Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56^bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α₄β₁ integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4⁺ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4⁺ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4⁺ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4⁺ T cells and the cytolytic activity of NK cells.Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system (CNS) (1) and one of the major causes of neurological disability in young adults (2). MS is considered to be a primarily antigen-driven T cell-mediated disease with a complex genetic background influenced by environmental factors (1, 3) that is caused by an imbalanced immune-regulatory network (4). Among other well-known players of this network such as regulatory T cells and tolerogenic dendritic cells (DCs) (1), natural killer (NK) cells have been recently identified as additional factors in controlling homeostasis of antigen-activated T cells (5, 6).Originally discovered as antigen receptor-negative innate lymphocytes that play an important role in controlling virus-infected and tumor cells (7), NK cells have also been shown to suppress activated T cells through secretion of anti-inflammatory cytokines and/or cytolytic function (5, 6, 8–12). NK cells lyse target cells in a complex process depending on cell surface expression of certain inhibitory and activating receptors on NK cells and the corresponding ligands on target cells (13). Several activating NK-cell receptors–in particular, NKG2D (CD314) (5, 8, 9, 11, 14), the receptor for MIC-A/B and ULBP1-6, and DNAM-1 (DNAX accessory molecule, CD226) (6, 12, 15), the receptor for Nectin-2 (CD112) and poliovirus receptor (PVR/CD155)−have been proposed to be involved in NK cell-mediated lysis of activated T cells. Of note, polymorphisms in the gene encoding for DNAM-1 have been consistently found in MS-association studies (16–18). Both major NK-cell subsets, namely the CD56^brightCD16^dim/− and the CD56^dimCD16⁺ subsets (here referred to as CD56^bright and CD56^dim, respectively), seem to be capable of killing activated T cells (19). CD56^dim NK cells are the major NK-cell subset in the peripheral blood (PB) (90% of NK cells) and kill target cells without prior sensitization but only secrete low levels of cytokines (7, 20, 21), whereas CD56^bright NK cells are more abundant in secondary lymphoid tissues and inflammatory lesions (75–95% of NK cells), where they produce high amounts of immune-modulating cytokines but acquire cytolytic functions only after prolonged activation (7, 20, 21).Immune-modulating therapies targeting NK-cell frequencies and cytolytic functions among others such as IFN-β (22–24), glatiramer acetate (25), natalizumab (26, 27), fingolimod (28, 29), and daclizumab (10, 30, 31) point to an immune-protective role of both NK-cell subsets in MS. Daclizumab, a humanized antibody directed against the IL-2 receptor (IL-2R) α-chain (CD25) (reviewed in ref. 4) is a promising MS therapy, which recently showed superior efficacy compared with IFN-β in a phase III study (32). Expansion of peripheral (10, 33) as well as intrathecal (34) CD56^bright NK cells under daclizumab treatment correlated positively with therapeutic response (10, 30, 35). Nevertheless, it still remains to be elucidated whether NK-cell immune-regulatory functions are impaired as part of the disease process and whether modulation of the IL-2R with daclizumab restores these deficits or simply boosts NK-cell activity (4). Furthermore, the distribution and function of NK cells in active MS lesions is still poorly understood. Resolving the molecular basis of NK cell-mediated immune control and its potential impairment in MS is important for a better understanding of the role of NK cells in MS pathogenesis and the mechanism of action of NK cell-modulating therapies.The aim of the current study was to characterize the role of NK cells in the pathogenesis of MS by investigating the presence, distribution, and function of NK cells in three different compartments [CNS, cerebrospinal fluid (CSF), and PB]. Furthermore, a potential deficit in NK-cell immune-regulatory function, its underlying molecular mechanism, and the impact of IL-2R modulation by daclizumab high-yield process (DAC HYP) were explored by studying PB mononuclear cells (PBMCs) derived from clinically stable therapy-naïve MS patients and MS patients receiving daclizumab treatment in comparison with those derived from healthy individuals.     

Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94

Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94–NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor–ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A⁺ NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR⁺ and NKG2A⁺ NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I–bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I.Natural killer (NK) cells play an important role in the immune response to viral infections and cancer. Their responses are determined by signals integrated from activating and inhibitory receptor–ligand interactions (1). In many situations inhibitory signals dominate activating signals. Therefore, releasing NK cells from inhibition is an important mechanism of enhancing their response to target cells. Inhibitory interactions are mediated by receptors for self-MHC class I. Most species have at least two discrete gene families of inhibitory receptors for MHC class I: the CD94–NKG2A C-type lectin-like receptor system and either the related Ly49 family of receptors or the unrelated killer cell Ig-like receptors (KIR) (2). The KIR family is important in humans and other primates, having undergone extensive diversification under positive selection. In contrast, the CD94–NKG2A system has remained relatively well conserved across the species with orthologous genes in primates and a closely related functional homolog in rodents (3, 4). Consistent with the coevolution of these families and their MHC class I ligands, KIR bind polymorphic MHC class I, HLA-A, -B, and -C molecules, whereas CD94–NKG2A binds the conserved oligomorphic HLA-E molecule or the rodent homolog Qa-1 (5, 6).Both receptor families are important in the immune response to viral infections. KIR are genetic determinants in the outcome of both HIV and hepatitis C virus (HCV) infection (7–10). Expression of CD94–NKG2A is up-regulated on NK cells in HIV and HCV infection and in the latter has been associated with a poor response to treatment (11, 12). Furthermore NKG2A⁺ NK cell clones lyse vaccinia-infected targets (13), and CD94 is important in clearing mouse pox infection (14). Both KIR and CD94–NKG2A respond to MHC class I down-regulation. One hypothesis is that the KIR have evolved to recognize MHC class I-specific down-regulation (15). However, because the majority of MHC class I leader peptides bind HLA-E and are inhibitory for NKG2A, the CD94–NKG2A system also is able to recognize down-regulation of most MHC class I alleles. It has been shown that KIR⁺ NK cells can be modulated by changes in the peptide bound by MHC class I, which confers additional functionality on the KIR system (16–18). In particular peptide antagonism is a potent mechanism for activating KIR⁺ NK cells (19, 20). The CD94–NKG2A receptor also is peptide selective, with receptor binding being particularly influenced by residues 5, 6, and 8 of the peptide bound by HLA-E (21–23). These residues interact primarily with the nonsignaling CD94 moiety, which occupies the majority of the HLA-E–binding interface. CD94–NKG2A seems to be a target for viral escape, with peptides derived from CMV, HCV, HIV, and EBV binding HLA-E and subsequently inhibiting NK cells (24–27). Viral peptides that inhibit at KIR also are identifiable (28), but their relevance likely is limited to the subset of individuals who have the relevant peptide-binding MHC class I allele. Understanding differences in how the KIR and NKG2 systems respond to peptide may be important for interpreting their roles in the immune response to viral infections and tumors. Therefore we explored how HLA-E–bound peptide can influence NK cell reactivity.     

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5⁺ ILC2 responses, and induced a dominant IL-13⁺ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ–deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR⁺ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Innate lymphoid cells (ILCs) and T cells represent critical populations of cells that have diverse roles in inflammation and protection (1, 2). Both cell populations consist of subsets that differ in cytokine expression and function. While T cells are important for viral clearance, they can also exacerbate lung immunopathology (1, 3–5) Among ILC subsets, ILC2s play a critical role in pulmonary immunity, particularly in maintaining the lung barrier surface (6–9). During infection, ILC2s respond to the epithelial cell–derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin, and produce the type 2 cytokine IL-5 (10–12). This, in turn, can lead to increased eosinophil recruitment and airway hyperreactivity (AHR) (8, 13–15). Like T cells, ILC2s can play both beneficial and detrimental roles during viral lung infection (6–8).It is known that host cytokines can regulate the activity of ILC and T cell subsets. For example, we previously found that interferon (IFN)-γ deficiency results in enhanced ILC2 activity and increased survival from challenge with the 2009 pandemic strain A/California/04/2009 (CA04) influenza A virus (8). However, our current studies have shown no effect of IFN-γ following challenge with the Puerto Rico/8/1934 (PR8) influenza A virus, a strain that is a commonly used model for the highly virulent 1918 pandemic influenza virus. Although both strains are H1N1 influenza A viruses, they have striking differences in expression of functional PB1-F2, a viral proapoptotic protein that is associated with immunopathology and mortality (16). While the PR8 viral strain expresses full-length PB1-F2, the PB1-F2 gene in the CA04 strain is truncated and nonfunctional (16–20). As a result, the PR8 virus exhibits significantly increased virulence compared to the CA04 viral strain. However, the impact of PB1-F2 on the lymphocyte function that is critical for protection during influenza is not known. A better understanding of the role of pathogen virulence factors in regulating immune cell activity during influenza may aid in designing future therapies for human use.We hypothesized that the PB1-F2 virulence protein can differentially regulate ILC2 and T cell activity in conjunction with host IFN-γ signaling. To test this hypothesis, we have investigated pulmonary immunity in wild-type (WT) and IFN-γ–deficient BALB/c mice infected with PB1-F2 gene reassortant PR8 and CA04 viruses. Our findings demonstrate that viral virulence genes, together with host factors, play critical roles in regulating both ILC2 and T cell responses during influenza, and this, in turn, determines host survival.     

CD1d-unrestricted NKT cells are endowed with a hybrid function far superior than that of iNKT cells

