Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK–DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues.Natural killer (NK) cells play an important role in protecting the host against viral infection and cancer. As well as having potent cytotoxic activity, NK cells are endowed with immunoregulatory activity (1, 2). For example, NK cell activation induces the production of chemokines, such as macrophage inflammatory protein-1α (MIP-1α) and IL-8, and proinflammatory cytokines, such as IFN-γ, GM-CSF, and TNF-α. These molecules regulate the recruitment and activity of numerous immune cell types (1, 2). Importantly, NK cells can promote development of T-cell responses via NK–dendritic cell (DC) interactions that favor both DC maturation and NK-cell activation (3–5), with NK cell-derived IFN-γ skewing T-cell differentiation toward the Th1 phenotype (6, 7).Cytotoxic activity and cytokine production are coupled to signaling pathways downstream of a repertoire of activating and inhibitory receptors; signals from activating receptors (including NKG2D, DNAM-1, and 2B4, as well as the natural cytotoxicity receptors NKp30, NKp44, and NKp46) compete with signals from inhibitory receptors such as the killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A heterodimers to regulate activation. In addition, NK cells express CD16, the low-affinity receptor for IgG, conferring antibody-dependent cellular cytotoxicity (8–10). Activation thus coordinates the killing of target cells, the induction of inflammation, and the promotion of adaptive immunity. This potent cytotoxicity and proinflammatory activity must be strictly controlled to minimize damage to healthy tissue. Functional competency of unstimulated NK cells is achieved via a process termed “licensing” or “education” (11–14). Licensing ensures that only those NK cells expressing inhibitory receptors for self-MHC class I can respond to target cells and NK cells that lack inhibitory receptors for self-MHC class I molecules are rendered hyporesponsive, preventing them from attacking healthy cells expressing normal levels of MHC class I molecules.We have analyzed the consequences of human NK cell activation by tumor cells. Our results reveal induction of the TNF superfamily member 14 (TNFSF14), also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT) (15). We show that activated NK cells produce TNFSF14 in response to different stimuli, that tumor cells induce TNFSF14 production by licensed NK cells, and that TNFSF14-producing NK cells aid DC maturation during NK–DC cross-talk.     

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.     

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPβ1 mediated by induction of antitumorigenic macrophages

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPβ1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell–killing activity of macrophages, however. Moreover, knockdown of SIRPβ1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPβ1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPβ1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPβ1 is a potential target for cancer immunotherapy.

Macrophages are innate immune cells that show phenotypic heterogeneity and functional diversity; and they play key roles in development, tissue homeostasis and repair, and in cancer, as well as in defense against pathogens (1–3). In the tumor microenvironment (TME), macrophages are exposed to a variety of stimuli, including cell–cell contact, hypoxia, as well as soluble and insoluble factors such as cytokines, chemokines, metabolites, and extracellular matrix components (2, 4). These environmental cues promote the acquisition by macrophages of protumorigenic phenotypes that facilitate tumor development, progression, and metastasis as well as suppress antitumor immune responses (2, 4). A high density of macrophages within tumor tissue is associated with poor prognosis in patients with various types of cancer, including that of the bladder or breast (5–7). Depletion of macrophages in the TME or the reprogramming of these cells to acquire antitumorigenic phenotypes has been shown to ameliorate the immunosuppressive condition and result in a reduction in tumor burden in both preclinical and clinical studies (2, 4, 8, 9). Macrophages within the TME have therefore attracted much attention as a potential therapeutic target for cancer immunotherapy.Signal regulatory protein α (SIRPα) is a transmembrane protein that possesses one NH₂-terminal immunoglobulin (Ig)-V–like and two Ig-C domains in its extracellular region, as well as immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic region (10, 11). The extracellular region of SIRPα interacts with that of CD47, another member of the Ig superfamily of proteins, with this interaction constituting a means of cell–cell communication. The expression of SIRPα in hematopoietic cells is restricted to the myeloid compartment—including macrophages, neutrophils, and dendritic cells (DCs)—whereas CD47 is expressed in most normal cell types as well as cancer cells (12, 13). The interaction of SIRPα on macrophages with CD47 on antibody (Ab)-opsonized viable cells such as blood cells or cancer cells prevents phagocytosis of the latter cells by the former (13–15), with this negative regulation of macrophages being thought to be mediated by SHP1, a protein tyrosine phosphatase that binds to the cytoplasmic region of SIRPα (14). Indeed, blockade of the CD47–SIRPα interaction by Abs to either SIRPα or CD47, in combination with a tumor-targeting Ab such as rituximab (anti-CD20), was found to enhance the Ab-dependent cellular phagocytosis (ADCP) activity of macrophages for cancer cells that do not express SIRPα, resulting in marked suppression of tumor formation in mice (15–19). Targeting of SIRPα in combination with a tumor-targeting Ab therefore provides a potential approach to cancer immunotherapy dependent on enhancement of the ADCP activity of macrophages for cancer cells. In contrast, the effect of Abs to SIRPα in the absence of a tumor-targeting Ab on the phagocytosis by macrophages of, as well as on tumor formation by, cancer cells that do not express SIRPα was minimal or limited.We have now further examined the antitumor efficacy of a monoclonal Ab (mAb) to mouse SIRPα (MY-1) (20) in immunocompetent mice transplanted subcutaneously with several types of murine cancer cells that do not express SIRPα. This Ab prevents the binding of mouse CD47 to SIRPα and cross-reacts with mouse SIRPβ1 (15). We found that monotherapy with MY-1 efficiently attenuated the growth of tumors formed by bladder or mammary cancer cells. In addition, MY-1 markedly promoted the induction of antitumorigenic macrophages able to target these cancer cells. Furthermore, our results suggest that SIRPβ1 on macrophages likely participated in the antitumorigenic effect of MY-1.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

