While investigators agree on the potential importance of ADCC as a mechanism for protection against a virus (3
), there is no generally accepted, standardized assay to measure ADCC. Assays for measuring HIV-1-specific ADCC must have three components: target cells that express HIV-1 antigens, effector cells such as natural killer (NK) cells, and a source of antibody such as serum, plasma, or monoclonal antibodies. The traditional radioactive chromium release assay is relatively insensitive and labor-intensive (1
). Gomez-Roman and colleagues have recently described a fluorescent killing assay that they term a “rapid fluorescent ADCC (RFADCC) assay” (9
). This assay measures a fluorescent dye that target cells release when they die. Other groups have explored intracellular cytokine staining-based assays for evaluating NK cell expression of effector molecules following activation by HIV-1 antigens (Ags) and anti-HIV-1 Abs (16
). It is not clear whether these different ADCC assays are comparable in what they measure.
The generally accepted target cells for HIV-1 ADCC assays are either virus-infected, mitogen-stimulated human peripheral blood mononuclear cells (PBMC) (18
) or CD4+
T cell lines that are resistant to lysis by NK cells in the absence of antibody (26
). The major limitation of the PBMC assay is donor-to-donor variability in the kinetics of HIV-1 replication in vitro
. An immortalized cell line is easier to work with and less expensive than PBMC, and its use allows for standardization of assays. CEM-NKr-CCR5 is an NK-resistant CD4+
T cell line that stably expresses CCR5 (26
). In the current study, we compared NK cell-mediated ADCC responses using 3 different target cells, including SIVmac251 gp130-coated CEM-NKr-CCR5 target cells, VSVg Env-pseudotyped SIVmac239-infected CEM-NKr-CCR5 target cells, or SIVmac251-infected CEM-NKr-CCR5 target cells. Peripheral blood mononuclear cells (PBMC) from uninfected donors that were enriched for NK cells were used as effector cells. The flow cytometry-based assay allowed us to gate on CD3−
cells in characterizing the function of NK cells following activation. The results of the present study show that, in contrast to neutralizing antibody activity, antibody-mediated killing of the SIV-infected cells in the presence of NK effector cells is detectable in monkeys as early as 3 weeks after SIVmac251 infection (2
). This finding suggests that HIV-1-specific antibody may play a role in the control of viremia during acute infection.
We have shown that SIV Env gp130-coated target cells are more sensitive than VSVg Env-pseudotyped SIVmac239-infected or SIVmac251-infected target cells for detection of ADCC. This can be explained, at least in part, by the percentage of target cells that are expressing SIV antigen. Target cells prepared by SIV Env gp130 coating have saturating amounts of gp130 that bind stably to target cells, with little shedding of antigen over a 6-hour time course—an appropriate length of time for a flow-based ADCC assay. This approach permits the analysis of ADCC activity directed solely against determinants of the gp130 molecule. VSVg-pseudotyped SIVmac239 infection results in a high percentage of infected target cells, as measured by staining target cells with plasma obtained from an SIVmac251-infected rhesus monkey. SIVmac251 infection resulted in the infection of fewer than 40% of target cells. ADCC activity was detected in plasma sampled from monkeys during acute viral infection, and lower-level responses were measured during chronic infection using these target cells. Therefore, SIV Env gp130-coated target cells greatly facilitate analyses of ADCC activity with a high degree of sensitivity and reproducibility. However, a recent study demonstrated that substantial ADCC activity against the Pol and Vpu proteins can be detected in the sera of HIV-infected individuals (24
). Therefore, further study of ADCC responses using Pol or Vpu protein-pulsed cell lines would better define the ADCC activity in the plasma specimens of SIVmac251-infected rhesus monkeys. Further, the use of primary cells rather than an immortalized cell line might provide useful data in an ADCC assay since cell lines and primary cells can differ in their expression of receptors and antigen-presenting machinery.
Since ADCC results in the lysis of target cells, we used CD107a as an indictor of the biological activity of the NK cells in the ADCC assays. Although much of the antiviral activity mediated by plasma antibody from infected animals is probably due to the death of the virus-infected cells, it is likely that noncytolytic mechanisms, such as the secretion of soluble molecules by activated NK cells, also play a role in reducing virus replication. HIV-1-specific antibody, in the presence of envelope-expressing target cells, augments chemokine and cytokine release from NK cells (5
). Thus, it is likely that cytokines, including IFN-γ and TNF-α, and the chemokine MIP-1β released from NK cells after Fc receptor stimulation were responsible for some of the antiviral activity detected in the ADCC assay. However, the role of cytokine release relative to cytotoxicity in inhibiting virus replication was not ascertained. In the current study, using polychromatic flow cytometry, we were able to simultaneously assess Ab-stimulated NK cell production of IFN-γ, TNF-α, MIP-1β, and CD107a. The results demonstrated a very strong positive correlation between the frequency of NK cells expressing CD107a and NK cell production of other molecules, including the activation-associated molecule CD69, cytokines IFN-γ and TNF-α, and the chemokine MIP-1β. A strong negative correlation was also detected between the proportion of NK cells producing CD107a and the downregulation of CD16. These results suggest that NK cells are able to recognize Ab bound to target cells through FcγRIIIa (CD16) that is expressed on 80 to 90% of peripheral blood NK cells. Following activation, NK cells enter a refractory period during which CD16 molecules are shed from their surfaces (10
). This loss of CD16 is thought to prevent chronic stimulation of NK cells and activation-induced cell death. The activated NK cells then produce a variety of functional molecules, including CD107a, IFN-γ, TNF-α, MIP-1β, and CD69. The strong positive correlation between Env-specific ADCC-triggered degranulation and the expression of cytokines and chemokines by activated NK cells suggests that measuring a single molecule expressed by effector NK cells, such as CD107a, is sufficient to evaluate the anti-SIV function of NK cells in the context of ADCC.
ADCC, like cytotoxic T lymphocyte (CTL) activity, mediates the death of infected cells in vitro. It is therefore plausible that ADCC may play a role in vivo in controlling SIV replication during primary infection. In the current study, we found that in contrast to neutralizing activity, antibody directed against infected cells and capable of inhibiting SIV replication in the presence of NK effector cells is detectable in infected monkeys as early as 3 weeks after infection. Furthermore, we found that the magnitude of the antiviral antibody response is inversely associated with plasma SIV RNA levels in monkeys with moderate to high levels of viral replication. These findings suggest that SIV-specific antibody could play a role in the control of viremia during SIVmac251 infection. Interestingly, relative low ADCC activity was observed in monkey A3V005, the animal with the lowest plasma viral RNA levels at day 100 after viral infection. This result is consistent with the possibility that certain levels of antigen may be needed to maintain high ADCC activity after viral infection.
We also evaluated the association between the development of SIVmac251 gp140 binding antibody and the NK cell-mediated ADCC responses in SIV-infected monkeys. We found that binding antibodies to gp140 as measured by ELISA were detectable at 4 weeks after viral infection, coincident with the development of measurable ADCC activity. In addition, the magnitude of this antibody response was associated with the level of ADCC activity. These results are consistent with the possibility that the antibodies that mediated ADCC are a cohort of the antibodies detected in the gp140 protein binding assay, and the titers of these antibodies reflect the immune competence of the infected monkeys.