Chapter 24 Detection of Complement Receptors 1 and 2 on Mouse Splenic B Cells Using Flow Cytometry Luke R. Donius and John H. Weis Abstract The complement receptor 2 (Cr2) gene is exclusively expressed in B cells and follicular dendritic cells (FDC) in mice and in humans. However, mice also express an alternative splice variant, CR1, of the Cr2 gene. CR2 and CR1 are receptors for the complement component 3 (C3) cleavage fragments C3d(g) and iC3b. Additionally, CR1 is a receptor for C3b and regulates complement convertase activity. CR1 and CR2 have various functions including antigen retention by FDC, regulation of surface complement convertases, and canonically as the B cell coreceptor in which CR2 acts to lower the threshold for B cell activation. Detection of CR1 and CR2 can be utilized to identify B cells and, depending on expression level, to delineate various B cell populations. This protocol describes methods for detecting CR1/2 expression on splenic B cell subsets via flow cytometry. Key words Complement receptor 1, Complement receptor 2, CR1, CR2, CD21, CD35, B cell coreceptor, B cell activation, Flow cytometry, Marginal zone B cell, Follicular mature B cell, Transitional 1 B cell, Transitional 2 B cell
1
Introduction Complement receptor 2 (CR2) and the alternative isoform CR1 are cellular receptors that bind enzymatic products of complement protein C3: C3d(g) and iC3b for CR2 as well as C3b and C4b for CR1 [1]. (It should be noted that CR1 and CR2 are also known by their cluster of differentiation (CD) designations, CD35 and CD21, respectively. Both names are used commonly and will be used in the following protocol.) CR2 reduces the threshold for B cell stimulation [2, 3], facilitates transport of immune complexes to immune follicles [4, 5], and retains immune complexes in immune follicles [6, 7]. CR1 has an additional role as a surface regulator of C3 convertases [1, 8, 9]. CR1 and CR2 are important markers for B cells and FDC and are a critical bridge for complement-driven immune modulation of the humoral immune
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_24, © Springer Science+Business Media New York 2014
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response [6, 10, 11]. Identification of CR1 and CR2 is common for identifying B cells and aiding in delineation of B cell subsets [12]. Antibody detection of CR1/2 by flow cytometry is a common method of detection and is an often included identification of B cell subsets. The protocol described in the chapter Detection of Complement Receptors 1 and 2 describes methods for detecting CR2 and CR1 on the surface of cells via flow cytometry.
2
Materials 1. FACS (Fluorescent Activated Cell Sorting) buffer: 0.1 % bovine serum albumin, 1× PBS, pH 7.4. 2. 75 × 12 mm Conical bottom tube. 3. 12 × 75 mm 5 ml Polystyrene round-bottom tube. 4. Red blood cell lysis buffer (ACK buffer): Combine 8.3 g NH4Cl, KHCO3 and 200 μl of 0.5 M EDTA pH 7.4 in 800 ml deionized H2O. Adjust pH to 7.4 and bring solution to 1 L total volume with deionized H2O. 5. Tube rack for holding 12 × 75 mm diameter tubes on ice. 6. Tabletop centrifuge with temperature control capable of maintaining 4 °C. 7. Rat anti-mouse CR2 (CD21/35) antibody, FITC conjugated, clone 7G6 (BD Pharmingen, San Diego, CA, USA). 8. Rat anti-mouse CD24 antibody, PE-Cy7 conjugated, clone M1/69 (eBioscience San Diego, CA, USA). 9. Rat anti-mouse CD23 antibody, Phycoerythrin (PE) conjugated, clone B3B4 (eBioscience San Diego, CA, USA). 10. Anti-human/mouse CD45R (B220) PerCP-Cy5.5- conjugated antibody, clone RA3-6B2. 11. Fc receptor block, anti-mouse CD16/32, purified antibody, clone 93. 12. 100 μm cell strainer. 13. 1 mM Stock solution of DAPI in deionized H2O. 14. Flow cytometer capable of exciting/detecting at 495/519 nm (FITC), 358/461 nm (DAPI), 480;565/578 nm (PE), 482/694 nm (PerCP-Cy5.5), 480;565/767 nm (PE-Cy7).
