Figure 4
Figure 4. Type I mAbs modulate CD20 from the cell surface through internalization, leading to mAb and CD20 degradation. (A) hCD20 Tg B cells were incubated in vitro with Rit m2a–Alexa 488 (Rit-488) or Tosit–Alexa 488 (Tosit-488) mAbs (5 μg/mL) for 1, 2, or 6 hours, washed, and then incubated in the presence or absence of anti–Alexa 488 quenching Ab. The fluorescence remaining after quenching indicates the proportion of internalized mAbs (histogram top panel). Bars represent mean with range for duplicate determinations, one of 3 similar experiments. (B) hCD20 Tg × γ−/− mice were administered Rit-488 or Tosit-488 (100 μg) intravenously, and the amount of fluorescence associated with splenic B cells was assessed 24 hours later (top panel), Rit-488-treated B cells accumulated approximately 4 times the fluorescence compared with that of Tosit-488-treated mice (n = 3 mice). Despite the Rit m2a-treated B cells having a higher level of B cell–associated fluorescence, the Tosit-treated B cells (blue) expressed more CD20 on their surface than Rit-488–treated cells (red; bottom panel), histograms from one of 3 similar experiments. (C) Left panel: In vivo treatment: 10 μm spleen sections from mice treated, as in panel B, were analyzed by confocal microscopy. Assessed at the same gain intensity, far greater mAb accumulation is evident after Rit m2a (top panels), with its more punctate staining apparent when Rit m2a and Tosit were compared at optimized gain intensity (bottom panels). Right panel, in vitro treatment: hCD20 Tg B cells treated with Rit-488 (5 μg/mL) in vitro and assessed by confocal microscopy with a maximal projection, Z section through the center of the cell, and overlaid versus the bright field (Bf). In both panels an HXC PL APO CS 100×/1.4 oil immersion lens, 2× optical zoom was used. (D) Internalization of Rit-488 or Tosit-488 on hCD20 Tg B cells under normal conditions (NT), at 4°C, in the presence of azide (Azd, 15mM) or latrunculin B (Lat B, 50μM). Bars represent mean with range for duplicate determinations, one of 3 similar experiments. (E) hCD20 Tg B cells treated with Rit-488 or Tosit-488 for 6 hours and assessed by Western blot for the expression of intact Alexa 488–IgG, CD20, and actin (as a loading control). (F) mRNA levels of CD20 and GAPDH (as a control) were assessed in the same cells.

Type I mAbs modulate CD20 from the cell surface through internalization, leading to mAb and CD20 degradation. (A) hCD20 Tg B cells were incubated in vitro with Rit m2a–Alexa 488 (Rit-488) or Tosit–Alexa 488 (Tosit-488) mAbs (5 μg/mL) for 1, 2, or 6 hours, washed, and then incubated in the presence or absence of anti–Alexa 488 quenching Ab. The fluorescence remaining after quenching indicates the proportion of internalized mAbs (histogram top panel). Bars represent mean with range for duplicate determinations, one of 3 similar experiments. (B) hCD20 Tg × γ−/− mice were administered Rit-488 or Tosit-488 (100 μg) intravenously, and the amount of fluorescence associated with splenic B cells was assessed 24 hours later (top panel), Rit-488-treated B cells accumulated approximately 4 times the fluorescence compared with that of Tosit-488-treated mice (n = 3 mice). Despite the Rit m2a-treated B cells having a higher level of B cell–associated fluorescence, the Tosit-treated B cells (blue) expressed more CD20 on their surface than Rit-488–treated cells (red; bottom panel), histograms from one of 3 similar experiments. (C) Left panel: In vivo treatment: 10 μm spleen sections from mice treated, as in panel B, were analyzed by confocal microscopy. Assessed at the same gain intensity, far greater mAb accumulation is evident after Rit m2a (top panels), with its more punctate staining apparent when Rit m2a and Tosit were compared at optimized gain intensity (bottom panels). Right panel, in vitro treatment: hCD20 Tg B cells treated with Rit-488 (5 μg/mL) in vitro and assessed by confocal microscopy with a maximal projection, Z section through the center of the cell, and overlaid versus the bright field (Bf). In both panels an HXC PL APO CS 100×/1.4 oil immersion lens, 2× optical zoom was used. (D) Internalization of Rit-488 or Tosit-488 on hCD20 Tg B cells under normal conditions (NT), at 4°C, in the presence of azide (Azd, 15mM) or latrunculin B (Lat B, 50μM). Bars represent mean with range for duplicate determinations, one of 3 similar experiments. (E) hCD20 Tg B cells treated with Rit-488 or Tosit-488 for 6 hours and assessed by Western blot for the expression of intact Alexa 488–IgG, CD20, and actin (as a loading control). (F) mRNA levels of CD20 and GAPDH (as a control) were assessed in the same cells.

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