Expression of ligands for activating natural killer cell receptors on cell lines commonly used to assess natural killer cell function.

BACKGROUND: Natural killer cell responses to virally-infected or transformed cells depend on the integration of signals received through inhibitory and activating natural killer cell receptors. Human Leukocyte Antigen null cells are used in vitro to stimulate natural killer cell activation through missing-self mechanisms. On the other hand, CEM.NKr.CCR5 cells are used to stimulate natural killer cells in an antibody dependent manner since they are resistant to direct killing by natural killer cells. Both K562 and 721.221 cell lines lack surface major histocompatibility compatibility complex class Ia ligands for inhibitory natural killer cell receptors. Previous work comparing natural killer cell stimulation by K562 and 721.221 found that they stimulated different frequencies of natural killer cell functional subsets. We hypothesized that natural killer cell function following K562, 721.221 or CEM.NKr.CCR5 stimulation reflected differences in the expression of ligands for activating natural killer cell receptors. RESULTS: K562 expressed a higher intensity of ligands for Natural Killer G2D and the Natural Cytotoxicity Receptors, which are implicated in triggering natural killer cell cytotoxicity. 721.221 cells expressed a greater number of ligands for activating natural killer cell receptors. 721.221 expressed cluster of differentiation 48, 80 and 86 with a higher mean fluorescence intensity than did K562. The only ligands for activating receptor that were detected on CEM.NKr.CCR5 cells at a high intensity were cluster of differentiation 48, and intercellular adhesion molecule-2. CONCLUSIONS: The ligands expressed by K562 engage natural killer cell receptors that induce cytolysis. This is consistent with the elevated contribution that the cluster of differentiation 107a function makes to total K562 induced natural killer cell functionality compared to 721.221 cells. The ligands expressed on 721.221 cells can engage a larger number of activating natural killer cell receptors, which may explain their ability to activate a larger frequency of these cells to become functional and secrete cytokines. The few ligands for activating natural killer cell receptors expressed by CEM.NKr.CCR5 may reduce their ability to activate natural killer cells in an antibody independent manner explaining their relative resistance to direct natural killer cell cytotoxicity.

Differential contribution of education through KIR2DL1, KIR2DL3, and KIR3DL1 to antibody-dependent (AD) NK cell activation and ADCC.

The engagement of activating NK receptors (aNKR) stimulates NK cell activity, provided that interactions between inhibitory NK receptors (iNKR) with their HLA ligands do not override them. Abs bound to target cells can also activate NK cells by engaging the CD16 aNKR. NK cell education status is an important factor for Ab-dependent NK cell activation (ADNKA) of some NK cell subsets. However, whether NK cell education also influences Ab-dependent cellular cytotoxicity (ADCC) levels is not fully known. ADCC-GranToxiLux (GTL) assays measured ADCC activity as the frequency of granzyme B positive (%GzB+ ) target cells. Target cells were anti-HIV Immunoglobulin G (HIVIG)-opsonized CEM-NKr.CCR5 (CEM) cells. Lymphocytes and sorted single positive (SP) NKG2A+ , KIR2DL1+ , KIR2DL3+ , and KIR3DL1+ NK cells, to self- and nonself HLA, were used as effectors in ADCC-GTL assays to examine how education status influenced ADCC activity. ADNKA activity was assessed by stimulating lymphocytes with HIVIG-opsonized CEMs and measuring the frequency of NK cell populations defined by their expression of iNKRs, along with IFN-γ, CCL4, and CD107a functions. ADCC: the %GzB+ CEM cells generated by self- versus nonself HLA-specific SPiNKR did not differ. ADNKA: More NK cells educated through KIR2DL1 and KIR3DL1, but not KIR2DL3, responded to ADNKA than their uneducated counterparts. CD16 engagement induced ADCC and ADNKA activity. With the proviso that groups' sizes were small, our results support the notion that NK cell education does not influence ADCC levels but does contribute to ADNKA activity.

Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4-) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4-) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

Natural Killer (NK) Cell Education Differentially Influences HIV Antibody-Dependent NK Cell Activation and Antibody-Dependent Cellular Cytotoxicity.

