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Disruption of the mucosal epithelium during lentivirus infections permits translocation of microbial products into circulation, causing immune activation and driving disease. Although the liver directly filters blood from the intestine and is the first line of defense against gut-derived antigens, the effects of microbial products on the liver are unclear. In livers of normal macaques, minute levels of bacterial products were detectable, but increased 20-fold in simian immunodeficiency virus (SIV)–infected animals. Increased microbial products in the liver induced production of the chemoattractant CXCL16 by myeloid dendritic cells (mDCs), causing subsequent recruitment of hypercytotoxic natural killer (NK) cells expressing the CXCL16 receptor, CXCR6. Microbial accumulation, mDC activation, and cytotoxic NK cell frequencies were significantly correlated with markers of liver damage, and SIV-infected animals consistently had evidence of hepatitis and fibrosis. Collectively, these data indicate that SIV-associated accumulation of microbial products in the liver initiates a cascade of innate immune activation, resulting in liver damage.

The progression to AIDS is influenced by changes in the biology of heterogeneous monocyte subsets. Classical (CD14++CD16–), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes may represent progressive stages of monocyte maturation or disparate myeloid lineages with different turnover rates and function. To investigate the relationship between monocyte subsets and the response to SIV infection, we performed microarray analysis of monocyte subsets in rhesus macaques at three time points: prior to SIV infection, 26 days postinfection, and necropsy with AIDS. Genes with a 2-fold change between monocyte subsets (2023 genes) or infection time points (424 genes) were selected. We identify 172 genes differentially expressed among monocyte subsets in both uninfected and SIV-infected animals. Classical monocytes express genes associated with inflammatory responses and cell proliferation. Nonclassical monocytes express genes associated with activation, immune effector functions, and cell cycle inhibition. The classical and intermediate subsets are most similar at all time points, and transcriptional similarity between intermediate and nonclassical monocytes increases with AIDS. Cytosolic sensors of nucleic acids, restriction factors, and IFN-stimulated genes are induced in all three subsets with AIDS. We conclude that SIV infection alters the transcriptional relationship between monocyte subsets and that the innate immune response to SIV infection is conserved across monocyte subsets.

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells. Although several new drugs for the treatment of MM have greatly improved survival, many patients are known to relapse and become refractory to all presently available therapies or experience treatment-related toxicities. Therefore, MM remains an unmet medical need and the development of additional novel therapies is required. NK cells play a crucial role in the control of multiple myeloma and accumulating evidence shows the presence of highly cytotoxic NK cells in the bone marrow of MM patients suggesting that targeting NK cells could provide a specific treatment modality to leverage NK cell cytotoxicity in myeloma. Furthermore, NK cells are the first lymphocytes population to reconstitute after autologous stem cell transplant (ASCT) providing an opportunity to target minimal residual disease (MRD) shortly after transplant. B-cell maturation antigen (BCMA) is an excellent target in MM because its restricted expression in normal and malignant plasma cells from untreated and relapsed myeloma patients, but absent in all other main bone marrow cell subsets. Our first-in-class NKp30xBCMA NK cells engager, CTX-4419, binds to BCMA on MM cells and to NKp30 and CD16A (FcγRIIIA) on NK cells, specifically redirecting NK cells towards tumor cells expressing BCMA. We demonstrate here that CTX-4419 retains activity in the presence of high levels of BCMA ligands and serum IgG and induces potent NK cytotoxicity against high and low BCMA expressing cell lines as well as patients (autologous) primary myeloma cells. Moreover, CTX-4419 does not require CD16A binding to kill tumor cells, a unique characteristic that overcomes the reduction or loss of activity of CD16A due to receptor shedding or downregulation in the tumor microenvironment. Furthermore CTX-4419 induces NK proliferation and cytokines/chemokines production by NK cells only in the presence of tumor cells providing sustainable anti-tumor specificity to NK cells. CTX-4419 activates in-vitro cynomolgus NK cells in presence of target cells expressing cynomolgus BCMA and, when given intravenously, CTX-4419 decreases plasma cell counts. CTX-4419 represents a novel class of NK-cell engagers that shows strong potency even in the absence of CD16A engagement and induces NK cell proliferation and lysis of tumor cells expressing low amount of antigen. CTX-4419 has strong activity in an autologous setting when tested in bone marrow samples of MM patients and shows efficacy in a non-human primate model of plasma-cell depletion. These data show that CTX-4419 is strongly differentiated from conventional therapeutic antibodies and is a promising candidate for MM treatment with the potential to be used as monotherapy or in combination with adoptive transfer of NK cells and/or other immuno-therapies. Citation Format: Monia Draghi, Jamie L. Schafer, Allison Nelson, Zach Frye, Amanda Oliphant, Sara Haserlat, jason Lajoie, Kenneth rogers, Francois Villinger, Michael Schmidt, Robert Tighe, Piotr Bobrowicz, Jennifer Watkins-Yoon, Thomas Schuetz. Preclinical development of a first-in-class NKp30xBCMA NK cell engager for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4972.

