Surface-associated antigen induces permeabilization of primary mouse B-cells and lysosome exocytosis facilitating antigen uptake and presentation to T-cells
- Department of Cell Biology and Molecular Genetics, University of Maryland, United States
- Immunology Division, Garvan Institute of Medical Research, Australia
- Immunology, Garvan Institute of Medical Research, Australia
Figures
Figure 1 with 6 supplements
BCR binding to surface-associated ligands causes B-cell PM permeabilization.
(A) Time-lapse images of a splenic B-cell incubated with αM-beads (1:2 cell:bead ratio) in the presence of PI (Video 1). (B) Percentages of B-cells bound to beads. (C) Percentages of PI-positive …
Figure 1—figure supplement 1
BCR binding to αM-beads causes localized PM permeabilization in B-cells.
(A) Live spinning-disk microscopy images of splenic B-cells incubated with αM- or Tf-beads before and after 60 min at 37°C in the presence of PI. The arrows point to bead-bound B-cells that became …
Figure 1—figure supplement 2
Identification of bead-bound B-cells by flow cytometry.
Splenic B-cells were incubated with αM-conjugated yellow-green fluorescence beads in the presence of PI and analyzed by flow cytometry. Representative dot plots of side scatter (SSC) versus forward …
Figure 1—figure supplement 3
BCR binding to αM-beads does not increase apoptosis in B-cells.
Splenic B-cells treated or not with staurosporine for 24 hr were incubated with αM- or Tf-beads for 30 min at 37°C, fixed, permeabilized, stained with antibodies against cleaved caspase-3, and …
Figure 1—figure supplement 4
Sudden increases in intracellular staining with the lipophilic FM dye in B-cells permeabilized by interaction with αM-PLB.
(A) Live spinning disk time-lapse images of splenic B-cells (permeabilized or non-permeabilized) after contact with αM-PLB in the presence of FM1-43 and PI at 37 °C. The arrows point to B-cell sites …
Figure 1—figure supplement 5
The lipophilic FM dye enters B-cells permeabilized by αM-PLB and stains the nuclear envelope.
The images show eight examples of FM4-64 nuclear envelope staining (arrows) in splenic B-cells permeabilized by αM-PLB after 60 min incubation at 37°C and imaged by live spinning disk fluorescence …
Figure 1—figure supplement 6
BCR cross-linking with soluble ligands does not permeabilize B-cells but induces a punctate form of FM uptake at the cell periphery that is distinct from the massive FM influx induced by surface-associated ligands.
Spinning disk time-lapse images of B-cells pre-labeled with soluble anti-BCR antibodies and FM1-43 (green) at 4°C and then imaged at 37°C after addition of secondary fluorochrome-labeled …
Figure 2
Extracellular Ponceau 4R quenches cytoplasmic CFSE in αM-PLB-permeabilized B-cells.
(A) Flow cytometry histograms of CFSE FI in B-cells incubated with or without SLO for 10 min in the presence or absence of Ponceau 4R, showing 8500 cells per condition. (B) Percentages of cells with …
Figure 3
BCR-mediated binding of HEL coupled to beads or expressed as a transmembrane protein on COS-7 cells causes B-cell PM permeabilization.
(A) Flow cytometry histograms of PI FI in WT or MD4 B-cells incubated with αM- or HEL-beads for 30 min by flow cytometry, showing 1000 cells per condition. (B) Percentages of WT and MD4 B-cells …
Figure 4 with 3 supplements
PM permeabilization induced by surface-associated antigen depends on high-affinity BCR-antigen binding, BCR signaling, and non-muscle myosin II (NMII) motor activity.
(A) Percentages of PI+ single bead-binding B-cells after incubation with HEL-, DEL-I- or Tf-beads (1:4 cell:bead ratio) for 30 min. Data points represent independent experiments (mean ± SD). (B) …
Figure 4—figure supplement 1
Impact of BCR-antigen affinity on B-cell-bead binding.