3
Methods 1. To assess cells for CR2 expression, obtain a single-cell suspension in FACS buffer from a source of interest. Cells commonly assessed in this manner are cells from the peripheral blood, the
Detection of CR2
307
spleen, the peritoneal cavity, and the bone marrow (see Note 1 and 2). This protocol describes the preparation and antibody staining of splenocytes, although cells from any of the preceding sources could be assessed in a similar manner. 2. Centrifuge the cells at 300 × g for 5 min at 4 °C and aspirate the supernatant. 3. Resuspend the cell pellet in 1 ml of 4 °C chilled ACK lysis buffer with gentle pipetting to lyse red blood cells and incubate for 5 min. 4. Centrifuge the cells at 300 × g for 5 min at 4 °C and aspirate the supernatant. 5. Resuspend the pellet in 10 ml of 4 °C chilled FACS buffer and place on ice. 6. Count the cells in the sample to determine the total cells per milliliter. Transfer 2 × 106 cells to a 75 × 12 mm conical bottom tube (see Note 3). Place the tube on ice and proceed with preparing the antibody mix for staining the cells. 7. Prepare the antibody mix of FITC-conjugated Rat anti-mouse CD21/35 (CR2) antibody diluted 1:200 (2.5 μg/ml) (see Note 4), PE-conjugated anti-mouse CD23 antibody diluted 1:200 (1 μg/ml), PE-Cy7-conjugated anti-mouse CD24 antibody diluted 1:600 (0.3 μg/ml), Rat anti-mouse CD16/32 antibody diluted 1:1,000 (0.5 μg/ml) (see Note 5), and PerCP-Cy5.5-conjugated B220 antibody diluted 1:200 (1 μg/ml) in FACS buffer (100 μl/sample). 8. Centrifuge the cells at 300 × g for 5 min at 4 °C and aspirate the supernatant. 9. Resuspend each sample of cells in 100 μl of the prepared antibody mix. Incubate the sample on ice in the dark for 20 min. 10. Dilute each sample with 1 ml of FACS buffer, centrifuge the cells at 300 × g for 5 min at 4 °C, and aspirate the supernatant. 11. Resuspend the cells in 1 ml of FACS buffer (see Note 6) and filter through a 100 μm cell strainer into round -bottom tube. Add 3 μl of DAPI (3 μM final concentration), gently vortex, and let sit for 5 min. Acquire fluorescence staining data using a flow cytometer (see Note 7). 12. Identification of transitional 1, transitional 2, marginal zone, and follicular mature B cells is done by gating on the DAPI negative (live) and B220 positive (B cells), and then separating the groups by CD23 expression (positive are the transitional 2 and follicular mature B cells and negatives are the transitional 1 and marginal zone B cells). Gating the B cells within the CD23 positive or CD23 negative groups by CD21/35 and CD24 expression can then be used to transitional 1 B cells
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Luke R. Donius and John H. Weis
CD23
CD21/35
T2
Live B220+ (B cells)
Follicular
CD24
FSC
CD21/35
Marginal Zone
T1
CD24 Fig. 1 Surface expression of CD23, CD24, and CD21/35 on the total live B220+ cells in the spleen can be used to identify splenic B cell subsets. These subsets include mature follicular B cells and marginal zone B cells as well as the immature B cell populations transitional 1 (T1) and T2
(CD23-/CD21/35Lo/CD24+), marginal zone B cells (CD23-/CD21/35Hi/CD24+), transitional 2 B cells (CD23+/ CD21/35Hi/CD24Hi), and follicular mature B cells (CD23+/CD21/35Lo/CD24Lo) (Fig. 1).
4
Notes 1. In order to assess cells for CR2 expression using this method the cells must be able to be processed through a flow cytometer. Lymphocytes are ideal for analysis by flow cytometry. However, of the two major cell types in the spleen that express CR1/2, B cells, and FDC, this protocol will result only in analysis of the former. FDC can be analyzed but FDC isolation protocols should be sought out in order to efficiently collect them. 2. This protocol has been written for detection of CR2 on the surface of mouse cells. A similar procedure could be used on human/primate cells with antibodies that recognize human epitopes.
