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. 2020 Jun 17;136(18):2065–2079. doi: 10.1182/blood.2020005571

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Figure 3. — ADCP-induced hypophagia is associated with rapid loss and differential reexpression of surface FcγRs. (A) Surface levels of indicated FcγRs were measured by flow cytometry at various times after coculture with thymocytes ± αCD90.2 to induce hypophagia. Representative histograms (left panels) show surface levels of the FcγRs on unfed (gray) and hypophagic (color) BMDMs after 2-hour coculture with thymocytes ± αCD90.2. Unstained control shown as dashed line. Graphs to the right of each histogram show relative mean fluorescence intensity (MFI) of each FcγR at different times after feeding BMDMs with αCD90.2-opsonized thymocytes. Data shown are mean ± standard error of the mean (SEM; n = 4; P values derived from unpaired 1-way analysis of variance with multiple comparisons correction). Gray and blue bars indicate BMDM surface levels on BMDMs cocultured with thymocytes without mAb or mAb without thymocytes, respectively. (B) Flow cytometric analysis of FcγRI and FcγRIIb surface levels on hMDMs cocultured with CLL cells in the absence (gray) or presence (color) of ALM mAb for 2 hours. Unstained control shown as dashed line. Graphs below show quantification of relative MFI for each receptor. Data shown are mean ± SEM (n = 3; P values derived from unpaired 2-tailed Student t test). (C) Flow cytometric analysis of CD11b surface levels on BMDMs after 2-hour coculture with thymocytes and αCD90.2 (n = 3; P values derived from unpaired 2-tailed Student t test). (D) Flow cytometric analysis of CD11b surface levels on hMDMs after 2-hour coculture with CLL cells and ALM mAb (n = 3; P values derived from unpaired 2-tailed Student t test). (E) BMDMs were cocultured with thymocytes ± anti-CD90.2 for 2 hours to induce hypophagia; targets and free Ab were then washed away and BMDMs cultured in fresh medium for 0 to 48 hours. Surface levels of FcγRI and FcγRIIb were measured by flow cytometry at the indicated times. Unstained control shown as dashed line. Representative histograms (left) and MFI quantification of FcγRI and FcγRIIb levels relative to unfed BMDM (right) are shown. Data shown are mean ± SEM (n = 3-6; P values derived from unpaired 2-tailed Student t test). (F) Relative surface levels of FcγRI and FcγRIIb from panel E for anti-CD90.2 conditions from 0-, 24-, and 48-hour time points were compared. Data shown are mean ± SEM (n = 3-6; P values derived from unpaired 2-tailed Student t test). (G) Representative flow cytometric analysis of FcγRI and FcγRIIb surface levels on KCs isolated from hCD2-iCre-tdTomato mice treated for 6 hours with 25 µg of αCD20 IV (red) or not (blue). Unstained KCs from untreated mice are shown in gray. (H) Quantification of FcγRI and FcγRIIb levels on KCs from f (n = 3 mice per group). **P < .01, ***P < .001, ****P < .0001. ns, not significant.

Figure 3. — ADCP-induced hypophagia is associated with rapid loss and differential reexpression of surface FcγRs. (A) Surface levels of indicated FcγRs were measured by flow cytometry at various times after coculture with thymocytes ± αCD90.2 to induce hypophagia. Representative histograms (left panels) show surface levels of the FcγRs on unfed (gray) and hypophagic (color) BMDMs after 2-hour coculture with thymocytes ± αCD90.2. Unstained control shown as dashed line. Graphs to the right of each histogram show relative mean fluorescence intensity (MFI) of each FcγR at different times after feeding BMDMs with αCD90.2-opsonized thymocytes. Data shown are mean ± standard error of the mean (SEM; n = 4; P values derived from unpaired 1-way analysis of variance with multiple comparisons correction). Gray and blue bars indicate BMDM surface levels on BMDMs cocultured with thymocytes without mAb or mAb without thymocytes, respectively. (B) Flow cytometric analysis of FcγRI and FcγRIIb surface levels on hMDMs cocultured with CLL cells in the absence (gray) or presence (color) of ALM mAb for 2 hours. Unstained control shown as dashed line. Graphs below show quantification of relative MFI for each receptor. Data shown are mean ± SEM (n = 3; P values derived from unpaired 2-tailed Student t test). (C) Flow cytometric analysis of CD11b surface levels on BMDMs after 2-hour coculture with thymocytes and αCD90.2 (n = 3; P values derived from unpaired 2-tailed Student t test). (D) Flow cytometric analysis of CD11b surface levels on hMDMs after 2-hour coculture with CLL cells and ALM mAb (n = 3; P values derived from unpaired 2-tailed Student t test). (E) BMDMs were cocultured with thymocytes ± anti-CD90.2 for 2 hours to induce hypophagia; targets and free Ab were then washed away and BMDMs cultured in fresh medium for 0 to 48 hours. Surface levels of FcγRI and FcγRIIb were measured by flow cytometry at the indicated times. Unstained control shown as dashed line. Representative histograms (left) and MFI quantification of FcγRI and FcγRIIb levels relative to unfed BMDM (right) are shown. Data shown are mean ± SEM (n = 3-6; P values derived from unpaired 2-tailed Student t test). (F) Relative surface levels of FcγRI and FcγRIIb from panel E for anti-CD90.2 conditions from 0-, 24-, and 48-hour time points were compared. Data shown are mean ± SEM (n = 3-6; P values derived from unpaired 2-tailed Student t test). (G) Representative flow cytometric analysis of FcγRI and FcγRIIb surface levels on KCs isolated from hCD2-iCre-tdTomato mice treated for 6 hours with 25 µg of αCD20 IV (red) or not (blue). Unstained KCs from untreated mice are shown in gray. (H) Quantification of FcγRI and FcγRIIb levels on KCs from f (n = 3 mice per group). **P < .01, ***P < .001, ****P < .0001. ns, not significant.