Rapid Degranulation of NK Cells following Activation by HIV-Specific Antibodies

Abstract

Ab-dependent cellular cytotoxicity (ADCC) Abs stimulate NK cell effector functions and play a role in protecting from and controlling viral infections. We characterized ADCC Abs in a cross-sectional cohort of 80 HIV-infected subjects not on antiretroviral therapy. We analyzed ADCC response by killing fluorescently labeled target cells, as well as expression of IFN-γ and the degranulation marker CD107a from activated NK cells as measured by a novel intracellular cytokine assay. HIV-specific ADCC directed toward Envelope proteins were present in the majority of 80 untreated HIV-infected individuals measured by killing function. Similarly, most subjects had HIV-specific Abs that mediated degranulation or cytokine expression by NK cells. Interestingly, there was a poor correlation between ADCC-mediated killing of fluorescently labeled whole Envelope protein-pulsed cell lines and Ab-mediated expression of IFN-γ by NK cells. However, in contrast to healthy donor NK cells, autologous patient NK cells more effectively degranulated granzyme B in response to ADCC activation. Activation of NK cells in response to stimulation by HIV-specific Abs occurs at least as rapidly as activation of Gag-specific CTLs. Our studies highlight the complexity of ab-mediated NK cell activation in HIV infection, and suggest new avenues toward studying the utility of ADCC in controlling HIV infection.

Immune escape from HIV-specific antibody-dependent cellular cytotoxicity (ADCC) pressure

“…The analyses included studying both IFN-γ and CD107a expression as markers of NK cell activation (an example is shown in Fig. 2A) because these NK cell effector functions can be activated differentially (10). In addition, we studied both CD56 + CD3 − NK lymphocytes and CD3 − CD2 + NK lymphocytes because CD56 expression by NK cells can be down-regulated in subjects with HIV infection (18).…”

“…To study evolution across HIV-specific ADCC epitopes, we first mapped a large series of linear HIV-specific ADCC epitopes. We used an intracellular cytokine staining (ICS)-based ADCC assay which measures NK cell activation by ADCC antibodies in the presence of 15-mer peptide pools corresponding to whole subtype B consensus HIV-1 proteins as previously described (10,11). Patient plasma samples giving positive responses to peptide pools spanning whole proteins were then tested against subpools of 30 peptides to define the region of the epitope more narrowly.…”

“…Viremic HIV-infected adults who were ART-naïve (n = 80) or who had ART-resistant HIV (n = 12) were recruited from the Melbourne Sexual Health Centre and the Alfred Hospital (Australia) to donate blood samples (10,24). All subjects provided informed consent.…”

“…Similarly, ADCC responses to specific epitopes may be more effective in controlling HIV replication than others. We recently developed a method to detect and map linear ADCC antibody epitopes by analyzing the ability of ADCC antibodies to activate natural killer (NK) cells in the presence of overlapping HIV peptide sets (10,11). The HIV antibodyinduced expression of degranulation markers by NK cells in this assay correlates with killing of infected cells in cytotoxicity-based ADCC assays.…”

Anti-HIV Antibody–Dependent Activation of NK Cells Impairs NKp46 Expression

“…As a source of HIV-specific Abs, plasma was prepared from whole blood samples from two HIV-infected clients of the Melbourne Sexual Health Center. These two individuals previously were demonstrated to have Abs capable of robustly activating NK cells through CD16 (33,34). Individual statistical tests reported throughout the article consist of assays completed with only one of the two plasma donors.…”