Splenic B-cells were incubated with HEL, DEL-I or Tf-beads at the indicated cell:bead ratios for 30 min at 37 °C and analyzed by flow cytometry. (A) Representative SSC versus FSC dot plots gated for …
Figure 4—figure supplement 2
B-cell binding to αM-PLB but not to Tf-PLB triggers BCR polarization first and PM permeabilization later.
(A) Splenic B-cells stained for surface BCR (green) were incubated with Tf-PLB (top panels) or αM-PLB (bottom panels) for 60 min at 37 °C in the presence of FM4-64 (red) and imaged by live spinning …
Figure 4—figure supplement 3
BCR and phosphorylated myosin light chain (pMLC) polarize toward αM-bead binding sites.
The images show several examples of splenic B-cells stained for surface BCRs with a Cy3-labeled Fab fragment of donkey anti-mouse IgM+G (red), incubated with αM (left, 5 examples)- or Tf (right, 5 …
Figure 5 with 2 supplements
Antigen-induced permeabilization triggers lysosomal exocytosis.
(A) Flow cytometry analysis of surface-exposed (no detergent permeabilization) and/or intracellular LIMP-2 (with detergent permeabilization) of bead-bound B-cells after incubation with αM- or …
Figure 5—figure supplement 1
BCR-mediated binding of αM-beads induces surface exposure of the LIMP-2 luminal domain at bead contact sites.
The images show several examples of splenic B-cells incubated with αM (left)- or Tf (right)-beads for 30 min at 37 °C, stained with LIMP-2-specific antibodies (green) at 4 °C without detergent …
Figure 5—figure supplement 2
Detection of lysosomal exocytosis by TIRF microscopy.
Splenic B-cells were added to αM-PLB and imaged by TIRF at eight frames/s. Live time-lapse XY images of individual SiR-Lyso puncta (top rows), their FI surface plots (bottom rows), and MFI (plots on …
Figure 6 with 2 supplements
Antigen-permeabilized B-cells reseal their PM in a lysosomal exocytosis-dependent manner.
(A) B-cells were incubated with αM-beads and permeabilized/resealed cells were assessed by flow cytometry of FM4-64 (added from the start) and SYTOX Blue (added in the last 10 min) FI, in the …
Figure 6—figure supplement 1
BEL does not affect the PM integrity and viability of B-cells.
Splenic B-cells were pretreated or not with BEL and incubated with αM-beads in the presence of FM4-64 and analyzed by flow cytometry. (A) Representative dot plots of side scatter (SSC) versus …
Figure 6—figure supplement 2
B-cell morphological changes occurring during permeabilization by surface-associated antigen are reversible.
Spinning disk time-lapse images of B-cells interacting with αM-PLB in the presence of PI (red). The dashed line indicates the maximum cell diameter initially reached by a B-cell that became …
Figure 7
Antigen-induced PM permeabilization promotes antigen internalization and presentation.
(A) Confocal live imaging of a B-cell interacting with fluorescent αM-beads (arrows, internalized αM). (B) Percentages of cells containing internalized αM or Tf, bound or not to αM- or Tf-beads, …
Videos
Video 1
BCR binding to αM-beads permeabilizes the PM of splenic B-cells.
Splenic B-cells were incubated with αM-beads at 4 °C and warmed to 37 °C in a live imaging chamber with 5 % CO2 in DMEM-BSA. Time-lapse images were acquired for 60 min at one frame/15 s in the …
Video 2
BCR binding to αM-beads causes localized PM permeabilization in A20 B-cells (cell line).
A20 B-cells were incubated with αM-beads in a live imaging chamber at 37 °C with 5 % CO2 in DMEM/BSA. Time-lapse images were acquired for 65 min at one frame/20 s in the presence of PI using a …
Video 3
Bead exchange between B-cells causes PM permeabilization.
Splenic B-cells were incubated with αM-beads in a live imaging chamber at 37 °C with 5 % CO2 in DMEM-BSA. Images were acquired for 60 min at one frame/30 s in the presence of PI using a spinning …
Video 4
Surface-associated ligand induces B-cell permeabilization and massive FM influx, while soluble ligand does not cause permeabilization but induces endocytosis, detected as puncta at the cell periphery.
