Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Abstract

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.

Fig 1

Fluorometric granzyme cytotoxicity assay effectively measures serum-mediated cytotoxicity against CD4⁺ T cells. (A) Gating strategy for granzyme B-positive and infected targets. Live CD4⁺ targets (LIVE/DEAD [L/D] violet stain positive) were selected from the negatively stained NK cell population (LIVE/DEAD far red stain negative). GrB cleaved GranToxiLux substrate, indicating active GrB present within live targets. Live, CD4⁺ targets were also fixed and stained for p24 to determine the frequency of infected targets. SSC, side scatter; FSC, forward scatter. (B) Cellular GrB content. (C) Elimination of HIV-infected cells. Representative data show active GrB in live targets or p24 in CD4⁺ cells. Some infected cells show downregulation of CD4. A reduction in the number of p24-stained cells is represented as ICE and is an indication of cell death. In the example described above, ICE_{infected-NK cells-serum} = [(42.5 − 30.7)/42.5] × 100 = 27.7%. (D) A novel human anti-CD3 Ab (OKT3-huIgG1) mediates delivery of GrB by NK cells to CD4⁺ T cell-infected targets.

Fig 2

Antibodies mediate ADCC in a dose-dependent manner. GrB delivery (A) and ICE (B) by serum at serial 5-fold dilutions starting at 1:100 for sera from 10 HIV⁺ patients, Bpool serum, and normal serum.

Fig 3

ADCC activity of sera does not correlate with clinical status, CD4⁺ T cell count, viral load, or NAb titers. The ADCC activity of sera from 20 HIV⁺ patients diluted 100-fold was compared by patient status for GrB activity (A) and ICE (B). ICE values for individual patients were also compared to CD4⁺ T cell count (number of cells/μl) (C), viral load (number of copies/ml plasma) (D), NAb titers (50% inhibitory dose [ID₅₀]) against HIV_SF162 (E), and the geometric mean of NAb titers (50% inhibitory dose) against 20 tier 2 primary isolates (F). Orange, LTNPs; black, progressors.

Fig 4

Serum exhibits breadth in mediating ADCC. Bpool serum mediates ADCC against clade B (SF162, THRO, YU2, 96ZM), clade C (CH162, CH067), and clade A/E (C1081, CM235) viruses. MLV is the pSG3^Δenv backbone with MLV env.

Fig 5

Serum-mediated ADCC is attributable to IgG; single anti-Env MAbs mediate only modest ADCC. (A) Standard neutralizing Abs were titrated in parallel with each other and Bpool serum IgG. (B) Anti-gp120 and gp41 NAbs isolated from two patients (patients 4 and 6), a combination of the four patient 4 MAbs, and purified patient 4 IgG were titrated in parallel.

Fig 6

ADCC correlates with surface-bound IgG. (A) Gating strategy to determine surface IgG binding. MFI of anti-IgG (B) and anti-IgG1 (C) bound to serum-coated infected cells; MFI of anti-IgG (D) and anti-IgG1 (E) bound to MAbs whose titers on infected cells were determined (D).

Fig 7

Correlation of ADCC with binding of serum IgG (A) or IgG1 (B) to infected targets. Cells were incubated with patient sera diluted 1:2,500.