Rituximab capping triggers intracellular reorganization of B cells

The antibody rituximab, which binds to the protein CD20 on the surface of B-cells, has been used to treat B-cell malignancies for several years. However, the molecular mechanisms underlying this treatment are not yet fully understood. One well established rituximab-induced mechanism, natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), has recently been described to involve the polarisation of bound rituximab and CD20 to one side of the B-cell. B-cells polarised this way were cleared more efficiently by NK-cells, which led us to further investigate the cellular events involved in the polarisation process. Using optical microscopy on rituximabtreated cells, we have found that the rituximab/CD20-rich, polarised side accumulated mitochondria and actin, whereas the nucleus was reorganised to the opposite side of the cell. Depleting actin via different methods correlated with a decrease in rituximab, mitochondria, and nucleus polarisation, suggesting polarisation to be actin-dependent, active process that triggers intracellular rearrangement. The influence of these intracellular rearrangements on the efficiency of NK-cell-mediated clearance of B-cell malignancies remains open for future investigation. Introduction Rituximab was the first Food and Drug administration (FDA) approved monoclonal antibody for use in cancer therapy and is now used to treat some non-Hodgkin lymphomas and rheumatoid arthritis. Further off-label use in systemic lupus erythematosus, multiple sclerosis, autoimmune haemolytic anaemia, and graft versus host disease exemplifies the importance of rituximab in current medicine [1]. Despite its long-standing use, its mechanism of action is not fully understood. Rituximab is binding to the protein CD20 on the surface of B-cells, and this seems to induce a combination of complementdependent cellular cytotoxicity (CDCC), “direct signalling” induced apoptosis, and antibodydependent cellular cytotoxicity (ADCC) to deplete malignant or autoreactive B-cells [2]. Recent research into the molecular basis of one of these mechanisms, ADCC, found that rituximab and CD20 polarised to one side of the target B-cell upon rituximab binding [3]. In contrast to other forms of antibody capping, this effect was found to be crosslinking and Fc-receptor-independent [3]. Interestingly, malignant B-cells polarised in this way were more likely to be cleared by NK cells than B-cells with homogeneously bound rituximab [3]. Objective Building on these findings, we set out to further investigate cellular changes upon rituximab polarisation since further understanding the underlying processes will ultimately allow harnessing this polarisation effect to increase rituximab treatment efficiency or to screen other monoclonal antibodies for triggering ADCC in various situations. We here aim to elucidate: (1) organelle repositioning upon rituximab-induced polarisation, and (2) involvement of the cortical actin cytoskeleton in rituximab-induced polarisation. Rituximab capping triggers intracellular reorganisation of B cells DOI: 10.19185/matters.201612000001 Matters (ISSN: 2297-8240) | 2 a Figure Legend Figure 1. (A) Representative confocal image of Raji-B-cells treated with Alexa Fluor-647-labelled rituximab (Rtx-Alexa647, red) highlighting rituximab-induced polarisation. (B)Nucleus repositioning is dependent on rituximab polarisation, not binding. Percentage of cells showing the polarisation of nucleus, as determined from dual-color confocal images of Rtx-Alexa647 and nucleus stain (NucBlue) in live Raji cells: asymmetric nucleus repositioning after Rtx-Alexa647 treatment, no nucleus repositioning after Rtx-Alexa647 treatment, and asymmetric nucleus repositioning without Rtx-Alexa647 treatment. n is number of total cells. All shown data represents pooling of at least 3 repeats. (C) Side view of a representative 3D confocal image of a Raji cell with polarised RtxAlexa647 (red), similar polarisation of mitochondria (labelled via MitoTracker Orange, yellow) and asymmetric polarisation of nucleus (NucBlue stain, blue). (D) Representative snapshots of the same kind of images of panel C for 1 h time-lapse recording. Top row shows the uniform Rtx-Alexa647 binding and symmetrically distributed nucleus and mitochondria, representative of the first fewminutes after addition of the antibody. Middle row shows gradual polarisation Rtx-Alexa647, mitochondria and nucleus, taking place at around 40 min. Bottom row shows complete Rtx-Alexa647 polarisation with reorganised mitochondria and nucleus at around 1 h after adding RtxAlexa647. (E)Actin disruption leads to decreased rituximab binding. Shown are absolute values of fluorescence intensity for surface-bound, non-polarised Rtx-Alexa647 after Latrunculin B or Cytochalasin D-mediated actin disruption in Raji cells, compared to cells with intact actin (untreated). Fluorescence intensity values (arbitrary unit) were taken from confocal microscopy