Natural killer (NK) cells are generally considered as the effector arm of nonspecific innate immunity, providing rapid recognition and lysis of virus-infected and tumor cells, but can also play significant regulatory roles in pregnancy, cooperate with T and B cell responses, and even exhibit independent adaptive memory. However, much like other lymphocyte populations, recent burgeoning evidence suggests that in chronic conditions NK cells can become functionally exhausted. T cell exhaustion is generally induced by constant exposure to persistent antigens resulting in a loss of effector functions and an overall decline in resistance to disease, and NK cell exhaustion appears to share many similar features. One of the best-studied examples of NK cell dysregulation is in HIV infection and in SIV-infected nonhuman primate models. Indeed, recent work from our laboratory found that NK cells in lymph nodes of SIV-infected rhesus macaques are continuously activated, likely by low-level ongoing virus replication [1]. This continuous activation resulted in in situ differentiation followed by functional anergy, as indicated by a failure to lyse target cells and upregulated Tim-3 expression. These data helped clarify empirical observations in both HIV and SIV infections of impaired cytotoxic function, altered cytokine production and impaired antibody-dependent cell-mediated cytotoxicity (ADCC) [2]. Indirect effects of HIV/SIV include the consistent bombardment of NK cells with target cells expressing dysregulated HLA and systemic cytokines, resulting in net exhaustion. Interestingly, similar indicators of upregulated Tim-3, PD-1 and lytic failure have all been found in CMV, Hepatitis C and Hepatitis B infections. Together, these data indicate NK cell exhaustion in chronic stages of viral disease may be directly related to a loss of control of virus replication and/or reactivation of latent virus reservoirs. In addition to chronic viral infections, NK cell exhaustion has also been observed in several human cancers and animal tumor models. Much like NK cells in SIV infection and previous descriptions of exhausted T cells, common features of NK cells in cancer and cancer models are high expression of inhibitory receptors including Tim-3, downregulated activating receptors, and decreased expression of transcription factors, EOMES and T-bet [3–5]. Furthermore, mouse models have demonstrated that NK cell dysfunction due to inefficient phosphorylation in activation signaling pathways arises only against MHC-deficient tumors, not against tumors that maintain MHC expression [6]. Indeed, just repeated exposure of NK cells in the presence of tumors can reduce cytotoxic activity [3]. While NK cell exposure to tumors can reduce cytotoxic activity, excess proliferation also contributes to NK cell dysfunction, as even homeostatic proliferation may induce an exhausted state in the absence of a tumor. Exhaustion of NK cells is also highly specific, as NK cell defects are far more severe in tumor-associated NK cells than in peripheral NK cells from the same individual or animal [3, 5, 6]. The clinical relevance of decreased NK cell function is also evident in disease progression, a prime example being breast cancer where NK cells with reduced function and high expression of inhibitory receptors are associated with more aggressive and invasive tumors [5]. NK cell exhaustion in chronic diseases appears to have many conserved features, including upregulation of inhibitory receptors and excess proliferation due to virus or cytokine-induced activation. Furthermore, NK cell recognition is highly dependent on ‘self’ and ‘missing-self’ discrimination through MHC binding, and repeated NK cell interaction with target cells where MHC is lacking, either by viral or oncogenic etiology, is a prominent cause of exhaustion. Although in its infancy, therapeutic restoration of NK cell numbers and functions are being developed. Preliminary success reverting NK cell exhaustion by therapeutic overexpression of EOMES [3], blocking of Tim-3 [4], or cytokine stimulation by IL-12, IL-18 or an IL-2 derivatives [6] have improved NK cell anti-tumor responses. Exogenous IL-12, IL-15 and/or IFN-α have also proven beneficial in reactivating NK cell function in nonhuman primate models [7]. Additionally, nonpathogenic hosts of SIV infection do not appear to exhibit evidence of NK cell exhaustion despite high virus replication [8], and these models could inform future strategies to augment NK cell function. In summary, these data highlight the concept that functional exhaustion of NK cells is associated with loss of control of chronic viral infections and inadequate elimination of neoplastic cells, but current strategies for augmentation of impaired NK cell function are underdeveloped and should be considered for future therapies.

