Document S1. Supplemental Experimental Procedures and

Immunity, Volume 38
Supplemental Information
Repression of the Transcription Factor Bach2
Contributes to Predisposition of IgG1 Memory B Cells
toward Plasma Cell Differentiation
Kohei Kometani, Rinako Nakagawa, Ryo Shinnakasu, Tomohiro Kaji, Andrei Rybouchkin, Saya
Moriyama, Koji Furukawa, Haruhiko Koseki, Toshitada Takemori, and Tomohiro Kurosaki
Figure S1 (Related to Figure 1)
Normal anti-NP IgM antibody production in the
absence of IgG1+ cells during secondary responses.
Figure S2
(Related to Figure 2) NP+IgG1 memory B cells derived from wild type
C57BL/6 mice exhibited a similar differentiation capability to those derived from B1-8
gene targeted mice.
Figure S3
(Related to Figure 3) Characterization of IgG1 B cells in chimeric mice
from IgG1-ES NTB2.
Figure S4
(Related to Figure 5) Generation of the Bach2flox/+ mice.
Figure S5
(Related to Figure 5) Expression of Bach2 in IgM memory B cells.
Figure S6
(Related to Figure 7) Verification of various inhibitors.
Supplemental Experimental Procedures
Supplemental References
Figure S1 Normal anti-NP IgM antibody production in the absence of IgG1+ cells
during secondary responses.
Cγ1-cre gene targeted mice (control) or Cγ1-cre x
iDTR (iDTR; IgG1) mice were immunized with NP-CGG in alum.
diphtheria toxin (DT) was injected i.p. for three consecutive days.
NP-CGG in PBS was injected i.p.
Each group consists of more than three mice
and representative data of two independent experiments are shown.
Related to Figure 1.
On day 60,
Sera were collected at the indicated time points and
anti-NP IgM was measured by ELISA.
mean ± SD.
Fifty six days later,
Data represent the
Figure S2
NP+IgG1 memory B cells derived from wild type C57BL/6 mice
exhibited a similar differentiation capability to those derived from B1-8 gene
targeted mice.
(A) NP+CD38+ IgG1 memory B cells were single cell sorted from
C57BL/6 mice immunized with NP-CGG in alum 30 days before.
synthesized and VH186.2-Cγ1 sequences were analyzed.
cDNA was
Fifty one samples were
sequenced and the number of mutations in VH186.2 gene are represented in the pie
chart.
The frequency of Trp33 to Leu substitution is indicated.
(B) Sorted
NP+CD38+ IgG1 memory B cells (~5000 NP-binding cells) were transferred to CGG in
alum-primed CD45.1 C57BL/6 mice and analyzed as described in Figure 2A.
As IgM
naive controls, in addition to the data of naive mature follicular NP+IgM B cells from
B1-8hi mice (already shown in Figure 2E), experiments were carried out by using those
B cells from gene targeted mice harboring B1-8 germline IgH gene (B1-8ge).
frequency of CD138+ cells among the donor cells was plotted in the graph.
represents the mean ± SD (n=3~4).
experiments.
Related to Figure 2.
The
Bar graph
Data are representative of two independent
Figure S3
Characterization of IgG1 B cells in chimeric mice from IgG1-ES NTB2.
(A) IgG1 and IgM expression in the bone marrow and spleen B cells of nuclear
transferred chimeric mice derived from IgG1-ES NTB2.
Cells are gated on BCR+.
The numbers in the plots indicate the percentages of each population in the gate.
(B) B
cell development of chimeric mice in the spleen was analyzed by flow cytometry.
(C)
IgH VDJ sequence of peripheral B cells derived from IgG1-ES (clone NTB2) B cells
was analyzed and compared with the canonical B1-8 (VH186.2-DFL16.1-JH2) germline
sequence.
(D) IgL VJ sequence of IgG1-ES NTB2 IgG1 B cells was analyzed and
compared with the Vλ1 and Jλ1 sequence.
(E) Bacterially expressed Fab of B1-8g1
IgH associated with IgG1-ES NTB2 Igλ1 (Figure S3D), B1-8hi, and B1-8ge associated
with germline type Igλ1 were titrated with NP-Cap.
Data were obtained at 25°C.
An
exothermic heat pulse observed after each injection of NP-Cap into the Fab solution is
shown in the insets.
Each area of this exothermic peak was integrated and the heat of
dilution of NP-Cap was subtracted from the integrated values.
The corrected heat was
divided by the moles of NP-Cap injected, and the resulting values were plotted as a
function of the molar ratio (NP-Cap/Fab).
nonlinear least–squares method.
The solid line represents best-fit using a
(F) The Ka (M-1) of each BCR for NP is listed.
Related to Figure 3.
