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-gamma 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-gamma 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.
Highlights
Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publicatio
We evaluated a rapid fluorescent ADCC” (RFADCC) assay in comparison to a novel intracellular
In the case of the RFADCC assay, when HIVϩ serum is cultured with healthy donor PBMC and a trimeric Env-protein derived from the HIV1NL4.3 strain pulsed onto the fluorescent CEM.NKr target cell line, there was a clear population of HIV-1 Env-pulsed CEM.NKr cells that still retained the membrane dye PKH26 but had lost intracellular CFSE, indicating target cell lysis (Fig. 1A)
Summary
HIV-infected adults (n ϭ 80) not on antiretroviral therapy were recruited to donate blood samples. Healthy donor PBMCs and plasma from the HIV-infected subjects were added to the labeled CEM-NKr cells for 6 h. Fluorescent Abs used in the ICS assays were CD3 (catalog number 347344, fluorescent label PerCP), CD2 (556611, FITC), CD56 (555516, PE), CD8␣ (335787 PE-Cy7), CD107a (624078, allophycocyanin), IFN-␥ (557995, Alexa700) (all from BD Biosciences) and granzyme B (3485-7, FITC; Mabtech) These assays were conducted using patient plasma and healthy donor PBMCs. To study the kinetics of the responses, we stopped the assay after varying intervals between 0 and 7 h, as previously described for T cell assays [18]. To analyze differences between autologous blood cells and donor blood cells, we incubated 50 l of plasma from the HIV-infected subjects with 150 l of fresh whole blood from healthy HIV-uninfected donors This ratio of plasma to donor blood cells was optimal for activation of NK cells and equivalent to using larger volumes of plasma on donor PBMC (not shown). To compare ADCC responses to different Ags and with different assays across the cohort we used double-sided non-parametric Mann-Whitney U tests and Fisher’s exact test for binary data
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