Immunotherapy using broadly neutralizing antibodies (bNAbs) endowed with Fc-mediated effector functions has been shown to be critical for protecting or controlling viral replication in animal models. In human, the RV144 Thai trial was the first trial to demonstrate a significant protection against HIV infection following vaccination. Analysis of the correlates of immune protection in this trial identified an association between the presence of antibody-dependent cellular cytotoxicity (ADCC) mediated by immunoglobulin G (IgG) antibodies (Abs) to HIV envelope (Env) V1/V2 loop structures and protection from infection, provided IgA Abs with competing specificity were not present. Systems serology analyses implicated a broader range of Ab-dependent functions in protection from HIV infection, including but not limited to ADCC and Ab-dependent NK cell activation (ADNKA) for secretion of IFN-γ and CCL4 and expression of the degranulation marker CD107a. The existence of such correlations in the absence of bNAbs in the RV144 trial suggest that NK cells could be instrumental in protecting against HIV infection by limiting viral spread through Fc-mediated functions such as ADCC and the production of antiviral cytokines/chemokines. Beside the engagement of FcγRIIIa or CD16 by the Fc portion of anti-Env IgG1 and IgG3 Abs, natural killer (NK) cells are also able to directly kill infected cells and produce cytokines/chemokines in an Ab-independent manner. Responsiveness of NK cells depends on the integration of activating and inhibitory signals through NK receptors, which is determined by a process during their development known as education. NK cell education requires the engagement of inhibitory NK receptors by their human leukocyte antigen ligands to establish tolerance to self while allowing NK cells to respond to self cells altered by virus infection, transformation, stress, and to allogeneic cells. Here, we review recent findings regarding the impact of inter-individual differences in NK cell education on Ab-dependent functions such as ADCC and ADNKA, including what is known about the HIV Env epitope specificity of ADCC competent Abs and the conformation of HIV Env on target cells used for ADCC assays.

NK Cells Expressing the Inhibitory Killer Immunoglobulin-Like Receptors (iKIR) KIR2DL1, KIR2DL3 and KIR3DL1 Are Less Likely to Be CD16+ than Their iKIR Negative Counterparts.

Natural Killer (NK) cell education, which requires the engagement of inhibitory NK cell receptors (iNKRs) by their ligands, is important for generating self-tolerant functional NK cells. While the potency of NK cell education is directly related to their functional potential upon stimulation with HLA null cells, the influence of NK cell education on the potency of the antibody dependent cellular cytotoxicity (ADCC) function of NK cells is unclear. ADCC occurs when the Fc portion of an immunoglobulin G antibody bridges the CD16 Fc receptor on NK cells and antigen on target cells, resulting in NK cell activation, cytotoxic granule release, and target cell lysis. We previously reported that education via the KIR3DL1/HLA-Bw4 iNKR/HLA ligand combination supported higher KIR3DL1+ than KIR3DL1- NK cell activation levels but had no impact on ADCC potency measured as the frequency of granzyme B positive (%GrB+) targets generated in an ADCC GranToxiLux assay. A lower frequency of KIR3DL1+ compared to KIR3DL1- NK cells were CD16+, which may in part explain the discrepancy between NK cell activation and target cell effects. Here, we investigated the frequency of CD16+ cells among NK cells expressing other iNKRs. We found that CD16+ cells were significantly more frequent among NK cells negative for the inhibitory KIR (iKIR) KIR2DL1, KIR2DL3, and KIR3DL1 than those positive for any one of these iKIR to the exclusion of the others, making iKIR+ NK cells poorer ADCC effectors than iKIR- NK cells. The education status of these iKIR+ populations had no effect on the frequency of CD16+ cells.

Antibody-Dependent Cellular Cytotoxicity Activity of Effector Cells from HIV-Infected Elite and Viral Controllers.

