Inhibition of cell death and reduced plasma vRNA after treatment with anti-FasL. Macaque PBMC specimens were collected 1 week before (−1) and at regular intervals after SIVmac239 infection (that occurred at week 0). The animals were treated with RNOK203 or a control IgG1 (Sigma #I-3889). Panels (a)–(c) show the proportion of dying (positive for 7-AAD staining) lymphocytes in the CD4+, CD8+, or CD20+ subsets. Open symbols represent the average values among four macaques treated with control IgG. Closed symbols represent the average values among four macaques treated with anti-FasL. Error bars show the standard error of the mean for each time point. The absolute cell counts for panels (a)–(c) are summarized in Table 2. Panel D shows the plasma SIVmac vRNA levels for each animal determined by b-DNA assays (conducted by Bayer Diagnostics, Emeryville, CA). The two animals with lowest set-point RNA were also the two that survived longest (Figure 4). Open symbols represent control animals and closed symbols represent macaques treated with anti-FasL. The limit of detection for this assay was 10³vRNA copies per mL of plasma.

Anti-FasL treatment prolongs survival in SIVmac-infected macaques. The fraction of surviving animals is plotted for up to 102 weeks after SIVmac239 infection. The thin line shows the fraction of animals surviving after control IgG treatment and SIV infection. All control animals died by 52 weeks after SIV infection. The thick line shows the effect of anti-FasL treatment (anti-FasL) in extending survival times. None of the anti-FasL-treated animals had died before 60 weeks after SIV infection, and the last surviving animal lived until 102 weeks. The two longest-lived animals also had lowest viral loads (Figure 2(d)).

The anti-FasL monoclonal antibody blocks MHC-unrestricted cytolysis and stains FasL on rhesus PBMC. (Panel A) Anti-FasL, the humanized NOK2 monoclonal (RNOK203), blocked MHC-unrestricted cytolysis in vitro using rhesus or human PBMC as effectors and human or rhesus B-LCL targets that expressed SIV envelope glycoprotein (transfected with pCDNA3/SIVenv). The four-hour chromium-release assay was done in the presence of 1 mM EGTA and varying amounts of anti-FasL at an ratio of as described [23]. Specific lysis was blocked completely at 20 g/mL of monoclonal antibody (shown) and not with a similar concentration of isotype control antibody (not shown). When no effector cells were added (No E), there was no specific lysis showing that 20 g/mL anti-FasL does not induce cell death on its own. These experiments were done 3 times with rhesus PBMC, 3 times with human PBMC, and 2-3 times cross species with the same results. The error bars represent the variation in 3 different experiments using rhesus cells. (Panel B) MHC-unrestricted cytolytic activity correlated with the mean fluorescence intensity (MFI) of cell surface FasL on circulating PBMC. PBMC specimensfrom rhesus macaques were screened by the MHC-unrestricted cytolysis assay as described [23]. Results from 10 rhesus macaques are shown here. Macaques were divided into high (≥15% specific lysis) and low (<10% specific lysis) groups, and stained with the anti-FasL antibody in an indirect immunofluorescence assay. The individual points, mean values, and standard deviations are shown for animals classified as having low or high cytolytic activity. Macaques with high levels of MHC-unrestricted cytolytic activity and correspondingly high levels of cell surface FasL were selected for this study.

Cellular and humoral immune responses to SIV are enhanced by anti-FasL treatment. MHC-restricted CTL to SIV p27 Gag protein (Panel A) were measured in a standard 4 hours. ⁵¹Cr-release assay [26]. Targets were syngeneic B-LCL (different lines for each animal) that were either uninfected or infected with VVgag as described [26]. Percent specific lysis at the 501 effector to target ratio was plotted. The mean and standard errors of the mean (SEM) are shown for four animals treated with anti-FasL (shaded bars) or four control macaques (solid bars) at the times indicated. The values for CTL activity were consistently higher among macaques treated with anti-FasL compared with controls, but the differences were not significant at 30 weeks after infection. Virus binding titers (Panel B) were measured by ELISA using plates coated with p27 Gag antigen as described [27]. Sera from weeks 12 (shaded bars) or 24 (solid bars) were diluted with normal saline, up to a maximum dilution of 1100,000. Macaques (A, B, C, D) on the left side of the figure had been treated with anti-FasL, and macaques (E, F, G, H) on the right side had been treated with control IgG. Anti-FasL treatment caused a substantial elevation in virus-binding antibodies in SIV-infected macaques, which have been shown to correlate with reduced disease progression [28].