Top: B-cells pre-labeled with FM1-43 (green) were added to αM-PLB (surface-associated ligand). Bottom: B-cells pre-labeled with FM1-43 (green) and anti-BCR antibodies followed by secondary …
Video 5
B-cell PM permeabilization during binding to αM-PLB enables membrane-impermeable Ponceau 4R to quench cytoplasmic CSFE fluorescence.
Splenic B cells pre-labeled with CFSE in the cytosol were added to αM-PLB in a live imaging chamber at 37 °C with 5 % CO2 in DMEM/BSA. Images were acquired for 60 min at one frame/10 s in the …
Video 6
Binding of MD4 B-cells to COS-7 cells expressing surface mHEL-GFP induces antigen clustering and PM permeabilization at interaction sites.
MD4 splenic B-cells were incubated with mHEL-GFP-expressing COS-7 cells cultured on fibronectin-coated coverslips at 37 °C with 5 % CO2 in DMEM/BSA. Images were acquired for 120 min at one frame/20 …
Video 7
Binding of WT B-cells to COS-7 cells expressing surface mHEL-GFP does not induce antigen clustering and PM permeabilization at interaction sites.
WT splenic B-cells were incubated with mHEL-GFP-expressing COS-7 cells cultured on fibronectin-coated coverslips at 37 °C with 5 % CO2 in DMEM/BSA. Images were acquired for 120 min at one frame/20 s …
Video 8
The BCR polarizes towards antigen-binding sites before PM permeabilization.
Splenic B-cells stained with anti-BCR antibodies were added to αM-PLB and imaged in a live imaging chamber at 37 °C with 5 % CO2 in DMEM/BSA. Images were acquired for 60 min at one frame/20 s in the …
Video 9
BCR and phosphorylated non-muscle myosin II (pMLC) polarize towards αM-bead-binding sites on a B-cell.
Shown is a 3D representation of co-polarization of the BCR (red) and pMLC (green) towards the site of αM-bead (white) binding in a splenic B-cell. Z-stack images were acquired using a Zeiss LSM710 …
Video 10
A lysosomal exocytosis event detected by total internal reflection fluorescence (TIRF) microscopy.
Splenic B-cells preloaded with SiR-Lyso were incubated with αM-PLB in a coverslip chamber at 37 °C with 5 % CO2 in DMEM/BSA for 30 min. Time-lapse images were acquired for 20 min at eight frames/s …
Video 11
B-cells exclude a second membrane-impermeable tracer after antigen-dependent permeabilization.
Splenic B-cells were added to αM-PLB and imaged in a live imaging chamber at 37 °C with 5 % CO2 in DMEM 2 % of FBS in the presence of SYTOX Green (green). Images were acquired for 4 hr at one …
Video 12
B-cell morphological changes occurring during permeabilization by surface-associated antigen are reversible.
Splenic B-cells were added to αM-PLB and imaged in a live imaging chamber at 37 °C with 5 % CO2 in DMEM without phenol red containing 2 % FBS in the presence of PI (red). Images were acquired for 4 …
Video 13
B-cell with polarized surface BCRs and containing fluorescent αM extracted from beads.