Detection of CR2
309
3. Ideally this protocol describes antibody staining of 2 × 106 cells. However, a range of 1–5 × 106 cells per sample may be labeled. Consistency between samples is the most important factor. 4. The CR2 monoclonal antibody clone 7G6 has been shown to bind both CR2 (CD21) and the isoform CR1 (CD35) [13]. No antibody is currently known to recognize only CR2. However, the anti-CD35 antibody (clone 8C12), available through BD Pharmingen, exclusively binds CR1. 5. Rat anti-CD16/32 is necessary to block the Fc Receptors so that cells expressing these receptors do not bind antibody via the Fc region in an epitope-independent manner. 6. The final resuspended volume of the cells to be analyzed by flow cytometry can be reduced in order to reduce data collection time if the total cell number is low. 7. All fluorescent parameters must be defined for the flow cytometer by single-color positive controls and an unstained negative control. All channels used must be compensated to eliminate cross-fluorescence in multiple detectors. Consult the manual or expert users for information on proper operation of your specific flow cytometer. References 1. Molina H, Kinoshita T, Webster CB, Holers VM (1994) Analysis of C3b/C3d binding sites and factor I cofactor regions within mouse complement receptors 1 and 2. J Immunol 153:789–795 2. Carter RH, Spycher MO, Ng YC, Hoffman R, Fearon DT (1988) Synergistic interaction between complement receptor type 2 and membrane IgM on B lymphocytes. J Immunol 141:457–463 3. Luxembourg AT, Cooper NR (1994) Modulation of signaling via the B cell antigen receptor by CD21, the receptor for C3dg and EBV. J Immunol 153:4448–4457 4. Whipple EC, Shanahan RS, Ditto AH, Taylor RP, Lindorfer MA (2004) Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice. J Immunol 173(4):2297–2306 5. Suzuki K, Grigorova I, Phan TG, Kelly LM, Cyster JG (2009) Visualizing B cell capture of cognate antigen from follicular dendritic cells. J Exp Med 206:1485–1493 6. Haas KM, Hasegawa M, Steeber DA, Poe JC, Zabel MD, Bock CB, Karp DR, Briles DE, Weis J, Tedder TF (2002) Complement receptors CD21/35 link innate and protective immunity during Streptococcus pneumoniae
7.
8. 9.
10.
11. 12.
infection by regulating IgG3 antibody responses. Immunity 17:713–723 Mattsson J, Yrlid U, Stensson A, Schön K, Karlsson MCI, Ravetch JV, Lycke NY (2011) Complement activation and complement receptors on follicular dendritic cells are critical for the function of a targeted adjuvant. J Immunol 187(7):3641–3652 Krych-Goldberg M, Atkinson JP (2001) Structure-function relationships of complement receptor type 1. Immunol Rev 180:112–122 Seregin SS, Aldhamen YA, Appledorn DM, Schuldt NJ, McBride AJ, Bujold M, Godbehere SS, Amalfitano A (2009) CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies. Gene Ther 16:1245–1259 Jacobson A, Weis J, Weis J (2008) Complement receptors 1 and 2 influence the immune environment in a B cell receptor-independent manner. J Immunol 180(7):5057–5066 Carroll MC, Isenman DE (2012) Regulation of humoral immunity by complement. Immunity 37:199–207 Su TT, Rawlings DJ (2002) Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J Immunol 168:2101–2110
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Luke R. Donius and John H. Weis
13. Molina H, Wong W, Kinoshita T, Brenner C, Foley S, Holers VM (1992) Distinct receptor and regulatory properties of recombinant
mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1. J Exp Med 175:121–129
1
Introduction Complement receptor 2 (CR2) and the alternative isoform CR1 are cellular receptors that bind enzymatic products of complement protein C3: C3d(g) and iC3b for CR2 as well as C3b and C4b for CR1 [1]. (It should be noted that CR1 and CR2 are also known by their cluster of differentiation (CD) designations, CD35 and CD21, respectively. Both names are used commonly and will be used in the following protocol.) CR2 reduces the threshold for B cell stimulation [2, 3], facilitates transport of immune complexes to immune follicles [4, 5], and retains immune complexes in immune follicles [6, 7]. CR1 has an additional role as a surface regulator of C3 convertases [1, 8, 9]. CR1 and CR2 are important markers for B cells and FDC and are a critical bridge for complement-driven immune modulation of the humoral immune
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_24, © Springer Science+Business Media New York 2014
305
306
Luke R. Donius and John H. Weis
response [6, 10, 11]. Identification of CR1 and CR2 is common for identifying B cells and aiding in delineation of B cell subsets [12]. Antibody detection of CR1/2 by flow cytometry is a common method of detection and is an often included identification of B cell subsets. The protocol described in the chapter Detection of Complement Receptors 1 and 2 describes methods for detecting CR2 and CR1 on the surface of cells via flow cytometry.
