Mice and cell lines
The mouse strain used in this study was C57BL/6; 129P2-Hvcn1Gt(RRN293)Byg/Mmcd (15990-UCD; from the Mutant Mouse Regional Resource Center (a strain repository funded by the National Center for Research Resources of the National Institutes of Health) and donated to the Mutant Mouse Regional Resource Center by the BayGenomics project funded by the National Heart, Lung and Blood Institute). The exogenous gene-trap vector sequence, containing an in-frame splice-acceptor site followed by coding sequence for a β-galactosidase–neomycin fusion protein, was inserted into a large intron of 17 kilobases at approximately 12 kilobases after exon 2, which contains the first translation initiation codon. Chimeric male mice were derived from sequence-verified RRN293 gene-trap embryonic stem cells and were used to establish heterozygous WT/RRN293 founders; homozygous offspring were generated from heterozygous mating pairs. Experiments used mice with a mixed background and mice backcrossed to the C57BL/6 strain for five generations. No substantial difference was detected between mixed and C57BL/6-backcrossed mice. Experimental procedures and husbandry were carried out according to the regulations of the Home Office Scientific Procedures Act, UK (1986), with prior Home Office approval.
For the generation of mixed chimeras, lethally irradiated μMT mice received a mixture of 80% bone marrow cells of μMT origin and 20% wild-type or HVCN1-deficient bone marrow cells38
. The μMT mutation prevents the generation of B cells, so the B cells in the mice that received HVCN1-deficient marrow would be HVCN1 deficient, whereas the recipients of wild-type marrow would have wild-type B cells. The 20/80 ratio favored reconstitution of all the other hematopoietic lineages from wild-type precursors.
The B lymphoma lines LK35.2 HyHEL10 (IgG2a, κ-chain; H-2kxd) and A20 D1.3 (IgG2a, κ-chain; H-2d) overexpressing recombinant mouse IgM receptors were a gift from F. Batista.
For analysis of T cell-independent responses, HVCN1-deficient and wild-type mice 8–12 weeks of age were injected intraperitoneally with 100 μg NP23-Ficoll (Biosearch Technologies). For analysis of T cell–dependent responses, 100 μg alum-precipitated NP23-KLH (Biosearch Technologies) was injected subcutaneously at the base of the tail. The μMT chimeric mice were immunized intraperitoneally with 100 μg alum-precipitated NP23-KLH at 8 weeks after bone marrow reconstitution.
Enzyme-linked immunosorbent assay
NP-specific antibodies were detected by enzyme-linked immunosorbent assay, and antibody end-point titers were used as a measure of relative concentration. For this assay, 96-well plates were coated overnight at 4 °C with NP18-BSA or NP3-BSA (10 μg/ml). Plates were then washed with 0.005% (vol/vol) Tween-20 in PBS, then they were blocked for 1 h at 37 °C with 2% (wt/vol) BSA in PBS. After washes, serially diluted serum samples were loaded onto the plates and incubated at for 1 h at 37 °C. NP-specific immunoglobulin subclasses were detected with alkaline phosphatase–conjugated secondary antibodies to IgM (1020-04), IgA (1040-04), IgG1 (1070-04), IgG2b (1090-04) and IgG3 (1100-04) and p-nitrophenyl phosphate substrate (all from Southern Biotech).
Staining of NP and IgD was detected as described49
. IgD was detected with sheep anti-IgD (PC013; The Binding Site) and NP-binding cells were identified with NP conjugated to rabbit IgG49
. The proportion of spleen sections occupied by GCs or plasmacytoid cells was determined by a published point-counting technique50
. NP-specific plasmacytoid and GC cells per mm2
were counted at a magnification of ×100 with serial sweeps of each spleen section by means of a 1-cm2
eyepiece graticule divided into areas of 100 mm2
to define the section area being counted.
Enzyme-linked immunospot assay
NP-specific antibody-forming cells were detected by enzyme-linked immunospot assay as described51
Purification and stimulation of mouse and human B cells
B cells were purified from spleens of 8- to 12-week-old mice either by negative selection with anti-CD43 magnetic beads, with a purity of ~95%, or by positive selection with anti-B220 and anti-CD19 magnetic beads (all from Miltenyi Biotech), with a purity of ~98%. Cells were cultured in RPMI complete medium containing 10% (vol/vol) FCS, penicillin and streptomycin, L-Glutamax and 50 μM 2-mercaptoethanol.