Invariant natural killer T (iNKT) cells to date represent the best example of cells known to have a hybrid function, representing both innate and adaptive immunity. Shared phenotypic similarities with NK cells together with a rapid response to a cytokine stimulus and a productive TCR engagement are the features that underline the hybrid nature of iNKT cells. Using these criteria, we provide molecular and functional evidence demonstrating that CD1d-independent (CD1d^ind) NKT cells, a population of CD1d-unrestricted NKT cells, are endowed with a hybrid function far superior to that of iNKT cells: (i) an extensive shared program with NK cells, (ii) a closer Euclidian distance with NK cells, and (iii) the ability to respond to innate stimuli (Poly:IC) with cytotoxic potential in the same manner as NK cells identify a hybrid feature in CD1d^indNKT cells that truly fulfills the dual function of an NK and a T cell. Our finding that CD1d^indNKT cells are programmed to act like NK cells in response to innate signals while being capable of adaptive responses is unprecedented, and thus might reemphasize CD1d-unrestricted NKT cells as a subset of lymphocytes that could affect biological processes of antimicrobial and tumor immunity in a unique way.Natural killer T (NKT) cells are increasingly regarded as cells endowed with a hybrid function between an NK cell and a T cell (1, 2). The current classification of NKT cells places them into three categories: type I, type II, and NKT-like cells (1). Type I comprises invariant NKT (iNKT) cells that recognize the glycolipid α-galactosylceramide (α-GalCer) loaded into the MHC class I molecule, CD1d, and contain an invariant TCR repertoire of Vα14-Jα18 (3–5). Type II NKT cells are also CD1d dependent but do not respond to α-GalCer in the same way as iNKT cells do (6, 7). NKT-like cells encompass all other NKT cells and are CD1d independent (CD1d^ind) (8); they are by far the most heterogeneous and the least characterized.Recent studies have increasingly shown a shared expression of NK cell-related receptors on other effector cells. CD8⁺ T cells are known to up-regulate NK markers, such as NK1.1, and can even respond quickly like NK cells (9). Other work has described NKT cells that express NKp46 (10), a marker selectively associated with conventional NK cells and NK22 cells in the gut (11). Moreover, γδ T cells have been shown to express NK markers and display an innate-like response (12). Collectively, these reports converge to raise the following key questions. What qualifies as an NKT cell? Do the cells need to express only NK1.1 and CD3 to be eligible for NKT nomenclature? With the continuous development of both NK and T-cell fields, the simplistic definition that NKT cells are subsets of T cells that express the NK1.1 marker is becoming increasingly misleading and even inaccurate. For instance, NK1.1 complex is expressed in the BALB/c strain but there are allelic divergences with the polymorphism leading to the PK136 antibody not reacting to the BALB/c NK.1.1 (NKrp1) complex (13). This definition is also limited in the C57BL/6 strain because of the discovery of NK1.1⁻CD1d⁺ NKT cells (14). Although phenotypic similarities can be misleading, the criteria that best describes an NKT cell is the ability to perform with a hybrid function between an NK cell and a T cell (2).Nonetheless, the concept of hybrid function is also an elusive notion allowing for a gradient of functions. A number of works refer to an NKT hybrid function as the ability of a T cell with phenotypic similarities to NK cells to perform with innate-like response. The best example of cells endowed with a hybrid NKT cell function are thought to be iNKT cells (2). In this study, we provide molecular and functional evidence demonstrating that CD1d^indNKT cells—a population of MHC-unrestricted T cells—are endowed with a hybrid function that associates them to the NK cell lineage in a manner far superior to the known link between NK and iNKT cells. An extensive shared program with NK cells, a similarity in the gene expression profile with NK cells, and their ability to respond (like NK cells) not only to cytokine signals (IL-12 plus IL-18) but also to innate stimuli [in vivo treatment with Poly:IC (Fisher)] with massive production of key effector players of the cytotoxic pathway collectively identify a hybrid feature in CD1d^indNKT cells that uniquely fulfills the function of an NK cell and a T cell.     

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8⁺ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C⁺CD57⁺FcεRIγ^–) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C⁺CD57⁺FcεRIγ^low–dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C⁺CD8⁺ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Cytomegalovirus (CMV) is life threatening for individuals with a compromised immune system, including solid organ and hematopoietic stem cell transplant patients. Additionally, infection or reactivation of CMV resulting in viremia in solid organ transplant patients has been associated with chronic graft rejection (1, 2). Through constant surveillance, natural killer (NK) and T cells cooperatively control CMV throughout an individual’s life. The antiviral drugs used prophylactically in transplant patients have significant side effects and toxicity, and there is no currently approved vaccine for CMV.We and others have identified a subpopulation of NK cells bearing the activating CD94-NKG2C receptor that preferentially respond to acute CMV infection in both solid organ (3) and hematopoietic stem cell transplant recipients (4–6). These NKG2C⁺ cells also express CD57, which marks a population of mature NK cells with a distinct phenotype and function (7, 8). These NKG2C⁺CD57⁺ NK cells are specific to CMV in that they do not respond to acute infection with Epstein–Barr virus during infectious mononucleosis (9) or herpes simplex virus (10). Moreover, these NK cells have been observed to be reactivated and persist over several years only in individuals who have been infected with CMV. These findings are in line with those from mouse models, in which Ly49H⁺ NK cells specifically respond to CMV infection (11–13) and have memory-like signatures (14), suggesting that in humans, NKG2C⁺CD57⁺ NK cells could include subsets with memory-like properties. Within this CMV-specific NKG2C⁺CD57⁺ NK cell population, we identified a unique subset of NK cells that do not express the FcεRIγ signaling subunit, which is expressed by all naïve NK cells. Rather, these FcεRIγ⁻ NK cells preferentially use the CD3ζ signaling adapter and ZAP70 tyrosine kinase for signal transduction mediated by the CD16 Fc receptor. These NK cells exhibit robust preferential expansion and an enhanced antibody-dependent cellular cytotoxicity (ADCC) response against CMV-infected cells in an antibody-dependent manner (15–17). In addition to NK cells, minor subsets of CD3⁺ T cells and γδ T cells, which express natural killer cell receptors (NKRs), are observed preferentially in CMV-seropositive patients (18, 19). Although such different lymphocyte subsets have been associated with immune response to CMV infection (20), in vivo kinetics of these immune-competent subsets over CMV infection remain limited.The aims of this study were to determine how specific subsets of human NK cells respond to CMV infection or reactivation in solid organ transplant recipients and to demonstrate the dynamic interactions between NK cells and T cells responding to CMV viremia in the same transplant patients. For this, we used mass cytometry to longitudinally analyze peripheral blood mononuclear cells (PBMCs) from renal transplant patients who underwent CMV infection or reactivation, followed by single-cell data analysis using clustering methods. Notably, our panel included markers such as NKG2C, CD57, FcεRIγ, Syk, and inhibitory killer cell immunoglobulin-like receptors (KIRs) for the purpose of in-depth phenotyping of the responding NK cells and T cells. This enabled us to identify different NK cell subsets, including memory-like NK subsets, to define the in vivo kinetics of the NK cell response over CMV infection at the single-cell level. Moreover, our study identified minor populations of cytotoxic T cells responding to CMV viremia and demonstrated the interplay between NK cells and T cells during CMV viremia. This study provides insights into how these immune-competent cells respond to CMV infection in vivo, may contribute to host protection, and potentially, influence graft survival.     