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses., Author Summary Natural killer (NK) cells recognize and kill infected cells without prior antigenic stimulation, and thus provide an important early defense against virus infection. NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. Inhibitory KIRs normally suppress NK cell responses through interactions with their MHC class I ligands on the surface of healthy cells. However, when these interactions are perturbed, this inhibition is lost resulting in NK cell activation and killing of the target cell. We investigated the functional implications of simian immunodeficiency virus (SIV) peptides bound by a common MHC class I molecule in the rhesus macaque that stabilize or disrupt binding to an inhibitory KIR. Whereas SIV peptides that stabilized KIR-MHC class I binding suppressed NK cell activation, peptides that disrupted this interaction did not and resulted in NK cell lysis. These findings demonstrate that viral peptides can modulate NK cell responses through KIR-MHC class I interactions, and are consistent with the possibility that human and simian immunodeficiency viruses may acquire changes in epitopes that increase the binding of MHC class I ligands to inhibitory KIRs as a mechanism to suppress NK cell responses.

Natural killer (NK) cells have traditionally been considered nonspecific components of innate immunity, but recent studies have shown features of antigen-specific memory in mouse NK cells. However, it has remained unclear whether this phenomenon also exists in primates. We found that splenic and hepatic NK cells from SHIV(SF162P3)-infected and SIV(mac251)-infected macaques specifically lysed Gag- and Env-pulsed dendritic cells in an NKG2-dependent fashion, in contrast to NK cells from uninfected macaques. Moreover, splenic and hepatic NK cells from Ad26-vaccinated macaques efficiently lysed antigen-matched but not antigen-mismatched targets 5 years after vaccination. These data demonstrate that robust, durable, antigen-specific NK cell memory can be induced in primates after both infection and vaccination, and this finding could be important for the development of vaccines against HIV-1 and other pathogens.

