1 Antibodies against HIV gp120 can trigger natural killer-mediated antibody-dependent cellular cytotoxicity lysis of HIV-1-infected autologous CD4+ T cells independently of MHC-I downregulation(a) Composite data from multiple donors measuring HIV-specific lysis of IIIB, NL4-3, or 96USHIPS9 (SHIP) infected autologous CD4+ primary T cells by peripheral blood mononuclear cells (PBMCs) following CpG-ODN 2216 stimulation at a 100 : 1 effector-to-target cell ratio in a standard 4-h chromium lysis assay. or absence of IFN- prestimulation. Results Plasma from HIV-1-infected patients and monoclonal antibodies against gp120 could trigger NK-dependent ADCC lysis of viral isolates that were resistant to direct NK cell lysis following IFN- stimulation. In contrast, viral isolates that exhibited potent MHC-I downregulation capacity could be lysed by NK cells through either IFN- stimulated direct cytotoxicity or through ADCC. When utilized in combination, IFN- prestimulation significantly augmented ADCC lysis of HIV-1-infected target FTI-277 HCl cells and increased NK cell CD107a degranulation against gp120-coated ADCC targets (<0.05, =6). Conclusion HIV-1 isolates with lower MHC-I downregulation capacity are resistant to direct lysis following IFN- stimulation but retain sensitivity to ADCC. IFN- presti-mulation can significantly increase NK-mediated clearance of HIV-1-infected target cells by both ADCC and/or direct cytotoxicity depending on MHC downregulation status. target cells expressing mismatched MHC-I ligands lead to reduced NK inhibitory receptor signaling that artificially increases their target cell sensitivity to NK cell lysis. In contrast, an NK assay system represents the most physiologically relevant in-vitro model for measuring NK activity due to the complete match between MHC-I alleles on target cells and inhibitory FTI-277 HCl receptors on NK effector cells [9C11]. However, previous research has shown that HIV-1-infected autologous CD4+ primary T cells remain largely resistant to direct NK lysis due to viral strategies of immune evasion such as selective MHC-I downregulation [9,12,13]. Following the reduction of inhibitory signals, NK cells also require the engagement of activating receptors to trigger the killing Snca of susceptible target cells. Examples of activating NK cell receptors include the NKG2D receptor that recognizes stress-induced ligands, activating KIRs lacking inhibitory motifs, the natural cytotoxicity receptor family (NKp46, NKp30, and NKp44) which directly recognize viral or cellular antigens, and the Fc-III receptor (CD16) which mediates antibody-dependent cellular cytotoxicity (ADCC) [14C17]. Cytokines such as IL-2, IL-12, IL-15, IL-21, or IFN- can also augment lysis of susceptible targets cells by preactivating NK cells [18C20]. We have previously shown that NK cytotoxicity against autologous HIV-1-infected CD4+ primary T cells can be brought on by IFN- pretreatment  in a MHC-I downregulation-dependent process that requires the NK-activating receptors NKp46 and NKG2D . Here, we identified that HIV-1 isolates with lower MHC-I downregulation capacity (IIIB and NL4-3) were resistant to direct lysis following IFN- stimulation FTI-277 HCl but retained sensitivity to ADCC. We FTI-277 HCl also identified that IFN- prestimulation could further increase NK-mediated ADCC lysis of autologous HIV-1-infected CD4+ primary T cells in the presence of antibodies against the HIV-1 gp120 viral envelope protein including the broadly neutralizing antibody VRC01 or plasma from HIV-1-infected patients. Materials and methods HIV-1 contamination and gp120 coating Peripheral blood mononuclear cells (PBMCs) were isolated from 20 healthy uninfected donors according to informed consent and Institutional Review Board approval from The Wistar Institute. PBMCs were stimulated for 3 days with 10 g/ml PHA-p (Sigma Aldrich, St. Louis, Missouri, USA) and 100 IU/ml hIL-2 (PeproTech, Rocky Hill, New Jersey, USA). CD4+ primary T cells were isolated by positive selection using anti-CD4+ magnetic beads as described by the manufacturer (Miltenyi Corporation, San Diego, California, USA). For coating experiments, 1 106 uninfected CD4+ primary T cells were coated with 1 g gp120 from the HIV-1 IIIB isolate (ProSpec Protein Specialists, New Jersey, USA) for 30 min. For HIV-1 infection, 5 106 activated CD4+ T cells were spinfected with 150 ng of p24 containing supernatant of the CXCR4-tropic HIV-1 isolates IIIB, NL4C3, or 96USH-IPS9 (SHIP) as previously described . If infection levels were not above 50% at day 4 postinfection (as determined by intracellular p24 positivity through.