Defining macrophage ADCP cytotoxic capacity in vitro and in vivo. (A) Representative live-cell epifluorescent images of TAMRA-SE–labeled BMDMs (red) showing engulfed targets as voids (left), detection of voids (middle, green), and detection of macrophages by masking (right, blue). Scale bar, 25 µm. (B) BMDMs were cocultured with thymocytes (orange) or splenic B cells (blue) at E:T of 1:10 and imaged for 10 minutes before addition of indicated mAbs (10 µg/mL final). Data shown are mean ± standard error of the mean (SEM; n = 3). (C) hMDM phagocytosis of CLL cells was carried out as in panel B using 10 μg/mL of the indicated mAbs (alemtuzumab [ALM], RTX). Data shown are mean ± SEM (n = 3). (D) Representative phase (upper) and epifluorescent (lower) images of CT Deep Red-labeled hMDM (violet) engorged 2 hours with αCD52-opsonized CLL cells (scale bar, 25 µm), and TAMRA-SE–labeled BMDM (red) engorged 2 hours with αCD90.2-opsonized thymocytes (scale bars, 25 µm). (E) BMDMs were cocultured with thymocytes for 2 hours in the absence (orange) or presence (crimson) of 10 µg/mL of αCD90.2. Free thymocytes and Ab were then washed away, and BMDMs were rechallenged with fresh thymocytes and 10 µg/mL of αCD90.2 for another 2 hours. Data shown are mean ± SEM (n = 6). (F) BMDM rechallenge assay performed as in panel E using splenic B cells opsonized with αCD20. Data shown are mean ± SEM (n = 3). (G) hMDMs were cocultured with CLL cells for 4 hours in the absence (light blue) or presence (dark blue) of 10 µg/mL of RTX. Free targets and Ab were then washed away, and hMDMs were rechallenged with fresh CLL cells and 10 µg/µL of RTX for another 4 hours. Data shown are mean ± SEM (n = 5). (H) hMDM rechallenge assay performed as in panel G using CLL cells opsonized with ALM. Data shown are mean ± SEM (n = 5). (I) BMDMs, hMDMs, and C57BL/6J pMacs were induced to undergo hypophagia and rechallenged with the indicated mAbs as in panels E and F, and the phagocytic index (relative void max) for each condition is shown for 3 to 5 independent experiments per condition. Data shown are mean ± SEM (n = 3; P values derived from unpaired 2-tailed Student t test). (J) BMDMs were cocultured with thymocytes and αCD90.2 for the times indicated on the x-axis. BMDMs were washed and rechallenged with fresh αCD90.2-opsonized thymocytes for 2 hours, and the phagocytic index was calculated for 3 to 4 per condition. Data shown are mean ± SEM (P values derived from unpaired 1-way analysis of variance [ANOVA] with multiple comparisons correction). (K) BMDMs cocultured with αCD20-opsonized B cells as in panel J. Data shown are mean ± SEM (n = 4; P values derived from unpaired 1-way ANOVA with multiple comparisons correction). (L) BMDMs cocultured with αCD90.2-opsonized thymocytes for 2 hours, washed, and cultured in fresh media to recover. At the indicated times, BMDMs were rechallenged with fresh αCD90.2-opsonized thymocytes for 2 hours, and the phagocytic index was calculated. Data shown are mean ± SEM (n = 3; P values derived from unpaired 2-tailed Student t test). (M) Flow cytometric analysis of KCs isolated from hCD2-iCre-tdTomato mice treated with or without 25 µg of αCD20 IV (clone 5D2). Top plots show representative data from analysis of KC engulfment of endogenous B cells (tdTomato⁺) at 6 hours. Lower panel shows time course of KC engulfment of B cells. Data shown are mean ± SEM (n = 2-3 mice per group). (N) Schematic of in vivo ADCP rechallenge experiment using hCD2-iCre-tdTomato mice. (O) hCD2-iCre-tdTomato mice were treated IV with 25 µg of αCD20 or not. Six hours later, both groups were then treated IV with 1 × 10⁷ splenic B cells from donor C57BL/6J mice labeled with eFluor670 and opsonized with 10 µg/mL of αCD20-opsonized before injection. Data shown are representative flow plots used to measure engulfment of tdTomato⁺ and/or eFlour670⁺ target cells by KCs 1 hour after injection of donor cells, with KCs gated on CD45⁺, Tim4^hi, SSC^hi. (P) KC engulfment of donor B cells (n = 8-11 mice per treatment group). Data shown are mean ± SEM for 3 independent experiments. P values indicated on graphs derived from unpaired 2-tailed t test. *P < .05, **P < .01, ***P < .001, ****P < .0001.