Natural killer cells in HIV controller patients express an activated effector phenotype and do not up-regulate NKp44 on IL-2 stimulation

Control of HIV replication in elite controller (EC) and long-term nonprogressor (LTNP) patients has been associated with efficient CD8⁺cytotoxic T-lymphocyte function. However, innate immunity may play a role in HIV control. We studied the expression of natural cytotoxicity receptors (NKp46, NKp30, and NKp44) and their induction over a short time frame (2–4 d) on activation of natural killer (NK) cells in 31 HIV controller patients (15 ECs, 16 LTNPs). In EC/LTNP, induction of NKp46 expression was normal but short (2 d), and NKp30 was induced to lower levels vs. healthy donors. Notably, in antiretroviral-treated aviremic progressor patients (TAPPs), no induction of NKp46 or NKp30 expression occurred. More importantly, EC/LTNP failed to induce expression of NKp44, a receptor efficiently induced in activated NK cells in TAPPs. The specific lack of NKp44 expression resulted in sharply decreased capability of killing target cells by NKp44, whereas TAPPs had conserved NKp44-mediated lysis. Importantly, conserved NK cell responses, accompanied by a selective defect in the NKp44-activating pathway, may result in lack of killing of uninfected CD4⁺NKp44Ligand⁺ cells when induced by HIVgp41 peptide-S3, representing a relevant mechanism of CD4⁺ depletion. In addition, peripheral NK cells from EC/LTNP had increased NKG2D expression, significant HLA-DR up-regulation, and a mature (NKG2A−CD57⁺killer cell Ig-like receptor⁺CD85j⁺) phenotype, with cytolytic function also against immature dendritic cells. Thus, NK cells in EC/LTNP can maintain substantially unchanged functional capabilities, whereas the lack of NKp44 induction may be related to CD4 maintenance, representing a hallmark of these patients.A benign disease course with long-term nonprogressing disease (LTNP) up and beyond 20 y is observed in a minority (<1–2%) of HIV-1–infected patients who maintain high CD4⁺ T-cell counts (>500 µL) with low-level viremia (<1,000 cp/mL) without progression to AIDS in the absence of antiretroviral treatment (ART). A subset of LTNPs is aviremic virus-controlling (<50–75 cp/mL) patients who are considered to represent a distinct clinical entity defined as elite controllers (ECs) because of their efficient and extensive spontaneous control of viral replication (1, 2). Understanding of the mechanisms that underlie the lack of disease progression in EC and LTNP patients has attracted relevant scientific focus over the years, with the ultimate goal to exploit this understanding for therapeutic or vaccination purposes.Viral replication may be decreased in LTNP/EC because of virus mutations or host genetic background conferring reduced CD4⁺ T-cell susceptibility. However, both an intact viral replication capacity and a conserved CD4⁺ T-cell susceptibility to HIV infection in vitro have recently been proven in most HIV controller patients (3–5). Among cytotoxic effector cells, an acknowledged role in the control of viremia and disease has been attributed to CD8⁺ cytotoxic T lymphocytes (CTLs), which in these patients, display an exceptionally high avidity and breadth against HIV epitopes (1, 2, 6, 7). Vigorous and effective CTL responses associated to HLA class I haplotype (e.g., B*57 and B*27 alleles) represent an example of genetic background positively affecting HIV control (1, 2, 6, 7). Also, HLA-C polymorphisms have been implicated in the control of HIV (8). Unique allele carriage is, however, not a feature uniquely characterizing LTNPs/ECs. HIV controllers may lack this genetic background, but they have CTL responses with high avidity and breadth against HIV_gag. Conversely, this immunogenetic background may be present in progressors who display poorer CTL response quality (5, 9–11). Also, HLA B*5701 LTNPs/ECs and HLA-matched progressors cannot be distinguished by the clonal composition of HIV-specific CD8⁺ T cells (12).The relevance of natural killer (NK) cell function in the setting of HIV controller status has been suggested by genetic studies showing the association between HLA-Bw4_80I DNA carriage and specific killer cell Ig-like receptors (KIRs; i.e., KIR3DL1/S1) (13, 14). NK cell-associated control of HIV replication in vitro occurs with KIR3DS1⁺ NK cells in a HLA-Bw4_80I⁺ target cell genetic background (15); however, this result has not been subsequently reproduced in vivo in EC/LTNP cohorts (16). Various combinations of these mechanisms seem to be involved in the successful control of HIV replication in some LTNP and EC patients; however, none of them taken alone can fully explain this condition, and it has not been shown to identify all of these patients.Involvement of the activating NK receptors in disease progression was suggested by the demonstration that HIV-1 infection was associated to profoundly decreased expression of natural cytotoxicity receptors (NCRs; i.e., NKp46, NKp30, and NKp44) (17). This decrease, in turn, leads to an impaired cross-talk between NK cells and dendritic cells (DCs), resulting in an altered DC editing (18). Moreover, rates of CD4⁺ T-cell loss after ART interruption are inversely associated with NCR expression on NK cells before ART discontinuation (19).Interestingly, in the AIDS-free HIV infection model of chimpanzees, peripheral NK cells have absent/low baseline expression of NKp30, which was, however, inducible on cytokine-mediated in vitro NK cell activation (20). In addition, activating NK cell receptor induction/modulation has been reported in vivo and in vitro during treatment of human HCV infection involving NKp30 (21) and DNAX-accessory molecule 1 (DNAM-1,CD226) (22), which are both involved in DC–NK cell cross-talk (23, 24). In addition, activating NK cell receptor ligands are lost in CD4⁺ T cells of infected patients, with the exception of NKG2D-Ligands (e.g., MHC class I polypeptide-related sequence A/B,MICA/B) (25). Furthermore, HIV_nef and HIV_vpu have been shown to directly target NKG2D and DNAM-1 ligands (i.e., MICA/B and poliovirus receptor, PVR) (26, 27). These immune evasion mechanisms are in line with the idea that NK cells may exert a critical control of HIV-1 infection. In this context, an as yet uncharacterized NKp44-L is reported to be induced in uninfected CD4⁺ T cells by an HIV_gp41 peptide inducing innocent CD4⁺ T-cell bystander lysis (28, 29). These observations, thus, raise the question of whether differences in NCR surface expression may help to explain the different disease course observed in HIV controllers—LTNPs, ECs, or both.Here, we report a study addressing the activating NK cell receptors expression, their modulation, and the consequences on NK cell function in a cohort of HIV controller (LTNP and EC) patients. The data provide evidence that differences in inducibility/modulation of NCR may offer clues on how successful disease-free HIV-1 control may be achieved in these patients.     

