Simian immunodeficiency virus (SIV)/immunoglobulin G immune complexes in SIV-infected macaques block detection of CD16 but not cytolytic activity of natural killer cells

Abstract

Natural killer cells are components of the innate immune system that play an important role in eliminating viruses and malignant cells. Using simian immunodeficiency virus (SIV) infection of macaques as a model, flow cytometry revealed a gradual loss of CD16+ NK cell numbers that was associated with disease progression. Of note, the apparent loss of NK cells was detected in whole-blood samples but not in isolated peripheral blood mononuclear cells (PBMC), suggesting that an inhibitor(s) of the antibody used to detect CD16, the low-affinity immunoglobulin G (IgG) receptor, was present in blood but was removed during PBMC isolation. (Actual decreases in CD16+ cell numbers in PBMC generally were not detected until animals became lymphopenic.) The putative decrease in CD16+ cell numbers in whole blood correlated with increasing SIV-specific antibody titers and levels of plasma virion RNA. With the addition of increasing amounts of plasma from progressor, but not nonprogressor, macaques to PBMC from an uninfected animal, the apparent percentage of CD16+ cells and the mean fluorescence intensity of antibodies binding to CD16 declined proportionately. A similar decrease was observed with the addition of monomeric IgG (mIgG) and IgG immune complexes (IgG-ICs) purified from the inhibitory plasma samples; some of the ICs contained SIV p27(gag) antigen and/or virions. Of interest, addition of purified IgG/IgG-ICs to NK cell lytic assays did not inhibit killing of K562 cells. These results indicate that during progressive SIV and, by inference, human immunodeficiency virus disease, CD16+ NK cell numbers can be underestimated, or the cells not detected at all, when one is using a whole-blood fluorescence-activated cell sorter assay and a fluorochrome-labeled antibody that can be blocked by mIgG or IgG-ICs. Although this blocking had no apparent effect on NK cell activity in vitro, the in vivo effects are unknown.

FIG. 1.

Changes in percentages of CD16⁺ cells in whole blood from macaques inoculated with SIVmac239 (A) or SIVmac239-Y/I (B). Circles or ovals indicate the intervals during which the CD16 MFI reached levels consistent with those of CD16⁻ cell populations. †, euthanasia as a result of SIV-induced disease.

FIG. 2.

Analysis of CD16⁺ cells in the lymphocyte gates of whole blood collected from macaque 99P041. The results are representative of those obtained from analyses of blood from all six animals. The times after infection when each FACScan profile was obtained are shown above each panel. The numbers in the upper left quadrants are percentages of CD16⁺ CD3⁻ NK cells and the MFI for CD16 staining in each quadrant. For FACScan profiles shown in all figures, MFIs are not provided if the number of events in a quadrant were too few and scattered, leading to aberrant MFIs.

FIG. 3.

Detection of CD16⁺ CD3⁻, CD8α⁺ CD3⁻, and CD7⁺ CD3⁻ cell populations in whole blood collected from macaque 99P006 (35 weeks). The putative percentages of NK cells with the indicated immunophenotypes are shown in the upper left quadrant of each FACScan plot. The CD3⁺ populations in the upper and lower right quadrants are T cells.

FIG. 4.

Comparison of CD16⁺ cell populations detected in whole blood and PBMC obtained from macaques 99P040 and 99P041 at 35 weeks after infection. Numbers in upper left and right quadrants indicate CD16 percentages and MFIs for cells in the two quadrants.

FIG. 5.

Expression of CD16 in NK cells enriched from PBMC of macaques with different percentages of CD16⁺ cells detectable in whole blood. The ratio of CD16 to β-actin was determined using areas under the curves of the respective bands, measured with an AlphaImager. The relative intensity or normalized ratio (CD16:β-actin ratio) for each animal was determined by dividing each ratio by the percentage of CD16⁺ cells in the enriched PBMC fractions and multiplying by 100.

FIG. 6.

Plasma inhibition of recognition and binding of MAb 3G8 to CD16. PBMC from macaques were incubated with increasing amounts of plasma from macaque 99P006 or 98P016, and the residual percentages of CD16⁺ cells (A) and the respective MFIs of CD16 (B) were evaluated. The data points represent mean values for plasma samples from each animal; plasma collected from 99P006 and 98P016 at 39 and 47 weeks after infection, respectively, was tested with PBMC from three different macaques. Plasma samples collected from 99P006 before infection were tested on two occasions with PBMC from three different macaques (n = 6).

FIG. 7.

Correlation between apparent absolute numbers of CD16⁺ cells in whole blood and either plasma viral RNA levels (A) or SIV antibody titers (B). Data obtained between 4 and 94 weeks for the three SIVmac239-infected animals and between 8 and 94 weeks (inclusive) for the three SIVmac239-Y/I-infected animals were combined for both analyses (Spearman correlation).

FIG. 8.

Inhibition of binding of MAb 3G8 to CD16 on the cell surface by purified IgG and IgG-ICs. Values shown are percentages of the value for the control (100%) to which no IgG was added. Total IgG and IgG-ICs were purified from plasma samples either from progressors (99P006 and 99P051), from a nonprogressor (98P016), or from 99P040 before infection (IgG040/0). These IgG preparations, designated by the last three digits of the animal identification number and the number of weeks after infection (e.g., IgG006/47), were tested by using PBMC from uninfected macaques and evaluating the percentages of CD16⁺ cells after incubation with different amounts of the purified IgG/IgG-ICs. The numbers of independent experiments performed using the various IgG/IgG-ICs are given below the plasma designations; the value for the percentage of CD16⁺ cells is the mean of values from all experiments with a particular IgG/IgG-IC preparation. For some IgG preparations, not all concentrations were tested the same number of times. Omission of a bar at a particular IgG concentration indicates that the concentration was not tested (not determined [ND]).