Figure S4
Generation of the Bach2flox/+ mice. (A) The targeted locus of Bach2 is
depicted before and after removal by Cre recombinase.
solid triangles.
The loxP sites are indicated as
Exon 4 is flanked by two LoxP sites (triangle).
LA: long arm, SA:
short arm, Neor: neomycin-resistance gene, and DTA: diphtheria toxin A.
indicate primer position for PCR shown in below.
(B) Genomic DNA was prepared
from tail and subjected to PCR analysis for detecting floxed allele.
5.
Arrows
Related to Figure
Figure S5
Expression of Bach2 in IgM memory B cells.
naive NP+IgM+ B cells were sorted from B1-8hi mice.
(A) Mature follicular
NP+CD38+B1-8hi IgG1 memory
B cells or NP+CD38+B1-8hi IgM memory B cells were generated as shown in Figure 2A.
IgM memory B cells were sorted as
CD45.1+B220+IgG1-IgG2a/b-IgG3-IgA-CD38+CD21intCD23hiCD273+ cells (Tomayko et
al., 2010). (B) The amount of each mRNA in these cells was measured by RT-qPCR.
The data were normalized to the amount of Gapdh.
SD.
Bar graph represents the mean ±
(C) The amount of Bach2 protein in NP+IgM+ naive B cells, NP+CD38+IgM+
memory B cells, and NP+CD38+IgG1+ memory B cells was measured by intracellular
flow cytometric analysis.
experiments.
The data are representative of three independent
Related to Figure 5.
Figure S6
Verification of various inhibitors.
(A) Purified C57BL/6 spleen B cells
were treated with each inhibitor for 60 min (mTOR: Rapamycin (20 nM), ERK:
PD98059 (50 µM), JNK: SP600125 (10 µM), p38: SB203580 (10 µM), NF-κB: IKK
inhibitor (10 µM), AKT: API-2 (20 µM)). These cells were further cultured with
anti-CD40 antibody (2 µg/ml), IL-4 (10 ng/ml), and anti-IgM antibody (1 µg/ml) for 6
hr in the presence or absence of each inhibitor.
The phosphorylation status or total
amount of indicated protein was analyzed by Western blotting.
(B) Purified C57BL/6
spleen B cells were treated with a Foxo1 inhibitor (AS1842856; 1 µM) for 24 h.
The
amount of Sell and Klf2, both of which are Foxo1 target genes, was measured by
RT-qPCR.
(C) Purified C57BL/6 spleen B cells were retrovirally transduced with a
mock empty vector or Foxo1-CA mutant.
Infected GFP+ cells were sorted and
followed by measuring the amount of each gene by RT-qPCR.
the mean ± SD.
experiments.
Bar graph represents
The data were the representative of at least two independent
**, P<0.01.
Related to Figure 7
Supplemental Experimental Procedures
Mice
B1-8hi gene-targeted mice, Cγ1-cre gene-targeted mice, iDTR mice, and Cd79a-cre
mice were provided by Dr. M. Nussenzweig, Dr. K. Rajewsky and Dr. S. Casola, Dr. A.
Waisman, and Dr. M. Reth and Dr. E. Hobeika, respectively.
targeted mice were purchased from Taconic Firm.
Rosa26-ERT2-cre gene
B1-8hi gene targeted mice were
crossed with CD45.2 C57BL/6 or CD45.1 C57BL/6 and used differently depending on
the recipient mice.
To generate B1-8 germline gene targeted (B1-8ge) mice, the
VH186.2-DFL16.1-JH2 sequence was amplified by PCR from the genomic DNA of
B1-8hi gene targeted mice.
method (Stratagene).
TTG of codon Leu33 was changed to TGG (Trp) by a PCR
The B1-8 germline fragment was flanked with two LoxP sites
and subcloned into a targeting vector containing short and long arms of IgH homology
regions.
The targeting vector was electroporated into Bruce4 ES cells.
targeted clones were identified by PCR.
Correctly
The neomycin resistance gene was removed
by transiently transfecting a Cre expression vector. Targeted ES clones were injected
into blastocysts from BALB/c mice to obtain chimeric mice.
These chimeric mice
were then crossed with C57BL/6 mice to obtain germline transmitted animals.
Bach2
flox mice were established by homologous recombination with floxed exon 4 and
chimeric mice were obtained as described in Figure S4.
was removed by crossing CAG-Flippase transgenic mice.
The neomycin resistance gene
To generate IgG1-ES mice,
NP+Igλ+CD38+ IgG1 memory B cells were isolated from NP-CGG in alum-immunized
C57BL/6 mice as described below.
The nuclei of memory B cells were transferred to
nucleus-expelled BDF1 unfertilized eggs.
ES cell lines were established from these
eggs and injected into blastocysts of BALB/c mice to generate chimeric mice.
mice were maintained under specific pathogen-free conditions.
