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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.

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.

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.

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.

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.sup.+ than KIR3DL1.sup.- NK cell activation levels but had no impact on ADCC potency measured as the frequency of granzyme B positive (%GrB.sup.+) targets generated in an ADCC GranToxiLux assay. A lower frequency of KIR3DL1.sup.+ compared to KIR3DL1.sup.- NK cells were CD16.sup.+, which may in part explain the discrepancy between NK cell activation and target cell effects. Here, we investigated the frequency of CD16.sup.+ cells among NK cells expressing other iNKRs. We found that CD16.sup.+ 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.sup.+ NK cells poorer ADCC effectors than iKIR.sup.- NK cells. The education status of these iKIR.sup.+ populations had no effect on the frequency of CD16.sup.+ cells.

Natural Killer (NK) cells direct anti-viral responses through a process dependent on the integration of signals received from inhibitory and activating NK receptors (iNKR and aNKR). NK cells can be activated by autologous HIV-infected CD4 T cells (iCD4) to inhibit HIV replication. While iNKR and the downregulation of their HLA ligands on iCD4s have been investigated, the contribution of aNKR and their ligands on iCD4 to NK cell activation and subsequent anti-viral responses remain unclear. Additionally, previous work from our lab showed that the HLA-null cell lines 721.221 (721) and K562 activate different frequencies and functional subsets of NK cells. These cell lines do not express iNKR ligands, but their aNKR ligand profile may differ in a manner that explains the how they activate NK cell differentially. In this thesis, I will describe experiments that characterized the aNKR profile of iCD4 and HLA null cells to improve our understanding of the way in which these cells activate NK cells. Using flow cytometry, I analyzed the expression of a panel of ligands to aNKR on iCD4, K562, and 721 cells. This panel included ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, MIC-B, CD48, CD80, CD86, CD112, CD155, ICAM-1, ICAM-2, HLA-E, HLA-F, the ligands to the aNKR NKp30, NKp44, NKp46, and KIR3DS1. I also compared the expression of HLA-A2 and HLA-C in uninfected CD4 and iCD4, which are differentially impacted by HIV infection. I found that, while K562 and 721 cells shared the expression of several aNKR ligands, K562 cells were characterized by the expression of ULBP-2/5/6, ULBP-3, CD112, CD155, and the ligand to NKp30 and 721 cells were characterized by the expression of MIC-B, CD48, CD80, CD86, the ligand to NKp44, and HLA-E. The aNKR ligand profile of 721 cells is capable of interacting with a greater breadth of aNKR on NK cells than that of K562 cells, which may in part explain why 721 cells can stimulate greater frequencies of NK cells. Additionally, I found that iCD4 T cells, compared to uninfected CD4 T cells, trended towards expressing a higher frequency and intensity, of aNKR ligands. I also observed that iCD4 that maintained CD4 expression at their surface expressed greater levels of aNKR ligands than iCD4 where membrane CD4 levels were downmodulated by the HIV Nef and Vpu (iCD4-). The levels of aNKR ligand expression in iCD4- were similar to those of uninfected CD4 T cells for ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A and CD80. In contrast, the levels of aNKR ligand expression in iCD4- were below those of uninfected cells for MIC-B, CD48, CD112, CD155, and ICAM-2. These findings suggest that ligands to aNKR are transiently upregulated in T cells upon HIV infection and that this is followed by a downregulation in what is thought to be the more productively infected iCD4-. The ligands that are downregulated to levels below those in uninfected cells are of particular interest for future research, as reducing the expression of these ligands and their subsequent interactions with NK cell receptors may confer a survival advantage to HIV infected cells.