Background Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. Methods and Findings Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I—presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. Conclusions These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells., An analysis from a cohort in South Africa reveals how the HIV virus may escape NK cell immunity by acquiring mutations in HLA-mediated epitopes, which affect binding to NK cell receptors., Editors' Summary Background Throughout life, our immune system—a complex network of cells, tissues, and organs—protects us from attack by viruses, bacteria, parasites, and fungi. The body’s first line of defense against these “pathogens” is the innate immune system, a collection of cells and proteins that is always ready to identify and kill a wide range of foreign invaders. As well as directly killing pathogens, the innate immune system activates the adaptive immune response, which recognizes and kills specific pathogens and is responsible for immunological memory. Most pathogens are dispatched quickly and effectively by the two arms of the immune system, but some infectious agents have found ways to evade the immune response. For example, infection with HIV-1, the virus that causes AIDS, results in prolonged, continuous viral replication even though the human body mounts a vigorous HIV-1-specific immune response. In large part, HIV-1’s evasion of the immune response reflects its ability to kill virus-specific CD4 lymphocytes, which are needed to help other immune system cells kill HIV-1-infected cells. In addition, the proteins on the surface of HIV-1 that are recognized by the human immune system (viral antigens) frequently acquire changes (mutations) that make it harder for the immune system to clear HIV-1 from the human body. Why Was This Study Done? Viruses evade immune surveillance in many ways, and if we understood the mechanisms underlying immune evasion better, it might be possible to develop targeted immune interventions to deal with viruses such as HIV-1. Here, the researchers investigate how natural killer (NK) cells, a type of lymphocyte that is an important component of the innate antiviral immune response, recognize HIV-infected cells and how HIV-1 evades NK-cell-mediated immune pressure. NK cell activation is determined by the integration of inhibitory and activating signals delivered to the cells by several different receptor families, including the family of killer-cell immunoglobulin-like receptors (KIRs). KIRs mainly bind to human leukocyte antigen (HLA) class I molecules (ligands) on their target cells. HLA class I proteins display fragments (epitopes; peptides recognized by the immune system) of pathogens present in infected cells on the cell surface so that the immune system knows that that cell needs destroying. The binding of distinct KIRs to HLA class I ligands depends on both the sequence of the HLA class I molecule and the sequence of the epitope presented by that HLA class I molecule. Thus, the researchers hypothesized that HIV-1 might evade NK-cell-mediated immune surveillance by acquiring mutations within epitopes presented by HLA class I molecules that enhance the engagement of inhibitory KIRs on NK cells, thereby inhibiting NK cell activity. What Did the Researchers Do and Find? To investigate this model, the researchers asked whether any polymorphisms (naturally occurring genetic variations) in the HIV-1 gene encoding the p24 Gag protein were selected on a population level in HIV-1-infected individuals expressing specific combinations of KIRs and HLA class I ligands. Using statistical methods to identify KIR/HLA combined genotypes in a large group of untreated HIV-1-infected individuals from South Africa, they showed that a specific sequence polymorphism in p24 Gag was selected for in individuals expressing both HLA-C*03:04 and KIR2DL3. Functional studies showed that the selection of this variant HIV-1 epitope resulted in better binding of KIR2DL3, an inhibitory KIR, to HLA-C*03:04 than the wild-type epitope. Moreover, the activation of KIR2DL3-positive NK cells from healthy donors in response to HLA-C*03:04-positive target cells presenting the variant epitope was significantly reduced compared to the activation of KIR2DL3-positive NK cells in response to target cells presenting the wild-type epitope. What Do These Findings Mean? Further studies are needed to assess the consequences of this and other viral sequence variants for viral fitness, the processing and presentation of the mutant epitope during natural infections, and the control of HIV-1 replication in patients. However, these findings provide new insights into how HIV-1 (and possibly other viruses that have a high mutation rate) might evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance the binding of inhibitory KIRs to HLA class I/peptide complexes. A better understanding of this molecular mechanism for evasion of immune surveillance should facilitate the development of targeted immune interventions (for example, the use of KIR-blocking antibodies, some of which are already being clinically tested for the treatment of cancer) to maximize the antiviral activities of NK cells. Additional Information This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001900. The US National Institute of Allergy and Infectious Diseases provides a simple description of the human immune system and information on all aspects of HIV infection and AIDS Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS; Avert also provides personal stories about living with HIV/AIDS The British Society for Immunology provides short articles about various aspects of immunology, including general information about host–pathogen interactions and immune evasion and specific information about HIV and immune evasion Wikipedia has pages on natural killer cells, KIRs, and HLA molecules (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