2
Materials 1. FACS (Fluorescent Activated Cell Sorting) buffer: 0.1 % bovine serum albumin, 1× PBS, pH 7.4. 2. 75 × 12 mm Conical bottom tube. 3. 12 × 75 mm 5 ml Polystyrene round-bottom tube. 4. Red blood cell lysis buffer (ACK buffer): Combine 8.3 g NH4Cl, KHCO3 and 200 μl of 0.5 M EDTA pH 7.4 in 800 ml deionized H2O. Adjust pH to 7.4 and bring solution to 1 L total volume with deionized H2O. 5. Tube rack for holding 12 × 75 mm diameter tubes on ice. 6. Tabletop centrifuge with temperature control capable of maintaining 4 °C. 7. Rat anti-mouse CR2 (CD21/35) antibody, FITC conjugated, clone 7G6 (BD Pharmingen, San Diego, CA, USA). 8. Rat anti-mouse CD24 antibody, PE-Cy7 conjugated, clone M1/69 (eBioscience San Diego, CA, USA). 9. Rat anti-mouse CD23 antibody, Phycoerythrin (PE) conjugated, clone B3B4 (eBioscience San Diego, CA, USA). 10. Anti-human/mouse CD45R (B220) PerCP-Cy5.5- conjugated antibody, clone RA3-6B2. 11. Fc receptor block, anti-mouse CD16/32, purified antibody, clone 93. 12. 100 μm cell strainer. 13. 1 mM Stock solution of DAPI in deionized H2O. 14. Flow cytometer capable of exciting/detecting at 495/519 nm (FITC), 358/461 nm (DAPI), 480;565/578 nm (PE), 482/694 nm (PerCP-Cy5.5), 480;565/767 nm (PE-Cy7).
3
Methods 1. To assess cells for CR2 expression, obtain a single-cell suspension in FACS buffer from a source of interest. Cells commonly assessed in this manner are cells from the peripheral blood, the
Detection of CR2
307
spleen, the peritoneal cavity, and the bone marrow (see Note 1 and 2). This protocol describes the preparation and antibody staining of splenocytes, although cells from any of the preceding sources could be assessed in a similar manner. 2. Centrifuge the cells at 300 × g for 5 min at 4 °C and aspirate the supernatant. 3. Resuspend the cell pellet in 1 ml of 4 °C chilled ACK lysis buffer with gentle pipetting to lyse red blood cells and incubate for 5 min. 4. Centrifuge the cells at 300 × g for 5 min at 4 °C and aspirate the supernatant. 5. Resuspend the pellet in 10 ml of 4 °C chilled FACS buffer and place on ice. 6. Count the cells in the sample to determine the total cells per milliliter. Transfer 2 × 106 cells to a 75 × 12 mm conical bottom tube (see Note 3). Place the tube on ice and proceed with preparing the antibody mix for staining the cells. 7. Prepare the antibody mix of FITC-conjugated Rat anti-mouse CD21/35 (CR2) antibody diluted 1:200 (2.5 μg/ml) (see Note 4), PE-conjugated anti-mouse CD23 antibody diluted 1:200 (1 μg/ml), PE-Cy7-conjugated anti-mouse CD24 antibody diluted 1:600 (0.3 μg/ml), Rat anti-mouse CD16/32 antibody diluted 1:1,000 (0.5 μg/ml) (see Note 5), and PerCP-Cy5.5-conjugated B220 antibody diluted 1:200 (1 μg/ml) in FACS buffer (100 μl/sample). 8. Centrifuge the cells at 300 × g for 5 min at 4 °C and aspirate the supernatant. 9. Resuspend each sample of cells in 100 μl of the prepared antibody mix. Incubate the sample on ice in the dark for 20 min. 10. Dilute each sample with 1 ml of FACS buffer, centrifuge the cells at 300 × g for 5 min at 4 °C, and aspirate the supernatant. 11. Resuspend the cells in 1 ml of FACS buffer (see Note 6) and filter through a 100 μm cell strainer into round -bottom tube. Add 3 μl of DAPI (3 μM final concentration), gently vortex, and let sit for 5 min. Acquire fluorescence staining data using a flow cytometer (see Note 7). 12. Identification of transitional 1, transitional 2, marginal zone, and follicular mature B cells is done by gating on the DAPI negative (live) and B220 positive (B cells), and then separating the groups by CD23 expression (positive are the transitional 2 and follicular mature B cells and negatives are the transitional 1 and marginal zone B cells). Gating the B cells within the CD23 positive or CD23 negative groups by CD21/35 and CD24 expression can then be used to transitional 1 B cells
308
Luke R. Donius and John H. Weis
CD23
CD21/35
T2
Live B220+ (B cells)
Follicular
CD24
FSC
CD21/35
Marginal Zone
T1
CD24 Fig. 1 Surface expression of CD23, CD24, and CD21/35 on the total live B220+ cells in the spleen can be used to identify splenic B cell subsets. These subsets include mature follicular B cells and marginal zone B cells as well as the immature B cell populations transitional 1 (T1) and T2
(CD23-/CD21/35Lo/CD24+), marginal zone B cells (CD23-/CD21/35Hi/CD24+), transitional 2 B cells (CD23+/ CD21/35Hi/CD24Hi), and follicular mature B cells (CD23+/CD21/35Lo/CD24Lo) (Fig. 1).