Decidual NK cells kill Zika virus–infected trophoblasts

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal–fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1^−/− pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.

In the first trimester of human pregnancy, when Zika virus (ZIKV) infection is most damaging to the developing fetus (1–3), ∼70% of maternal immune cells at the maternal–fetal interface are decidual natural killer cells (dNK), which are in close contact with extravillous trophoblasts (EVT), placental cells of fetal origin which invade the decidua to orchestrate placentation. T lymphocytes and natural killer (NK) cells generally do not kill EVT, which do not express the main classical HLA molecules responsible for T cell activation (HLA-A and B) (4–7) but do express HLA-C and nonclassical HLA-E and -G, which help inhibit NK cell activation. HLA-G, whose expression is mostly restricted to the placenta, helps maintain dNK tolerance of fetal cells. There is important cross-talk between dNK and EVT—EVT regulate the maturation of dNK precursor cells into tolerant dNK (8) and dNK promote EVT migration and invasion of spiral arteries, processes essential for placentation (9–13). Invading fetal EVT are susceptible to infection by a variety of pathogens, including human cytomegalovirus (HCMV), Toxoplasma gondii, and ZIKV (1, 14–17). dNK in the placenta face a difficult problem—they must tolerate the semiforeign fetus but still protect against infection. Although dNK contain the machinery needed to recognize and kill, they have reduced cytolytic activity against classical NK target cells recognized by peripheral blood NK cells (pNK) and are not known to kill infected trophoblasts (18–23). However, dNK degranulate and secrete cytokines, including interferon (IFN)-γ, in response to HCMV-infected maternal decidual stromal cells in equal levels to pNK (19, 20). We recently demonstrated that dNK can eliminate intracellular Listeria infection in trophoblasts without degranulating or killing the host cells (24). Although ZIKV infects the placenta and fetus and can cause severe birth defects including microcephaly, brain atrophy, and subcortical and chorioretinal and optic nerve atrophy, little is known about the response of decidual immune cells to ZIKV and its effect on maternal–fetal transmission (25).     

Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1–dependent NK cell-mediated lysis of infected cells

Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2–infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.Alphaherpesviruses constitute the largest subfamily of the herpesviruses, comprising closely related and important pathogens like herpes simplex virus (HSV) in man, pseudorabies virus (PRV) in pigs, and bovine herpesvirus 1 (BHV-1) in cattle.Natural killer (NK) cells play a central role in the defense against viral infections and cancer development. Functional NK cells are of particular importance in preventing herpesviruses from causing aggravated disease, including life-threatening encephalitis for alphaherpesviruses like HSV and varicella-zoster virus (1–3). The significance of the NK cell response against herpesviruses is also reflected by the various mechanisms that these pathogens have evolved to evade or delay it (4). Indeed, for beta- and gammaherpesviruses, a variety of molecular mechanisms avoiding the NK-mediated antiviral activity have been defined (4–16). Remarkably and paradoxically, such mechanisms have remained largely elusive for the alphaherpesviruses (17).Identifying and understanding these mechanisms is of particular relevance for alphaherpesviruses also in view of the potential therapeutic applications of HSV. Indeed, a limiting factor in HSV vector-based oncotherapy is the premature clearance of the viral vector by NK cells (18).NK cell activity is regulated by an array of germline-encoded activating and inhibitory surface receptors capable of transducing signals upon engagement by their respective ligands (19, 20). The sum of these signals determines the outcome of NK cell effector responses including cytotoxicity against NK-sensitive targets (20). A variety of NK activating receptors are involved in recognition of virus-infected cells (18, 21–26). One of the important NK activating receptors is DNAX accessory molecule 1 (DNAM-1), which binds to CD112 (nectin-2) and CD155 (poliovirus receptor, PVR), whose expression can be induced in both virus infected and tumor cells (5, 26–28).Interestingly, the viral gD envelope glycoproteins of certain human and animal alphaherpesviruses, including HSV-2, PRV, and BHV-1, interact with CD112 and/or CD155 to facilitate viral entry (29, 30). HSV-1 gD does not typically display substantial affinity for CD112, except for particular HSV-1 isolates, including some retrieved from patients with encephalitis (31).In the current study, we demonstrate that the significance of these virus ligand-cellular receptor interactions can stretch beyond virus entry and can influence immune recognition. We report that expression of gD of PRV and HSV-2 reduces DNAM-1–mediated cell lysis by NK cells through suppression of CD112 levels in infected and transfected cells. The gD/CD112/DNAM-1 interplay identified here may have consequences for the development of medical applications ranging from vaccination to oncolytic virotherapy.     

CD81 costimulation skews CAR transduction toward naive T cells

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Genetic manipulation of T cells has enabled adoptive T cell therapy to be translated to the clinic (1–10). Chimeric antigen receptor (CAR) therapy has evoked recent enthusiasm upon mediating curative outcomes in aggressive, refractory B cell malignancies (1–7, 11–15), leading to Food and Drug Administration approval (16–18). The process of ex vivo transduction and expansion of T cells to express CARs influences the phenotype, function, and ultimate fate of the final CAR T cell product (19–23). Preclinical data in animal models indicate that selecting specific T cell subsets for CAR transduction improves efficacy (21, 22, 24–26). Naive-derived T cells have been shown to exhibit greater replicative capacity, persistence, and antitumor function, compared with both effector- and memory-derived T cells (19, 20, 27). Naive CD4⁺ T cells, specifically, have a critical role in enhancing the cytotoxic effect of the CD8⁺ cooperating central memory cell subset (21). Furthermore, the translational CAR experience demonstrates that the presence of cells consistent with the naive and early memory phenotype in premanufactured T cell products correlates with successful clinical responses in both pediatric and adult B cell leukemia (28–30). Here, we explore if selective activation of naive T cells can result in skewing of transduction toward this specific T cell subset without the need for physical subset sorting.CAR constructs rely on intrinsic costimulatory signals, such as the intracellular domains of CD28 or 41BB, for efficacy (1–9). Here we focus on exogenous costimulatory signals necessary to induce proliferation and permit viral-mediated gene transfer. Prior to CAR transduction and antigen encounter, the majority of T cells are in a state of rest. Resting T cells mandate primary and costimulatory signals for activation (31, 32). CD28 is best known for its ability to costimulate T cells (33–38) and along with CD3 activation renders T cells susceptible to viral transduction (1, 39). CD81 is a member of the tetraspanin family that physically associates with CD4 and CD8 on the surface of T cells. CD81 was shown to have independent costimulatory properties and, when used with anti-CD3 and -CD28 antibodies, preferentially activates naive T cells as compared with effector and memory T cells, despite conserved surface CD81 expression across T cell subsets (40). Tetraspanins have no known cell-surface ligands, and therefore antibodies are used to engage and stimulate them. CD81 is the only tetraspanin whose complete three-dimensional structure has been solved (41). Moreover, the crystal structure of 5A6, the anti-CD81 antibody used in our study, in complex with CD81 has also been most recently solved (42). These authors demonstrate that engagement by this antibody changes the conformation of the large extracellular loop of the CD81 molecule. This conformational change may affect the interaction of CD81 with its associated CD4 and CD8 molecules.Here, we costimulate purified T cells with CD81 as a proof of principle to illustrate that the in vitro activation window prior to CAR transduction can be leveraged to favor transduction of a specific T cell subset.     

