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. 2023 Jan 15;210(2):204–215. doi: 10.4049/jimmunol.2200157

FIGURE 5.

FIGURE 5.

AL008 activity depends on FcγR engagement. (A) Human monocyte-derived macrophages were treated overnight with increasing concentrations of AL008, AL008-LALAPS, or control Ab. Cell surface SIRPα was detected by flow cytometry with a fluorescent anti-human SIRPα Ab, as previously described. Relative expression levels of SIRPα were normalized to the isotype-treated cells. Graph depicts aggregated data from six different human donors; average ± SEM is shown. (B) Macrophages from two separate donors (donor 38683, left panel; donor 38680, right panel) were treated as previously described and cocultured with CFSE-labeled Raji cells opsonized with anti-CD20. ADCP of Raji cells was measured by flow cytometry gating on the percentage of CD14+/CFSE+ macrophages. A two-tailed t test was used to determine the p values. **p < 0.01, ***p < 0.001. (C) Cell surface FcγR2A was detected by flow cytometry with a fluorescent anti-human FcγR2A Ab (clone IV.3). Relative expression level of FcγR2A was normalized to the control-treated cells. Each symbol represents macrophages from a different human donor. (D) Human macrophages were preincubated with blocking Abs against FcγR2A (clone IV.3) or FcγR2B (clone 2B6) either alone or in combination. Subsequently, macrophages were treated with isotype control or AL008 and cocultured with CFSE-labeled tumor cell lines. Phagocytosis of tumor cells was measured by flow cytometry gating on the percent of CD14+/CFSE+ macrophages. Results were normalized to the isotype-treated cells. Graph depicts aggregated data from four different human donors; average ± SEM is shown. A two-tailed t test was used to determine the p values. *p < 0.05.