostratin (PRO) and bryostatin-1 (BRY), for their impact on the function of natural killer (NK) cells, the major effectors of innate immunity whose potential in HIV-1 eradication has emerged in recent clinical trials. Using NK cells of healthy donors, we found that exposure to either PRO or BRY potently activated NK cells, resulting in upmodulation of NKG2D and NKp44 activating receptors and matrix metalloprotease-mediated shedding of CD16 receptor. Despite PRO and BRY affected NK cell phenotype in the same manner, their impact on NK cell function was diverse and showed considerable donor-to-donor variation. Altogether, in most tested donors, the natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells were either improved or maintained by PRO, while both activities were impaired by BRY. Moreover, we analyzed the effect of these drugs on the capacity of treated NK cells to kill autologous latently infected CD4+ T cells reactivated via the same treatment. First, we found that PRO but not BRY increased upmodulation of the ULBP2 ligand for NKG2D on reactivated p24+ cells. Importantly, we showed that clearance of reactivated p24+ cells by NK cells was enhanced when both targets and effectors were exposed to PRO but not to BRY. Overall, PRO had a superior potential compared with BRY as to the impact on key NK cell functions and on NK-cell-mediated clearance of the HIV-1 reservoir. Our results emphasize the importance of considering the effects on NK cells of candidate "shock-and-kill" interventions. With respect to combinative approaches, the impact on NK cells of each LRA should be re-evaluated upon combination with a second LRA, which may have analogous or opposite effects, or with immunotherapy targeting NK cells. In addition, avoiding co-administration of LRAs that negatively impact ADCC activity by NK cells might be essential for successful application of antibodies or vaccination to "shock-and-kill" strategies.

NK cells of HIV-1-infected patients with poor CD4+ T-cell reconstitution despite suppressive HAART show reduced IFN-γ production and high frequency of autoreactive CD56bright cells.

HIV-1-infected patients failing to recover CD4+ T-cell count despite HAART (immunological non-responders, NRs), are at increased risk of disease progression and death. To better understand the NR status, we performed a comprehensive assessment of NK cells in NR patients as compared to immunologic responders. NRs exhibited an accumulation of CD56bright NK cells inversely correlated with CD4+ counts. Both CD56bright and CD56dim NK cells of NRs displayed unimpaired degranulation ability, but poorly responded to cytokine stimulation in terms of NKp44 up-regulation and IFN-γ production that may explain the susceptibility of NRs to infections and tumors. Notably, CD56bright NK cells from NRs showed higher cytotoxicity against autologous activated CD4+ T cells. Moreover, NRs had reduced Treg cell counts that showed an inverse correlation with autoreactive CD56bright cells. These data suggest that accumulation of CD56bright NK cells, possibly linked to decreased homeostatic control by Tregs, contributes to poor immune reconstitution in NRs.