Use of an anti-CD16 antibody for in vivo depletion of natural killer cells in rhesus macaques

Abstract

Non-human primates serve as key animal models for a variety of viral infections. To evaluate the contribution of natural killer (NK) cells to the immune-mediated control of these viruses in macaque monkeys, we have described a method for depleting NK cells in vivo by administration of anti-human CD16 mouse monoclonal antibody. Using a fluorometric NK-cell cytotoxicity assay, we show that most NK-cell cytotoxicity in rhesus monkey peripheral blood mononuclear cells resides in the CD16(+) and/or CD159A(+) subset of lymphocytes. The anti-human CD16 antibody, 3G8, binds to subsets of rhesus monkey lymphocytes and monocytes but not to neutrophils. Intravenous administration of 10-50 mg/kg of 3G8 to normal rhesus monkeys resulted in anti-CD16 antibody persistence in the plasma for 1-3 weeks. This treatment also depleted 80-90% of CD3(-) CD159A(+) lymphocytes, putative NK cells, from blood for at least 1 week and was associated with the loss of NK-cell cytotoxicity when evaluated by in vitro assays. Using this method, transient depletion of NK cells from two rhesus monkeys chronically infected with simian immunodeficiency virus failed to cause changes in virus replication. These studies describe a non-human primate model for in vivo NK-cell depletion and suggest a limited role for cytotoxic CD16(+) NK cells in controlling AIDS virus replication during chronic infection.

Figure 1

Validation of fluorometric natural killer (NK)-cell cytotoxicity assay. (a) Calcein-loaded K562 target cells were lysed by NK-92 effector cells in a dose-dependent manner. (b) Rhesus peripheral blood mononuclear cells (PBMC) were fractioned into NK-cell-depleted or NK-cell-enriched using anti-CD16 and anti-CD159A antibodies with immunomagnetic beads. The efficiency of cell fractionation is shown. (c) Lysis of K562 target cells by unfractionated, sham-sorted or NK-cell-depleted PBMC. Depletion of cells expressing CD16 and CD159A eliminated K562 cell lytic activity. (d) Lysis of K562 cells by the NK-cell-enriched PBMC fraction. CD16⁺ and/or CD159A⁺ effector cells lysed target cells at low effector : target (E : T) ratios.

Figure 2

Anti-CD16 monoclonal antibody, 3G8, binds to a subset of rhesus lymphocytes and monocytes but not to neutrophils. Binding of 3G8 to blood leucocytes was assessed after staining with 3G8 using a whole blood lysis technique. Lymphocytes, monocytes and neutrophils were identified based on light-scatter characteristics. Antibody 3G8 bound to a subset of lymphocytes and monocytes but not to neutrophils.

Figure 3

Effect of anti-CD16 monoclonal antibody on natural killer (NK) cell number. Normal rhesus monkeys, one monkey per dose, were administered 10 mg/kg, 20 mg/kg, or 50 mg/kg of mouse monoclonal antibody, 3G8. NK-cell number, defined as CD3^– CD159A⁺, was determined in whole blood specimens. All dosages resulted in 80–90% depletion of NK cells for at least 1 week.

Figure 4

Persistence of 3G8 in plasma and emergence of anti-mouse immunoglobulin antibodies. Antibody persistence in plasma was dose-dependent. Anti-mouse immunoglobulin antibodies first appeared at approximately 1 week post-treatment irrespective, of dosage.

Figure 5

In vitro depletion of CD3^– CD159A⁺ lymphocytes after administration of 3G8 correlated with loss of K562 lytic activity. Natural killer (NK) -cell activity was measured in peripheral blood mononuclear cells (PBMC) of a rhesus monkey treated with 3G8 (20 mg/kg). Loss of NK-cell lysis of K562 cells was associated with depletion of the CD3^– CD159A⁺ subset (Fig. 3). The pretreatment value represents the mean of two assays performed during the 4 weeks before antibody treatment.

Figure 6

Natural killer (NK) cell depletion of macaques chronically infected with simian immunodeficiency virus (SIV) did not affect virus replication. (a) Change in CD3^– CD159A⁺ NK-cell number following treatment with 3G8 or control antibody. (b) Plasma virus level in chronically SIV-infected macaques during NK-cell depletion.