Structure of NKp65 bound to its keratinocyte ligand reveals basis for genetically linked recognition in natural killer gene complex

The natural killer (NK) gene complex (NKC) encodes numerous C-type lectin-like receptors that govern the activity of NK cells. Although some of these receptors (Ly49s, NKG2D, CD94/NKG2A) recognize MHC or MHC-like molecules, others (Nkrp1, NKRP1A, NKp80, NKp65) instead bind C-type lectin-like ligands to which they are genetically linked in the NKC. To understand the basis for this recognition, we determined the structure of human NKp65, an activating receptor implicated in the immunosurveillance of skin, bound to its NKC-encoded ligand keratinocyte-associated C-type lectin (KACL). Whereas KACL forms a homodimer resembling other C-type lectin-like dimers, NKp65 is monomeric. The binding mode in the NKp65–KACL complex, in which a monomeric receptor engages a dimeric ligand, is completely distinct from those used by Ly49s, NKG2D, or CD94/NKG2A. The structure explains the exceptionally high affinity of the NKp65–KACL interaction compared with other cell–cell interaction pairs (K_D = 6.7 × 10⁻¹⁰ M), which may compensate for the monomeric nature of NKp65 to achieve cell activation. This previously unreported structure of an NKC-encoded receptor–ligand complex, coupled with mutational analysis of the interface, establishes a docking template that is directly applicable to other genetically linked pairs in the NKC, including Nkrp1–Clr, NKRP1A–LLT1, and NKp80–AICL.Natural killer (NK) cells are a fundamental component of innate immunity against tumors and virally infected cells. The cytolytic activity of NK cells is regulated by a dynamic interplay between activating and inhibitory signals transmitted by distinct classes of receptors that recognize both MHC and non-MHC ligands on the surface of target cells (1–3). In humans, these receptors are encoded in two distinct genomic regions: the leukocyte receptor complex (LRC) on chromosome 19 (4) and the NK gene complex (NKC) on chromosome 12 (5). The LRC codes for receptors belonging to the Ig superfamily. These include killer Ig-like receptors (KIRs), leukocyte Ig-like receptors, and the natural cytotoxicity receptor NKp46. The NKC codes for ∼30 cell-surface glycoproteins belonging to the C-type lectin-like superfamily (6). These receptors are expressed on NK and other immune-related cells, whose activity they regulate in various ways depending on cellular environment.NKC genes have been subdivided into killer cell lectin-like receptor (KLR) genes and C-type lectin receptor (CLEC) genes (6). KLR genes encode molecules expressed on NK cells, whereas CLEC genes encode molecules expressed on other cell types (e.g., CLEC2B and CLEC9A are expressed on myeloid and dendritic cells, respectively). The KLR family includes NKG2D and CD94/NKG2A (human and rodent) and rodent Ly49s. These receptors bind classical MHC class I (MHC-I) molecules or their structural relatives and thereby facilitate detection of stressed cells or cells exhibiting aberrant MHC-I expression (5).In addition, the KLR family includes receptors that do not engage ligands with an MHC-like fold, but instead interact with CLEC2 glycoproteins that are also members of the C-type lectin-like superfamily. These KLR and CLEC2 molecules, whose genes are intermingled in the telomeric subregion of the NKC, function as genetically linked receptor–ligand pairs. In mice, for example, the activating KLR family receptor Nkrp1f binds the CLEC2 family member Clrg, whereas the inhibitory receptor Nkrp1d binds Clrb (7, 8). Tumorigenesis and genotoxic stress down-regulate Clrb expression and thus promote NK cell-mediated lysis (8, 9). Corresponding Nkrp1–Clr receptor–ligand pairs have also been identified in humans. Thus, the inhibitory NK receptor NKRP1A (CD161), the human homolog of mouse Nkrp1d, engages the CLEC2 family member LLT1, which is expressed by activated dendritic and B cells, thereby negatively modulating NK-cell-mediated cytotoxicity (10–13). Another CLEC2 family member, AICL, is recognized by the activating NK receptor NKp80, which is genetically linked to AICL in the human NKC (14). Whereas NKp80 is found exclusively on NK cells, AICL is expressed on monocytes. The NKp80–AICL interaction promotes NK cell-mediated cytolysis of malignant myeloid cells and also mediates cellular cross-talk between NK cells and monocytes (14).The most recent addition to the human CLEC2 family is keratinocyte-associated C-type lectin (KACL or CLEC2A), whose expression is almost exclusively restricted to the skin, in marked contrast to the broad expression of other CLEC2 family members in hematopoietic cells (15). The receptor for KACL is NKp65, a distant relative of NKp80, which is encoded adjacent to KACL in the NKC in a tail-to-tail orientation (16). Similarly to NKp80 and AICL, no related sequences for NKp65 and KACL are present in rodents, although homologs of NKp80 and KACL exist in chimpanzee, rhesus macaque, and cow (15, 17). NKp65 stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of ectopic KACL or of KACL on freshly isolated keratinocytes. The amino terminus of the cytoplasmic domain of NKp65 contains a hemi-ITAM motif that is required for NKp65-mediated cytotoxicity (16). This Syk kinase-recruiting motif is also found in other NKC-encoded activating receptors, including dectin-1, Clec1b, and NKp80 (17–19). The genetically linked NKp65–KACL receptor–ligand pair may fulfill a dedicated role in the immune surveillance of human skin through specific recognition of keratinocytes (16, 17).Considerable progress has been made in the structural analysis of NKC-encoded C-type lectin-like receptors that recognize MHC or MHC-related ligands (20). These structures include Ly49A bound to H-2D^d (21), Ly49C bound to H-2K^b (22, 23), NKG2D in complex with MICA (24), and NKG2A/CD94 in complex with HLA-E (25, 26). In addition, we determined the structure of killer cell lectin-like receptor G1 (KLRG1) bound to E-cadherin, a non-MHC ligand that is down-regulated in metastatic tumors (27). By contrast, no structural information is available for any of the NKC-encoded receptor–ligand pairs identified to date (Nkrp1f–Clrg and Nkrp1d–Clrb in rodents and NKRP1A–LLT1, NKp80–AICL, and NKp65–KACL in humans), except for the structures of mouse Nkrp1a and Clrg in unbound form (28, 29). To understand genetically linked recognition by C-type lectin-like receptors in the NKC at the atomic level, we determined the structure of NKp65 in complex with its keratinocyte ligand KACL.     