Splenic B cells were stimulated for various times at 37 °C with goat anti-mouse IgM F(ab′)2 fragment (20 μg/ml; 115-005-075; Jackson Immunoresearch). For inhibition with DPI or sodium stibogluconate, cells were incubated at 20 °C with 5 μM DPI (Sigma Aldrich) or sodium stibogluconate (Calbiochem) before being activated with anti–mouse IgM F(ab′)2 fragment. Whole-cell lysates were sonicated briefly, then were resolved and analyzed by immunoblot with antibody to phosphorylated tyrosine (4G10; Millipore) and anti-actin (AC-15; (Sigma Aldrich) as a loading control. Antibodies to phosphorylated Syk (C87C1), Akt (D9E) and Erk (E10), and to total Syk (2712) and total Erk (L34F12), were from Cell Signaling Technology; anti-Akt (C20) was from Santa Cruz Biotechnology. Human peripheral B cells were obtained from buffy coat provided by the Sheffield Blood Bank with approval from the ethical committee of University Hospitals Leicester. Human B cells were purified from peripheral blood mononuclear cells obtained by Ficoll centrifugation of whole blood. B cells were purified by either negative selection or CD19 positive selection with magnetic beads (Miltenyi Biotech). Human CD27− and CD27+ B cells were separated with CD27 magnetic beads (Miltenyi Biotech) from B cells previously isolated by negative selection.
Mouse B cell proliferation
Cells were stimulated with the following: anti-IgM F(ab′)2 (1–20 μg/ml), anti-CD40 (2 μg/ml; HM40-3; BD Biosciences), mouse IL-4 (20 ng/ml; Peprotech) or lipopolysaccharide (10 μg/ml; Sigma Aldrich). Purified B cells were labeled for 5 min with CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen) and were cultured for various times thereafter. Cells were then analyzed on a FACSCanto (BD) with DIVA software.
Generation of antiserum to HVCN1
The HVCN1-specific antibody (generated by CovalAb) was a polyclonal antiserum raised in rabbits immunized with a peptide from the amino-terminal domain of human HVCN1 (amino acids 26–46). The 21-amino acid peptide has 86% homology with the mouse sequence; therefore, the antibody is expected to recognize both species, albeit with a lower affinity for the mouse homolog. For immunoblot and immuno-fluorescence analysis, anti-HVCN1 was affinity-purified from rabbit serum (specificity control, Supplementary Fig. 1
Tissue and cell immunostaining
Tonsil tissue was cleared of paraffin and rehydrated by incubation in xylene and then was placed in industrial methylated spirits, followed by distilled water. Slides were subjected to heat-induced epitope retrieval in Tris-EDTA buffer (16 mM Tris and 6 mM EDTA, pH 9.0) and were allowed to cool gradually in ultrapure H2O. Tissue was blocked for 10 min in 3% (vol/vol) H2O2 and was washed in PBS. Antibodies applied to the sections were anti-HVCN1; anti-CD20 and anti-CD3 (LP1 and F7.2.38, respectively; both from Dako Cytomation); and anti-CD68 (generated by the K. Pulford laboratory). Slides were analyzed with a Zeiss laser-capture microscope. Use of human tissue was with approval from the ethical committee of University Hospitals Leicester.
For immunostaining of purified human B cells, cells were washed in ice-cold PBS after activation at 37 °C and were allowed to adhere to poly-L-lysine-coated slides for a few minutes before fixation in 4% (vol/vol) paraformaldehyde. After being washed, cells were incubated with primary antibodies (rabbit polyclonal anti-HVCN1 (generated in-house) and anti-EEA1 (610456), anti-HLA-DR (G46-6) and anti-HLA-DM (MaP.DM1; BD Pharmingen)) in PBS containing 10% (vol/vol) rat serum and 0.1% (wt/vol) saponin and then fluorescence-labeled secondary antibodies (Invitrogen). The DNA-intercalating dye DAPI (4,6-diamidino-2-phenylindole) was used to label nuclei before samples were mounted with ProLong Gold antifade reagent (Invitrogen). Cells were analyzed on a Zeiss laser-capture microscope at a magnification of ×100.