Objective The aim of this study was to assess the consequence of sequence variations in HLA-C03:04-presented HIV-1 p24 Gag epitopes on binding of the inhibitory natural killer (NK) cell receptor KIR2DL2 to HLA-C03:04. Design HIV-1 may possibly evade recognition by KIR+ NK cells through selection of sequence variants that interfere with the interactions of inhibitory killer cell immunoglobulin-like receptors (KIRs) and their target ligands on HIV-1 infected cells. KIR2DL2 is an inhibitory NK cell receptor that binds to a family of HLA-C ligands. Here, we investigated whether HIV-1 encodes for HLA-C03:04-restricted epitopes that alter KIR2DL2 binding. Methods Tapasin-deficient 721.220 cells expressing HLA-C03:04 were pulsed with overlapping peptides (10mers overlapped by nine amino acids, spanning the entire HIV-1 p24 Gag sequence) to identify peptides that stabilized HLA-C expression. The impact that sequence variation in HLA-C03:04-binding HIV-1 epitopes has on KIR2DL2 binding and KIR2DL2+ NK cell function was determined using KIR2DL2-Fc constructs and NK cell degranulation assays. Results Several novel HLA-C03:04 binding epitopes were identified within the HIV-1 p24 Gag consensus sequence. Three of these consensus sequence peptides (Gag144-152, Gag163-171 and Gag295-304) enabled binding of KIR2DL2 to HLA-C03:04 and resulted in inhibition of KIR2DL2+ primary NK cells. Furthermore, naturally occurring minor variants of epitope Gag295-304 enhanced KIR2DL2 binding to HLA-C03:04. Conclusion Our data show that naturally occurring sequence variations within HLA-C03:04-restricted HIV-1 p24 Gag epitopes can have a significant impact on the binding of inhibitory KIR receptors and primary NK cell function.

The identification of MHC class I ligands for rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to our basic understanding of KIR and MHC class I coevolution and to the study of NK cell responses in this nonhuman primate model for AIDS and other viral diseases. In this study, we show that Mamu-KIR3DL01, which is expressed by ∼90% of rhesus macaques, recognizes MHC class I molecules with a Bw4 motif. Primary NK cells expressing Mamu-KIR3DL01 were identified by staining with a mAb which, in this study, was shown to bind Mamu-KIR3DL01 allotypes with an aspartic acid at position 233. The cytolytic activity of Mamu-KIR3DL01+ NK cells was suppressed by cell lines expressing the Bw4 molecules Mamu-B*007:01, -B*041:01, -B*058:02, and -B*065:01. The Bw4 motif was necessary for Mamu-KIR3DL01 recognition because substitutions in this region abrogated Mamu-KIR3DL01+ NK cell inhibition. However, the presence of a Bw4 motif was not sufficient for recognition because another Bw4 molecule, Mamu-B*017:01, failed to suppress the cytolytic activity of these NK cells. Replacement of three residues in Mamu-B*017:01, predicted to be KIR contacts based on the three-dimensional structure of the human KIR3DL1-HLA-Bw4 complex, with the corresponding residues at these positions for the other Mamu-Bw4 ligands restored Mamu-KIR3DL01+ NK cell inhibition. These results define the ligand specificity of one of the most polymorphic and commonly expressed KIRs in the rhesus macaque and reveal similarities in Bw4 recognition by Mamu-KIR3DL01 and human KIR3DL1, despite the absence of an orthologous relationship between these two KIRs or conservation of surface residues predicted to interact with MHC class I ligands.

No summer evening outdoors seems complete without a mosquito bite (or ten). While these bites are often just a nuisance, they can also serve as the point of transmission for many mosquito-borne diseases: West Nile virus, dengue virus, yellow fever virus, malaria, and the filarial nematodes that cause elephantiasis, to name a few. Efforts to control the spread of these diseases, many of which cannot be curbed by vaccination, have lately looked to the intracellular bacterium Wolbachia pipientis for help. Wolbachia naturally infect many insects including some mosquitoes, and strains have recently been developed to infect Aedes aegypti, the vector for dengue, and Anopheles gambiae, malaria's vehicle of choice. Infection by Wolbachia may limit the spread of these diseases by efficiently becoming established in insect populations, where they shorten the lifespan of their hosts or directly interfere with the pathogens the insects carry.