All the
The protocols for
animal experiments were approved by the RIKEN Animal Research Committee.
Immunization
For primary responses, mice were injected intraperitoneally (i.p.) with 100 µg of
NP-CGG precipitated with Imject alum (Thermo Fisher Scientific).
For recall
responses, 50 µg of soluble NP-CGG in PBS was injected i.p.
In vivo cell depletion
Mice were injected i.p. with 100 ng of diphtheria toxin (4 ng DT/g body weight; Sigma)
for three consecutive days.
The efficiency of depletion was measured by flow
cytometry 2 days after the last injection.
Adoptive transfers
For generating memory B cells, B cells were purified from spleens of CD45.1+B1-8hi
gene targeted mice by magnetic cell depletion using anti-CD43 microbeads and the
AutoMACS system (Miltenyi Biotech).
Purified B cells containing 1x105 NP-binding
B cells were transferred intravenously (i.v.) into C57BL/6 mice.
µg of NP-CGG precipitated with Imject alum was injected i.p.
On the next day, 100
Thirty days later,
spleen cells were collected and labeled with biotin-conjugated anti-IgM, IgD, DX5 (BD
Bioscience ), F4/80, Gr-1, TER119, CD90.2 (BioLegend), and CD5 (eBioscience)
antibodies followed by incubation with Streptavidin-microbeads (Miltenyi Biotech).
The labeled cells were depleted using a MACS column system (Miltenyi Biotech) and
NP-specific IgG1 memory B cells were sorted as
CD45.1+B220+IgG2a/b-IgG3-IgA-CD38+CD21intCD23hi cells using a FACSAria (BD
Bioscience).
In the case of C57BL/6 mice, NIP monomer-Dylight649 (Iris Biotech)
was used to purify NP-specific memory B cells.
NP-binding naive B cells were sorted
from B1-8 gene targeted mice as
B220+IgG1-IgG2a/b-IgG3-IgA-Igκ-CD38+CD21intCD23hi cells.
An aliquot of the cells
was stained with NP to calculate the frequency of NP-binding cells contained in the
sorted cells.
Sorted NP-binding cells (about 5000) were transferred to recipient mice
i.v. and the mice were boosted with 50 µg of soluble NP-CGG in PBS i.p. on the next
day.
Flow cytometric analysis
Single cell suspensions lysed of red blood cells were stained with
fluorochrome-conjugated antibodies.
(BD Bioscience).
Stained cells were analyzed using FACSCantoII
Anti-B220 (RA3-6B2), Fas (Jo2), IgM (II/41), IgG1 (A85-1)
IgG2a/b (R2-40), Igκ (187.1), Igλ (R26-46), Ly9.1 (30C7), CD21 (7G6), CD138
(281-2) antibodies were purchased from BD Bioscience.
Anti-CD93 (AA4.1), GL7
(GL7), MHC class II (M5/114.15.2), CD273 (122), CD23 (B3B4) antibodies were
purchased from eBioscience.
BioLegend.
Anti-CD80 (16-10A1) antibody was purchased from
NP-binding B cells were detected using NIP-BSA-PE, NIP-APC, or NIP
monomer-Dylight649.
surface molecules.
For intracellular staining, cells were first stained with cell
After washing, the cells were fixed and permeabilized using a
Foxp3 staining kit (eBioscience) followed by staining with anti-Pax5 (Santa Cruz
Biotechnology), anti-Bach2 antibody (established in our laboratory using GST-fusion
mouse Bach2131-291 protein as an immunogen), or phosho-S6 ribosomal protein (Cell
Signaling Technology).
The stained cells were analyzed by FACSCantoII (BD
Bioscience).
In vitro culture
Naive B cells were purified from spleens of C57BL/6 mice by magnetic cell depletion
using anti-CD43 microbeads and the AutoMACS system (Miltenyi Biotech).
cells were pre-treated with various inhibitors for 60 min.
These
The cells were further
cultured with anti-CD40 antibody (2 µg/ml; BioLegend), IL-4 (10 ng/ml; R&D), and
anti-IgM antibody (1 µg/ml; Jackson ImmunoResearch LABORATORIES) at 37°C
under 5% CO2 for 6 or 24 hr in the absence or presence of each inhibitor.
For
retrovirally gene transfer experiments, purified B cells were stimulated with above
stimulants for one day and infected with retrovirus produced from pMx-ires-gfp (mock)
or pMx-Foxo1-CA-ires-gfp plasmid using Plat-E packaging cells.
The cells were
further cultured for 48 hr and infected GFP+ cells were sorted for the assay using
FACSAria.