Flow cytometry analysis and flow sorting
Mouse cells were collected from the spleen, inguinal lymph nodes, bone marrow and peritoneal cavity. Cell phenotype was assessed by staining of cells for 1 h on ice with the following antibodies: fluorescein isothiocyanate-conjugated anti-CD21 (eBio8D9), anti-CD5 (53-7.3), anti-IgM (II/41) and anti-IgD (11–26); phycoerythrin-indotricarbocyanine-conjugated anti-CD23 (B3B4); and phycoerythrin-conjugated anti-IgM (II/41) and anti-B220 (RA3-6B2; all from eBioscience). Stained cells were analyzed on a FACSCanto (BD) with DIVA software. For analysis of HVCN1 protein expression, B cell populations were sorted on a BD FACSAria II (BD). Pro-B cell, pre-B cell and immature B cell populations were sorted from bone marrow cells purified with anti-CD19 microbeads and were stained for IgD, IgM, B220 and CD25. IgD+IgM+ mature B cells were excluded. Marginal zone cells were sorted from splenic cells purified with anti-CD19 microbeads and were stained for CD21 and CD23. B-1a cells were sorted from peritoneal cavity cells purified with anti-CD19 microbeads and were stained for IgM and CD5.
Cells were lysed for 15 min on ice in a solution of 1% (wt/vol) CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate hydrate), 20 mM HEPES, pH 7.6, 137 mM NaCl, 2.5 mM NaF, 1 mM Na3O4, 2 mM EDTA and a protease inhibitor `cocktail' (Sigma Aldrich). After pelleting of nuclei and cell debris, supernatants were cleared with protein G Sepharose beads (GE Healthcare) and/or protein G Sepharose beads conjugated to mouse or rat IgG (Santa Cruz Biotechnology) before being incubated with anti-Myc (9E10; Cell Signaling Technology) or antibody to immunoglobulin-associated-β (AT107-2; Serotec) conjugated to protein G Sepharose beads. Beads were washed extensively with lysis buffer before being resuspended in 2× Laemmli buffer and analyzed by immunoblot. Nonreducing conditions were used to avoid overlap of immunoglobulin-associated-β and HVCN1 bands with light chains derived from the antibodies used for the immunoprecipitation.
Detection of ROS
Mouse B cells were prewarmed at 37 °C before addition of the chemiluminescent reagent Diogenes (National Diagnostics). After 10 min of incubation at 37 °C, phorbol 12-myristate 13-acetate (1 μM) or anti-IgM F(ab′)2 (20 μg/ml; Jackson Immunoresearch) was added to the cells and luminescence was monitored on a luminometer at 5-minute intervals. Alternatively, total ROS were measured by incubation of cells for 20 min at 20 °C in the dark with 10 μM DCFDA (2′,7′-dichlorodihydrofluorescein diacetate; Invitrogen). ROS emission was measured at 488 nm on a FACSCanto (BD) with DIVA software. For treatment with rotenone and DPI, cells were incubated with these reagents for 10 min at 20 °C before DCFDA staining. ROS production in the vicinity of BCR was detected by conjugation of an anti-IgM F(ab′)2 fragment to OxyBURST Green H2DCFDA, succinimidyl ester, according to the manufacturer's instructions (Invitrogen). Cells were then incubated for 30 min on ice with F(ab′)2 anti-IgM (20 μg/ml), then were washed and allowed to become activated at 37 °C. ROS emission was measured at 488 nm on a FACSCanto (BD) with DIVA software.
mobilization in splenic wild-type and HVCN1-deficient B cells was measured as described52
Oxidation of SHP-1
SHP-1 oxidation was analyzed as described29
, except in place of incubation with pervanadate for 1 h at 4 °C, samples were incubated with 0.4 mM PEO-iodoacetyl–biotin (PEO-IAA; Pierce). Oxidized immunoprecipitated SHP-1 was detected with horseradish peroxidase–streptavidin (GE Healthcare).
Oxygen consumption and glycolysis
The oxygen consumption rate and extracellular acidification rate were measured with a XF24 analyzer36
according to the manufacturer's recommendations (Seahorse Bioscience). Cells were activated for 24 h with F(ab′)2
anti-IgM and were cultured in a humidified incubator at 37 °C.
Student's t-test was used for statistical analysis unless otherwise specified. The software GraphPad Prism 5 was used for this analysis.