Lytic immune synapse function requires filamentous actin deconstruction by Coronin 1A

Lytic immune effector function depends upon directed secretion of cytolytic granules at the immunological synapse (IS) and requires dynamic rearrangement of filamentous (F)-actin. Coronin 1A (Coro1A) is the hematopoietic-specific member of the Coronin family of actin regulators that promote F-actin disassembly. Here, we show that Coro1A is required for natural killer (NK) cell cytotoxic function in two human NK cell lines and ex vivo cells from a Coro1A-deficient patient. Using superresolution nanoscopy to probe the IS, we demonstrate that Coro1A promotes the deconstruction of F-actin density that facilitates effective delivery of lytic granules to the IS. Thus, we show, for the first time to our knowledge, a critical role for F-actin deconstruction in cytotoxic function and immunological secretion and identify Coro1A as its mediator.Natural killer (NK) cell cytotoxicity is a finely controlled process that integrates signals from activating and inhibitory receptors to eliminate virally infected and tumorigenic cells sensitively and specifically. The importance for NK cells in immune function is underscored by the severe virus infections and malignancies suffered by patients with NK cell deficiency (1). A dynamic filamentous (F)-actin cytoskeleton is required for NK cell cytotoxicity because disruption of F-actin polymerization by pharmacological inhibitors or mutation of actin-nucleating factors results in impaired NK cell function (2–5). Actin nucleators, such as actin-related proteins 2 and 3 complex (Arp2/3), Wiskott–Aldrich syndrome protein (WASp), WIP, DOCK8, and WAVE2, serve well-defined critical roles in the formation and function of the NK cell immunological synapse (IS) (4–10).Killing of a susceptible target follows tightly regulated steps of NK cell immune synapse formation and lytic granule exocytosis (3). Although cortical F-actin has long been considered a barrier to exocytosis of granule-like organelles (11) in some cell types, ligation of NK cell activating and adhesion receptors results in the formation of conduits in F-actin that permit and actually facilitate NK cell degranulation (12–14). This finding suggests that fine regulation and deconstruction of the synaptic F-actin meshwork is required for the formation of granule-permissive–sized clearances (12).Coronin 1A (Coro1A) is the hematopoietic cell-specific isoform of the highly conserved Coronin family of actin regulators. Coronins contain a series of WD-repeat domains that form an F-actin–binding β-propeller domain, and thus bind F-actin directly (15–17). In addition, Coro1A binds to and inhibits the Arp2/3 complex (17, 18) required for actin branching and can enhance the activity of cofilin to promote actin disassembly in in vitro reconstituted systems (19–21). Coro1A localizes with actin-rich structures in immune cells, including phagocytic cups in neutrophils and macrophages, and at the leading edge of T cells (22–25). T cells from Coronin 1^−/− mice have defects in migration and cell survival attributed to impaired T-cell receptor signaling, Ca²⁺ flux, Rac activation, and subcellular Arp2/3 localization (26–28). Mutations in Coro1A lead to T⁻B⁺NK⁺ combined immunodeficiency and susceptibility to severe viral infections, including life-threatening varicella infection and EBV-driven lymphoproliferation (26, 29, 30).By manipulating expression of Coro1A in human NK cells, we show that Coro1A is required for cytotoxic function. Using superresolution nanoscopy, we define a requirement for Coro1A in F-actin deconstruction and subsequent delivery of lytic granules to the synaptic membrane. In addition, we have specifically evaluated cytotoxic function in a Coro1A-deficient patient and find that NK cell function is severely impaired. Further, we demonstrate the same F-actin structural defect in patient cells as in two Coro1A-deficient cell lines. Thus, with superresolution imaging, we identify, for the first time to our knowledge, a critical role for actin deconstruction in immunity and human host defense.