The mechanism of anti-CD20-mediated B cell depletion revealed by intravital imaging

Abstract

Anti-CD20 Ab therapy has proven successful for treating B cell malignancies and a number of autoimmune diseases. However, how anti-CD20 Abs operate in vivo to mediate B cell depletion is not fully understood. In particular, the anatomical location, the type of effector cells, and the mechanism underlying anti-CD20 therapy remain uncertain. Here, we found that the liver is a major site for B cell depletion and that recirculation accounts for the decrease in B cell numbers observed in secondary lymphoid organs. Using intravital imaging, we established that, upon anti-CD20 treatment, Kupffer cells (KCs) mediate the abrupt arrest and subsequent engulfment of B cells circulating in the liver sinusoids. KCs were also effective in depleting malignant B cells in a model of spontaneous lymphoma. Our results identify Ab-dependent cellular phagocytosis by KCs as a primary mechanism of anti-CD20 therapy and provide an experimental framework for optimizing the efficacy of therapeutic Abs.

Figure 1. The liver is a major site for anti-CD20–mediated B cell depletion.

(A) C57BL/6 mice were treated with a single injection of anti-CD20. The percentage of remaining B cells (compared with that in untreated mice) was measured over time in the indicated organs. (B) Mice were subjected to partial hepatectomy, splenectomy, or left untreated. The efficacy of B cell depletion was calculated as the fraction of B cells remaining in the blood 16 hours after anti-CD20 injection relative to the same values in mice subjected to surgery but not injected with anti-CD20. *P < 0.05. (C) Recipient Rag2^–/– mice were transferred with a 1:1 mixture of untreated and PTX-treated splenocytes and injected with anti-CD20. For each subset, the percentage of B cells at 6 hours after anti-CD20 injection (compared with that in recipients that did not receive anti-CD20) is shown for the indicated organs. (D and E) B cells circulating in the liver arrest immediately following anti-CD20 administration. B cells are indicated by white circles. Mice with RFP-expressing B cells were subjected to intravital imaging of the liver. (D) Representative time-lapse images and (E) quantification before or immediately after injection of anti-CD20 Ab. Each horizontal line represents an individual B cell, and red squares represent the time period during which the B cell is visible in the imaging field. Scale bar: 10 μm.

Figure 2. Tracking KCs using the MAFIA mouse model.

(A) Mice were injected intravenously with clodronate (lip-CL) or control liposomes (lip-PBS). After 24 hours, depletion of KCs (F4/80⁺CD11b^lo) by lip-CL was confirmed by flow cytometry. Numbers in A and D correspond to the percentage of cells falling in the indicated gate. (B) B cell depletion following anti-CD20 injection was quantified as in Figure 1 in the blood in mice treated with clodronate or control liposomes. ***P < 0.001. (C) Flow cytometric analysis of liver cells in MAFIA mice. Data are gated on CD45⁺ cells. Red and black histograms were obtained using the corresponding gates shown in the leftmost panel. (D) MAFIA mice were injected intravenously with clodronate or control liposomes. After 24 hours, liver cells were analyzed by flow cytometry. (E) Sessile GFP⁺ cells in the livers of MAFIA mice correspond to KCs. MAFIA mice were injected intravenously with PE-conjugated anti-F4/80 Ab and subjected to intravital imaging of the liver 10 minutes later. Time-lapse images showing that sessile spindle-shaped GFP⁺ cells with typical KC morphology were all stained with F4/80 Ab. In contrast, motile GFP⁺ cells (indicated by white arrowheads) were all F4/80^–. Scale bar: 10 μm.

Figure 3. KCs mediate the arrest and engulfment of normal and malignant B cells during anti-CD20 therapy.

(A–D) Rag2^–/– MAFIA mice were adoptively transferred with dye-labeled B cells and subjected to intravital imaging of the liver. (A) Representative time-lapse images recorded before or immediately after anti-CD20 injection. B cells are indicated by white circles. (B) B cell behavior and contact with KCs before or after anti-CD20 injection. Each line represents an individual B cell, red squares represent the time period during which B cells are present in the imaging field without contacting KCs, and green squares indicate the time period during which B cells are associated with a KC. (C) Impaired B cell arrest in FcRγ^–/– mice treated with anti-CD20. Rag2^–/–FcRγ^–/– mice were transferred with dye-labeled B cells and injected with anti-CD20. (D) Rag2^–/– MAFIA mice were injected with a YFP-expressing B cell tumor line. Representative time-lapse images show that tumor cells were rapidly engulfed by KCs upon anti-CD20 injection. Engulfed B cell tumors are indicated by white circles. (E and F) KCs are effectors of anti-CD20 therapy in a model of spontaneously developing B cell lymphoma. Csfr1^gfp/+Cd19^cre/+Rosa26^RFP/+Eμ-myc^+/– mice were subjected to intravital imaging of the liver following anti-CD20 injection. (E) Representative time-lapse images showing the engulfment and subsequent digestion of RFP-expressing lymphoma cells by KCs. (F) Quantification of B cell engulfment by KCs following anti-CD20 therapy. Scale bar: 10 μm.