images and correlate with surface concentration of bound Rtx-Alexa647. Rituximab capping triggers intracellular reorganisation of B cells DOI: 10.19185/matters.201612000001 Matters (ISSN: 2297-8240) | 3 (F) Actin disruption decreases rituximab polarisation. Shown is the likelihood of rituximab polarisation upon rituximab binding. This decreases as actin is disturbed by LatB or CytD. Raji cells were pre-incubated with LatB or CytD for 30 min before adding Rtx-Alexa647. Data determined from confocal microscopy images. (G)Representative confocalmicroscopic images of giant plasmamembrane vesicles (GPMVs) from Raji cells treated with Rtx-Alexa647, showing no rituximab induced polarisation. GPMVs have no intact actin cytoskeleton. (H) Representative dual-color confocal image of Rtx-Alexa647 (red) and PIP2 (green, labelled via PH-PLC-GFP) in live Raji cell, showing that PIP2 co-localises with RtxAlexa647. PIP2 plays a role in actin polymerisation and tethering and is usually distributed evenly in the plasma membrane. (Scale bars are 20 μm. Error bars are standard deviation of the mean. Statistical significance is unpaired t-test.) Rituximab labelling 100 μg rituximab (Invivogen, anti-hCD20-hIgG1) was labelled using Alexa Fluor® 647 Monoclonal Antibody Labelling Kit (Invitrogen) following the protocol provided. After that, labelled rituximab was added to Raji cells (ATCC CCL-86) and Jurkat cells separately to test for CD20 specificity. Different concentrations of rituximab were tested to determine the minimum concentration required to saturate the cell surface with rituximab via comparison of geometrical means of different fluorescence intensities. Raji cell culture Raji cells were maintained in 90% RPMI media, 10% FCS (fetal calf serum) supplemented with 1% L-glutamine at 37°C, 5% CO2. Cells were split every 2–3 days to keep them at appropriate confluence. Labelling of cells 15 μL of cells (around 7×106 cells/mL) were put in 75 μL cell-complete media (described above). 10 μL (0.1mg/mL) of labelled rituximabwas added onto the cells in an Eppendorf tube. This mix was then incubated for 1 h at 37°C, 5% CO2. Simultaneously, μ-Slide 8 well glass-bottom imaging chambers (Ibidi) were coated with 1 mg/mL BSA for 30 min and washed twice with 200 μL phenol red-free L15 medium, leaving 200 μL L15 in each well. Following the 1 h antibody incubation, 500 μL L15 was added to dilute unbound antibody. Cells were subsequently spun down (2000 rpm, 5 min, “Eppendorf mini-spin” centrifuge), supernatant was discarded, and the pelleted cells taken up in either (i) 300 μL PBS and transferred into the coatedmicroscopywell (removing the L15 from thewell) for imaging of labelled antibody only; or (ii) 100 μL L15 for other experiments. In case (ii), depending on the experiment, one or more of the following reagents were added: nuclear staining1 drop NucBlue® Live cell stain (Life Technologies) and incubation at RT for 5 min; mitochondrial staining0.5 μL (1 μM) MitoTracker® Orange CMTMRos (Thermofisher, #M7510) during antibody incubation and incubation for 30 min at 37°C. Thereafter, the mix was transferred into the imaging chamber containing 200 μL L15 media. Subsequent imaging was carried out on a Zeiss LSM 780 confocal microscope. Note: L15 serum-free media may be used to minimise cross-reactivity of antibody with serum. However, this comes at the cost of cell health and increased cell death. We hence recommend using cell media. Note: BSA coating prevents cell attachment to the well bottom. However, cell attachment in BSA-uncoated wells did not influence the number of polarised cells. Actin disruption with Latrunculin B or Cytochalasin D Before labelling, 1 μL (0.1 mM) (final concentration 1 μM) of Latrunculin B or Cytochalasin D was added to 75 μL complete-cell media and rituximab was subsequently added right away or after 30 min at 37°C, 5% CO2. Visualization of PIP2 In order to visualise PIP2, the PH-PLCD1 domain from the PH-PLCD1-GFP plasmid (Addgene plasmid # 51407) was cloned into the standard lentiviral vector Phr. PH-PLCD1 domain integrity was verified by sequencing. Subsequently lentivirus was obtained by transfecting HEK293T cells with pQ8.91 0.5 μg, pMD-G 0.5 μg, pHR-PH-GFP 0.5 μg, Genejuice (Merck) 4.5 μL, DMEM (Sigma-Aldrich) 150 μL, milliQ water 20 μL. 48 h after transfection, supernatant was harvested and spun down to remove particulates. 0.5 mL of viral suspension was then added to 1.5 mL of 70% confluent Raji suspension. After 3 days, localisation of PIP2 was imaged after labeling with Rtx-Alexa647, as described Rituximab capping triggers intracellular reorganisation of B cells DOI: 10.19185/matters.201612000001 Matters (ISSN: 2297-8240) | 4 previously, using the Zeiss LSM 780 confocal microscope. Preparation of GPMVs Raji cells were washed twice with 1 mL of hypotonic GPMV buffer (50 mM NaCl, 2 mM CaCl2, 10 mMHEPES; adjusted to pH 7.4 with HCl or NaOH).Then, 1 mL isotonic GPMV buffer (150mMNaCl, 2 mMCaCl2, 10mMHEPES; adjusted to pH 7.4 with H