4
Notes 1. In order to assess cells for CR2 expression using this method the cells must be able to be processed through a flow cytometer. Lymphocytes are ideal for analysis by flow cytometry. However, of the two major cell types in the spleen that express CR1/2, B cells, and FDC, this protocol will result only in analysis of the former. FDC can be analyzed but FDC isolation protocols should be sought out in order to efficiently collect them. 2. This protocol has been written for detection of CR2 on the surface of mouse cells. A similar procedure could be used on human/primate cells with antibodies that recognize human epitopes.
Detection of CR2
309
3. Ideally this protocol describes antibody staining of 2 × 106 cells. However, a range of 1–5 × 106 cells per sample may be labeled. Consistency between samples is the most important factor. 4. The CR2 monoclonal antibody clone 7G6 has been shown to bind both CR2 (CD21) and the isoform CR1 (CD35) [13]. No antibody is currently known to recognize only CR2. However, the anti-CD35 antibody (clone 8C12), available through BD Pharmingen, exclusively binds CR1. 5. Rat anti-CD16/32 is necessary to block the Fc Receptors so that cells expressing these receptors do not bind antibody via the Fc region in an epitope-independent manner. 6. The final resuspended volume of the cells to be analyzed by flow cytometry can be reduced in order to reduce data collection time if the total cell number is low. 7. All fluorescent parameters must be defined for the flow cytometer by single-color positive controls and an unstained negative control. All channels used must be compensated to eliminate cross-fluorescence in multiple detectors. Consult the manual or expert users for information on proper operation of your specific flow cytometer. References 1. Molina H, Kinoshita T, Webster CB, Holers VM (1994) Analysis of C3b/C3d binding sites and factor I cofactor regions within mouse complement receptors 1 and 2. J Immunol 153:789–795 2. Carter RH, Spycher MO, Ng YC, Hoffman R, Fearon DT (1988) Synergistic interaction between complement receptor type 2 and membrane IgM on B lymphocytes. J Immunol 141:457–463 3. Luxembourg AT, Cooper NR (1994) Modulation of signaling via the B cell antigen receptor by CD21, the receptor for C3dg and EBV. J Immunol 153:4448–4457 4. Whipple EC, Shanahan RS, Ditto AH, Taylor RP, Lindorfer MA (2004) Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice. J Immunol 173(4):2297–2306 5. Suzuki K, Grigorova I, Phan TG, Kelly LM, Cyster JG (2009) Visualizing B cell capture of cognate antigen from follicular dendritic cells. J Exp Med 206:1485–1493 6. Haas KM, Hasegawa M, Steeber DA, Poe JC, Zabel MD, Bock CB, Karp DR, Briles DE, Weis J, Tedder TF (2002) Complement receptors CD21/35 link innate and protective immunity during Streptococcus pneumoniae
7.
8. 9.
10.
11. 12.
infection by regulating IgG3 antibody responses. Immunity 17:713–723 Mattsson J, Yrlid U, Stensson A, Schön K, Karlsson MCI, Ravetch JV, Lycke NY (2011) Complement activation and complement receptors on follicular dendritic cells are critical for the function of a targeted adjuvant. J Immunol 187(7):3641–3652 Krych-Goldberg M, Atkinson JP (2001) Structure-function relationships of complement receptor type 1. Immunol Rev 180:112–122 Seregin SS, Aldhamen YA, Appledorn DM, Schuldt NJ, McBride AJ, Bujold M, Godbehere SS, Amalfitano A (2009) CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies. Gene Ther 16:1245–1259 Jacobson A, Weis J, Weis J (2008) Complement receptors 1 and 2 influence the immune environment in a B cell receptor-independent manner. J Immunol 180(7):5057–5066 Carroll MC, Isenman DE (2012) Regulation of humoral immunity by complement. Immunity 37:199–207 Su TT, Rawlings DJ (2002) Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J Immunol 168:2101–2110
310
Luke R. Donius and John H. Weis
13. Molina H, Wong W, Kinoshita T, Brenner C, Foley S, Holers VM (1992) Distinct receptor and regulatory properties of recombinant
mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1. J Exp Med 175:121–129