The surface BCRs of splenic B-cells were labeled with Cy3-Fab-donkey anti-mouse IgM+ G at 4 °C. Labeled B-cells were incubated with AF488-αM-beads at 37 °C with 5 % CO2 for 60 min and then fixed. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | A20 | ATCC | TIB-208 | B-cell lymphoma |
| Cell line (Mus musculus) | 3A9 | ATCC | CRL-3293 | T-cell hybridoma |
| Cell line (Cercopithecus aethiops) | COS-7 | ATCC | CRL-1651 | Kidney fibroblasts |
| Biological sample (Mus musculus) | WT (C57BL/6) | Jackson Laboratories | 000664 | Primary B-cells freshly isolated from C57BL/6’ spleen |
| Biological sample (Mus musculus) | MD4 (C57BL/6-Tg (IghelMD4)4Ccg/J) | Jackson Laboratories | 002595 | Primary B-cells freshly isolated from C57BL/6-Tg (IghelMD4)4Ccg/J’s spleen |
| Biological sample (Mus musculus) | B10.BR-H2K2 H2-T18a/ SgSnJJrep | Jackson Laboratories | 004804 | Primary B-cells freshly isolated from B10.BR-H2K2 H2-T18a/ SgSnJJrep’s spleen |
| Antibody | Anti-phosphotyrosine mAb 4G10 (mouse monoclonal) | Millipore | 05–321 | 1:500 |
| Antibody | AF488-anti-mouse IgG2b (goat polyclonal) | Thermo Fisher Scientific | A-21141 | 1:500 |
| Antibody | AF647-anti-goat IgG (H + L) (donkey polyclonal) | Invitrogen | A21447 | 10 µg/ml |
| Antibody | Anti-cleaved caspase-3 (Asp175) (rabbit polyclonal) | Cell Signaling | 9661T | 1:500 |
| Antibody | Cy5-Fab anti-mouse IgG (donkey polyclonal) | Jackson ImmunoResearch | 715-175-151 | 5 µg/ml |
| Antibody | AF488-anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (donkey polyclonal) | Thermo FisherScientific | A-21206 | 1:200 |
| Antibody | Anti-BTK (rabbit monoclonal) | Cell Signaling | 8,547 | 1:1,000 |
| Antibody | Anti-phospho-BTK (rabbit monoclonal) | Abcam | 68,217 | 1:500 |
| Antibody | HRP-anti-rabbit (goat polyclonal) | Jackson Immune Research | 111-035-144 | 1:1,000 |
| Antibody | Cy3-Fab-anti– mouse IgM+ G (goat polyclonal) | Jackson Immune Research | 115-165-166 | 1:200 |
| Antibody | Anti-phosphorylated myosin light chain (pMLC) (rabbit polyclonal) | Cell Signaling | 3,671 S | 1:50 |
| Antibody | AF633-anti-rabbit IgG (goat polyclonal) | Invitrogen | A-21070 | 1:500 |
| Antibody | Anti-LIMP-2 (rabbit polyclonal) | Sigma-Aldrich | SAB3500449- 100UG | 1:200 |
| Antibody | AF488 donkey-anti-rabbit IgG (donkey polyclonal) | Life technology | A32790 | 1:200 |
| Antibody | Anti-CD90.2 (rat monoclonal) | Biolegend | 105,310 | 1 µl/ 2 × 106 cells |
| Antibody | αM (F(ab’)2 goat-anti-mouse IgM+ G) (goat polyclonal) | Jackson Immune Research | 115-006-068 | Binds to BCR |
| Antibody | AF488-αMAffiniPure F(ab')₂ fragments of anti- mouse IgG (H + L) (goat polyclonal) | Jackson Immune Research | 115-546-003 | Binds to BCR |
| Antibody | Biotin-SP (long spacer)-conjugated Fab fragments of anti-mouse IgG (H + L) (goat polyclonal) | Jackson Immune Research | 115-067-003 | |
| Commercial assay or kit | SiR-Lysosome and Verapamil | Cytoskeleton | CY-SC012 | Lysosome probe1 µM and 10 µM |
| Commercial assay or kit | IL-2 ELISA kit | Biolegend | 431,804 | |
| Commercial assay or kit | BCA kit | Thermo Fisher Scientific | 23,235 | Protein measurement during bead preparation |
| Software, algorithm | Volocity Suite | PerkinElmer | https://ir.perkinelmer.com/news-releases/news-release details/perkinelmer- launches-volocityr- 60-high-performance- 3d-cellular | |
| Software, algorithm | NIH Image J | NIH | https://imagej.nih.gov/ij/ | |
| Software, algorithm | MATLAB | MathWorks | https://www.mathworks.com/products/matlab.html | |
| Software, algorithm | Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Chemical compound, drug | Staurosporine | Abcam | 120,056 | Apoptosis induction(1 µM) |