Enzyme-linked immunosorbent assay (ELISA)
Ninety six-well flat bottom plates were coated with 2 µg/ml of NP20-BSA followed by
blocking with 0.5% BSA in PBS.
overnight.
Serially diluted sera were incubated at 4°C
After washing with PBS containing 0.5% Tween-20, horseradish
peroxidase-conjugated anti-mouse IgM or IgG1 antibodies (SouthernBiotech) were
added to the wells.
SureBlue (KPL) was used as the substrate and absorbance at 450
nm was measured using a microplate reader (Bio-Rad).
For detecting high affinity
anti-NP IgG1 antibody, NP1-BSA was used as the capture antigen.
Quantitative RT-PCR
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's
instructions.
DNaseI (Invitrogen)-treated RNA was reverse transcribed using Super
Script III (Invitrogen).
Quantitative PCR was performed using SYBR Green
(Invitrogen) and the ABI StepOnePlus realtime PCR system (Applied Biosystems).
The following primers were used: 5’-atggtgaaggtcggtgtgaacggatttggc-3’,
5’-agcttcccattctcggcctggactgttctg-3’(gapdh); 5’-ccatcaggacaggacatggag-3’,
5’-ggcaagttccactatcctttgg-3’(pax5); 5’-catctcttcctctgcccagt-3’,
5’-agacatgccgttcaaaccat-3’(bach2); 5’-ctgcagcggcctgttctaca-3’,
5’-aaggtgctgagcgggagatg-3’(bcl6); 5’-ggctccactacccttatcctg-3’,
5’-gttgctttccgtttgtgtga-3’(prdm1); 5’- atgaacttggagacgggcagccggggctcag-3’,
5’-ctggcttgtcgatcccttctcggaacttgc-3’(irf4); 5’-tgacgaggttccagaggtg-3’,
5’-tgcagaggtgcacatagtctg-3’(xbp1).
RNAi gene silencing assay
For generating the gene silencing vector, the target sequence was inserted to the pMYs
retroviral vector using the BLOCK-iT polII miR RNAi system (Invitrogen).
For
generating retroviruses, the plasmid was transfected to Plat-E packaging cells using
FuGENE6 (Roche).
The medium was replaced over the next two days and the culture
supernatants were used for transductions.
For transduction, B1-8hi gene targeted mice
were injected with 2 mg of soluble NP-CGG and 8 h later spleen B cells were purified
using anti-CD43 magnetic beads and cultured with 2 µg/ml of anti-CD40 antibody for
24 h.
Then, the cells were transduced with retroviral culture supernatant containing 6
µg/ml of polybrene (SIGMA) by centrifugation at 1800 x g, 32°C for 90 minutes.
The
cells were cultured for two additional days and then transferred i.v. into C57BL/6 mice.
On the next day, the mice were injected i.p. with100 µg NP-CGG in alum and the cells
were analyzed four days later.
The transduced cells were detected as GFP+ cells and
analyzed by flow cytometry.
Bacterial expression of anti-NP Fabs
To examine NP-binding abilities of BCRs, their Fab regions were bacterially expressed.
Genes corresponding to their variable region were obtained from their cDNAs using
PCR with primers 5’-cagccatggcccaggtccaactgcagcag-3’ and
5’-cagccatggcccaggtccaactgcagcag-3’, and 5’-cagccatggcccaggtccaactgcagcag-3’ and
5’-cagccatggcccaggtccaactgcagcag-3’, for VH and VL gene, respectively.
The
amplified genes were cloned into expression plasmid, the details of which were
described previously.
The production and purification procedures for the Fabs were
the same as those previously described (Kuba et al., 2008).
Isothermal titration calorimetry (ITC)
ITC experiments were carried out on a MicroCal VP-ITC isothermal titration
calorimeter.
Fab proteins and NP-Cap (Biosearch Technologies) were dissolved in
degassed 50 mM sodium phosphate buffer, pH 7.0, containing 200 mM NaCl.
NP-Cap solution was injected into the Fab solution at 25°C.
The
The heat for each
injection was subtracted from the heat of dilution of the injectant, which was measured
by injecting the NP-Cap solution into the buffer used.
Each corrected heat value was
divided by the mole amounts of NP-Cap injected and the data were analyzed with
Origin software (OriginLab).
Statistical Analysis
Statistical analyses were performed using a two-tailed unpaired Student’s t-test.
Supplemental References
Kuba, H., Furukawa, A., Okajima, T., and Furukawa, K. (2008). Efficient bacterial
production of functional antibody fragments using a phagemid vector. Protein Expr.
Purif. 58, 292–300.
Tomayko, M.M., Steinel, N.C., Anderson, S.M., and Shlomchik, M.J. (2010). Cutting
Edge: Hierarchy of Maturity of Murine Memory B Cell Subsets. J. Immunol. 185,
7146–7150.

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