Growth factors and medium hyperglycemia induce Sox9+ ductal cell differentiation into β cells in mice with reversal of diabetes

We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9⁺ (Sox9⁺) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9⁺ ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9⁺ ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300–450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9⁺ ductal cell differentiation into β cells in adult mice.Autoimmune type 1 diabetes (T1D) results from autoimmune attack on insulin-secreting β cells and subsequent insulin deficiency (1). Cure of T1D requires both reversal of autoimmunity and resupply of insulin-secreting β cells by islet transplantation or augmentation of endogenous β-cell regeneration (2). Because of the lack of donors for islet transplantation and because islet grafts only last for ∼3 y (3), augmentation of endogenous β-cell regeneration would be the more favorable approach. We previously reported that combination therapy of induction of mixed chimerism and administration of gastrin and epidermal growth factor (GE) not only reversed autoimmunity, but also augmented β-cell neogenesis and replication and subsequently cured late-stage T1D in autoimmune nonobese diabetic (NOD) mice (4). However, the origin of neogenesis remains unknown.It has been proposed that β-cell neogenesis in adult mice can derive from pancreatic ductal cells (5, 6), cells in the islet (7, 8), transdifferentiation from glucagon-producing α cells (9, 10), or from acinar cells (11–13). Although it has been consistently reported that pancreatic ductal epithelial cells give rise to insulin-producing β cells during embryonic development (14–17), whether the pancreatic ductal progenitors can give rise to insulin-producing β cells in neonates and adult mice remains controversial. Using a Cre-based lineage tracing with a human carbonic anhydrase II (CAII) promoter, Inada et al. reported that pancreatic ductal cells were able to give rise to insulin-producing β cells in neonates and in pancreatic duct ligation (PDL)-treated adult mice (5). Using a cyclization recombinase (Cre)-based lineage tracing model with a neurogenin 3 (Ngn3) promoter, Xu et al. also found that cells in the pancreatic ductal lining could give rise to β cells in PDL-treated adult mice (6). Conversely, using a lineage-tracing model with a hepatocyte nuclear factor 1-β (Hnf1β) promoter, Solar et al. found that pancreatic ductal cells did not give rise to β cells in neonates, PDL-treated adult mice, or Alloxan-induced diabetic adult mice treated for 1 wk with GE (14). Using a lineage-tracing model with a SRY (sex-determining region Y)-box 9 (Sox9) promoter, Kopp et al. also showed that Sox9⁺ ductal cells did not give rise to β cells postnatally after β-cell ablation or after PDL (17, 18). Similarly, Furuyama et al. reported that Sox9⁺ pancreatic ductal cells were not able to give rise to β cells in PDL-treated adult mice or streptozotocin (STZ)-induced diabetic mice (16). These reports suggest that PDL injury with normal glycemia, hyperglycemia alone, or hyperglycemia plus short-term (1 wk) administration of GE is not able to augment pancreatic Sox9⁺ ductal cell differentiation into insulin-producing β cells in adult mice. Actually, how hyperglycemia regulates progenitor differentiation into β cells remains largely unknown, although it has been reported that hyperglycemia is toxic to β cells (19, 20).Several reports have shown that a subpopulation of cells in the pancreatic ducts of adult mice is clonogenic and can give rise to insulin-producing β cells in various in vitro culture systems (21, 22). One recent study, which used in vitro semisolid medium culture, showed that Sox9⁺CD133⁺ pancreatic ductal cells from adult mice are able to give rise to three cell lineages, including insulin-producing β cells, ductal epithelial cells, and acinar cells (23, 24). These results indicate that Sox9⁺CD133⁺ pancreatic ductal cells from adult mice have the potential to differentiate into insulin-producing β cells in culture, if induced by specific conditions.Because treatment with GE was reported to induce β-cell neogenesis from adult pancreatic ductal cells in vitro (25), and because we observed neogenesis in mixed chimeric late-stage diabetic NOD mice after long-term (8 wk) administration of GE (4), in the present studies, we tested whether long-term administration of GE was able to induce Sox9⁺ pancreatic ductal cell differentiation into insulin-producing β cells in diabetic C57BL/6 mice, using a Cre-based lineage-tracing construct driven by Sox9 regulatory sequences. The same strain of mice was used as in the studies of Kopp et al. (17), but a founder with higher recombination efficiency was used in the present studies. We found that islets consisting of β cells differentiated from Sox9⁺ ductal cells in adult mice with hyperglycemia (>300 mg/dL) and medium hyperglycemia (300–450 mg/dL) in combination with long-term administration of GE synergistically augments the differentiation of Sox9⁺ ductal cells into β cells with reversal of hyperglycemia. These results indicate that Sox9⁺ pancreatic ductal cells in adult mice can differentiate into insulin-producing β cells under specific conditions, which include medium hyperglycemia and long-term administration of low-dose GE, as used in previous reports (26).     

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.     

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.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

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.