Ig-transgenic MD4 (Jax 2595) mice were originally
purchased from the Jackson Laboratory. Cd79acre/cre
mice on the B6 background were kindly provided
as a gift by M. Reth. The Efnb1fl/fl line was backcrossed to B6 for more than 10 generations before
inter-breeding with other relevant alleles on the
B6 background. Relevant mice were interbred to
obtain dsRed-expressing OT-II mice, CFP-expressing
MD4 mice, Cd79a+/creEfnb1fl/y or Cd79a+/creEfnb1fl/fl
B6 mice, CFP-expressing Cd19+/creEfnb1fl/y or
Cd19+/creEfnb1fl/fl MD4 mice. All mice were maintained under specific pathogen-free conditions,
and used in accordance of governmental and institutional guidelines for animal welfare.
For sheep red blood cell (SRBC, Zhengzhou
Baiji, China) immunization, 1 ml of SRBC suspension was washed three times in cold phosphate-buffered saline (PBS) and intraperitioneally
injected into each mouse in a volume of 300 ml.
For some experiments, mice were immunized
intraperitoneally with 100 mg of NP-KLH (NP20
or higher conjugate ratios, Biosearch Technologies) mixed with 1 mg of lipopolysaccharide
(LPS) (Sigma) in alum (Thermo Scientific). For
measuring GC responses by MD4 and OT-II cells,
30 mg of HEL-OVA conjugate antigen made by
the Solulink cross-linking kit (17) or 130 mg of
conjugate antigen made by glutaraldehyde (57)
was mixed with 1 mg of LPS in alum and then
used to subcutaneously immunize the mice.
Cell isolation, culture and
B and CD4 T lymphocytes were isolated from
pooled spleens and lymph nodes by CD19 or CD4
Microbeads (Miltenyi Biotec) and activated with
1 mg/ml LPS or plate-bound anti-CD3 and anti-
CD28 (BioXcell), respectively. One or 2 days after
activation, T or B cells were spin-infected (1500g)
in the six-well plate at 7.5 × 106 cells per well with
appropriate viral supernatants in the presence of
1 mg/ml polybrene (Sigma) for 2hours at 32°C.
Infected T cells were expanded in 40 U/ml IL-2
(Peprotech) for 3 days before being used for adoptive transfer. Infected B cells were directly used
for conjugated assay.
EPHB4, EPHB6, and EFNB1
overexpression and knockdown
The EFNB1, EPHB4, and EPHB6 cDNA were cloned
from B6 splenocytes or the NIH3T3 cell line into the
murine stem cell virus (MSCV)–based, GFP-, or RFP-tagged expression vectors as used previously (7, 27).
The microRNA-based, Ametrine-tagged, shRNA
expression MSCV-LMP vector was a kind gift
from S. Crotty and Y.-C. Liu. The gene-specific
shRNA sequences were obtained from the CSHL
website ( http://katahdin.cshl.org/homepage/siRNA/
RNAi.cgi?type=shRNA), and knockdown efficien-cies were validated by quantitative RT-PCR. Verified oligonucleotidess are as follows: EPHB4
EPHB4 R′: TCCGAGGCAG TAGGCAAAGAGACC-CTG-TTGAACACAAATACATCTGTGGCTTCACT;
EPHB6 oligo F′: TGCTGTTGACA-GTGAGCGACC-TGATACTCTCCAGGCTGAAAGTGAAGCCACAG-ATGT; EPHB6 oligo R′: TCCGAGGCAGTAGG-CAGCCTGATACTCTCCAGGCTGAATA-CATCTG-TGGCTTCACT.
To measure T cell dynamics by live imaging or distribution in and around EFNB1-deficient or -sufficient
GCs by static imaging, 8 × 105 Cd19+/creEfnb1fl/y or
Cd19+/creEfnb1+/y nonfluorescent or CFP-expressing
MD4 B cells were cotransferred with 105
GFP-expressing OT-II cells to B6 recipients, which were
then immunized as described above. When retrovirally transduced OT-II T cells were involved, GFP-or Ametrine-expressing T cells were enriched by
flow sorting of Ficoll-isolated live cells of the infected T cell culture. Sorted cells were rested in
complete RPMI-1640 for 2 hours at 37°C before
intravenous injection of 106 transduced OT-II
cells and 5 × 105 MD4 B cells per B6 recipient.
These cells were rested in recipient mice for at
least 1 day before HEL-OVA immunization as
The splenic or lymph node single-cell suspension
was incubated in MACS buffer [PBS supplemented
with 1% fetal bovine serum (FBS) and 5mM
Lu et al., Science 356, eaai9264 (2017) 19 May 2017 7 of 10
Fig. 4. EFNB1 regulates plasma cell generation. (A) Typical CD4 T cell distributions in follicles
containing GCs after NP-KLH immunization (left) and GFDRs (right); data pooled from two
independent experiments; **P < 0.01. (B to D) GC, SPPC, and BMPC formation after NP-KLH
immunization of mice of the indicated genotypes; shown are FACS profiles (left) and quantitation
(right) of GCs on day 14 (B), SPPCs on day 14 (C), and BMPCs on day 21 (D); each symbol represents
one mouse, with data pooled from three independent experiments; *P < 0.05 by Mann-Whitney tests.
(E and F) Representative FACS profiles (E) showing competitive contributions by CD45.1 wild-type cells
and CD45.2 Cd79a+/creEfnb1fl/y or CD45.2 Cd79a+/creEfnb1+/y cells to non-GC B cell (CD4–CD19+GL7–Fas–),
total B cell (CD4–CD19+), GC (CD4–CD19+GL7+Fas+), SPPC (CD4–CD19+B220loCD138+), or
BMPC (IgM–IgD–Gr-1–B220loCD138+) compartments 21 days after NP-KLH immunization, and
(F) competitive competencies of CD45.2 cells in the two types of mixed chimera contributing to
different B cell compartments; the competitive competency is defined as the CD45.2/CD45.1
ratio in the indicated compartment normalized against the same ratio in the non-GC mature B cell
compartment; data pooled from two independent experiments; lines denote the mean values;
*P < 0.05 by Mann-Whitney tests.