| Chemical compound, drug | PP2 | Millipore-Sigma | 529,573 | Src kinase inhibitor(5 µM) |
| Chemical compound, drug | AVL-292 | Selleckchem | S7173 | BTK inhibitor(10 nM) |
| Chemical compound, drug | BEL (Bromoenol lactone) | Sigma-Aldrich | B1552 | 12 µM |
| Chemical compound, drug | Blebbistatin | Sigma-Aldrich | B0560 | 50 µM |
| Chemical compound, drug | Latex NH2-beads | Polysciences | 17145–5 | |
| Chemical compound, drug | HEL (hen egg lysozyme) | Sigma-Aldrich | L6876 | Binds to BCR from MD4 mice |
| Chemical compound, drug | DEL-1 (duck egg lysozyme) | David B. Langley and Daniel Christ laboratory | Binds to BCR from MD4 mice | |
| Chemical compound, drug | Tf (holo- transferrin) | Sigma-Aldrich | T0665-50MG | Binds to transferrin receptor |
| Chemical compound, drug | Biotinylated transferrin (Tf-PLB) | Sigma-Aldrich | T3915-5MG | |
| Chemical compound, drug | Streptavidin- conjugated Yellow- Green latex beads | Polysciences | 24159–1 | |
| Chemical compound, drug | Propidium iodide | Sigma-Aldrich | P4170-10MG | 50 µg/ml |
| Chemical compound, drug | FM1-43FX | Thermo Fisher Scientific | F35355 | 10 µg/ml |
| Chemical compound, drug | FM4-64FX | Thermo Fisher Scientific | F34653 | 10 µg/ml |
| Chemical compound, drug | SYTOX Blue | Invitrogen | S11348 | 300 nM |
| Chemical compound, drug | SYTOX Green | Invitrogen | S7020 | 300 nM |
| Chemical compound, drug | Guinea pig complement | Innovative Research | IGGPCSER | 100 µl/ 4 × 107 cells |
| Chemical compound, drug | 1,2-dioleoyl-sn- glycero-3-phosphocholine | Avanti Polar Lipids | 850375 P | 5 mM (PLB) |
| Chemical compound, drug | 1,2-dioleoyl-sn-glycero-3- phospho ethanolamine-cap-biotin | Avanti Polar Lipids | 870273 C | 50 µM (PLB) |
| Chemical compound, drug | Ponceau 4R | Sigma-Aldrich | 18,137 | 1 mM |
| Chemical compound, drug | CFSE | Thermo Fisher Scientific | C34553 | 1 µM |
| Chemical compound, drug | Lipofectamine 3,000 | Thermo Fisher Scientific | L3000008 | |
| Chemical compound, drug | Bovine fibronectin | Millipore | 341,631 | 5 mg/ml |
| Chemical compound, drug | AF88-Tf (transferrin from human serum, Alexa Fluor 488 conjugate) | Thermo Fisher Scientifc | T13342 | Binds to transferrin receptor |
| Transfected construct (Cercopithecus aethiops) | mHEL-GFP | Michael R. Gold laboratory | Wang et al., 2018a (DOI: 10.1007/978-1-4939-7474-0_10) | Wild-type HEL protein, the complete EGFP protein, the transmembrane region of H-2Kb, and the 23-amino acid cytoplasmic domain of H-2Kb |
Additional files
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/66984/elife-66984-transrepform1-v2.pdf
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Source code 1
iBTK inhibits BTK phosphorylation in activated B-cells.
Western blot analysis of pBTK (A) and BTK (B, regular exposure image; C, overexposed image) in mouse splenic B-cells incubated with HEL-beads in the presence or absence of a BTK inhibitor (iBTK) for 30 min.
- https://cdn.elifesciences.org/articles/66984/elife-66984-supp1-v2.zip
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Source code 2
iBTK inhibits BTK phosphorylation in activated B-cells.
Western blot analysis of pBTK (A) and BTK (B, regular exposure image; C, overexposed image) in mouse splenic B-cells incubated with HEL-beads in the presence or absence of a BTK inhibitor (iBTK) for 30 min.
- https://cdn.elifesciences.org/articles/66984/elife-66984-supp2-v2.zip
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Source code 3
iBTK inhibits BTK phosphorylation in activated B-cells.
- https://cdn.elifesciences.org/articles/66984/elife-66984-supp3-v2.zip