Carriage of alleles encoding certain inhibitory natural killer (NK) cell receptor/HLA ligand KIR3DL1/HLA-B combinations is associated with protection from HIV infection and slow time to AIDS, implicating NK cells in HIV control. NK cells also mediate antibody-dependent cellular cytotoxicity (ADCC). ADCC has been identified as a correlate of protection in secondary analyses of the modestly protective RV144 Thai HIV vaccine trial. In ADCC, HIV envelope (Env)-specific antibodies (Abs) bridge HIV-infected or gp120-coated target cells and NK cells expressing CD16 receptors for Ab Fc domains. CD16 engagement activates NK cells to secrete cytokines/chemokines, degranulate, deliver granzyme B (GrB) to target cells, and cytolysis. A subset of HIV+ subjects, known as slow progressors (SPs), maintains low-level viremia without treatment. HIV+ SPs versus progressors have higher titers and/or a greater breadth of ADCC-competent Abs. Investigations of the functional capacity of NK effector cells following CD16 engagement in HIV+ subjects are lacking. We used the ADCC-GranToxiLux (ADCC-GTL) assay to assess the frequency of GrB+ (%GrB+) cells generated by effector cells from 37 HIV+ SPs and 15 progressors to gp120-coated CEM.NKr.CCR5 target cells in the presence of anti-Env Abs. Subject groups were stratified according to whether or not they carried educating KIR3DL1/HLA-B combinations able to confer NK cells with functional potential. No differences were observed in %GrB+ target cells generated by effector cells from carriers of educating versus noneducating KIR3DL1/HLA-B pairs. The absence of an effect of NK cell education on this readout may be due to loss of the ability of educated NK cells from SPs to respond to Ab-dependent stimulation and/or the lower frequency of KIR3DL1+ than KIR3DL1- NK cells that coexpress CD16. That KIR/HLA genotypes have minimal impact on interindividual differences in ADCC potency has relevance for therapeutic interventions that target ADCC for HIV control.

Natural killer cell education does not affect the magnitude of granzyme B delivery to target cells by antibody-dependent cellular cytotoxicity.

OBJECTIVE: Interest in the role of antibody-dependent cellular cytotoxicity (ADCC) in protection from HIV infection has grown since analyses of the RV144 HIV vaccine trial results found ADCC correlated with protection. Natural killer (NK) cells are among the effector cells that mediate ADCC. The level of antibody-induced NK cell activation depends on NK cell education through inhibitory NK cell receptor human leukocyte antigen (HLA) ligand interactions. Here, we investigated the impact of NK cell education on the delivery of Granzyme B (GzB) to target cells. DESIGN: Lymphocytes from 50 HIV-uninfected [30 Bw4 (Bw4) and 20 Bw4 (Bw6)] KIR3DL1 homozygote persons were used as effectors and cocultured with gp120-coated target cells in the presence of a single source of anti-HIV gp120 antibody to ascertain whether NK cell education status influenced the level of GzB delivered to target cells. METHODS: The GTL assay assessed the frequency of GzB-positive (%GzB) CEM.NKr.CCR5 target cells generated by effectors from each individual. The frequency of CD107a, interferon (IFN)-γ and CCL4 NK cells was assessed as a measure of antibody-induced NK cell activation. RESULTS: KIR3DL1 NK cells from the Bw4 group were more functional than KIR3DL1 NK cells. Despite this, the %GzB target cells generated in the GTL assay did not differ according to the KIR3DL1-HLA-B genotype of the effector cells. The %GzB cells positively correlated with the frequency of CD16KIR3DL1 NK cells in the effector population. CONCLUSION: ADCC potency does not depend on NK cell education.

A Higher Frequency of NKG2A+ than of NKG2A- NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1.

UNLABELLED: Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on which NK cell populations, as defined by the inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4(+) (iCD4) T cells. NK cells acquire antiviral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (here, 3DL1), engaging their ligands. NKG2A interacts with HLA-E, and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV-infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA-null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A(+)/NKG2A(-) 3DL1(+)/3DL1(-) (NKG2A(+/-) 3DL1(+/-)) population and to measure the frequency of all possible combinations of CD107a expression and gamma interferon (IFN-γ) and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A(+) 3DL1(+) NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 cells were those expressing NKG2A; coexpression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as by the trifunctional CD107a(+) IFN-γ(+) CCL4(+), CD107a(+) IFN-γ(+), total CD107a(+), and total IFN-γ(+) functional subsets. These results indicate that the NKG2A receptor has a role in NK cell-mediated anti-HIV responses. IMPORTANCE: HIV-infected CD4 (iCD4) cells activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 cells stimulate to develop antiviral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKRs) NKG2A and 3DL1, that respond to iCD4 cells. NKG2A and 3DL1 are involved in priming NK cells for antiviral functions upon encountering virus-infected cells. A higher frequency of NKG2A(+) than NKG2A(-) NK cells responded to iCD4 cells by developing antiviral functions such as CD107a expression, which correlates with NK cell killing, and secretion of gamma interferon and CCL4. Coexpression of 3DL1 on the NKG2A(+) and NKG2A(-) NK cells did not modulate responses to iCD4 cells. Understanding the mechanisms underlying the interaction of NK cells with iCD4 cells that lead to HIV control may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.