when O T-II TFH recruitment and retention were
exaggerated in the EFNB1-deficient MD4 GC (Fig.
2), the abundance of total OT-II cells (fig. S5A)
and fractional abundance of TFH cells (fig. S5B),
particularly when defined by PD-1 levels, also
became significantly higher, consistent with in-
creased antigen exposure and higher levels of ac-
tivation locally. Indeed, CXCR5 expression on CD4
T cells is highly responsive to TCR signaling (35)
and can be enhanced by increased antigen stim-
ulation (36). Similarly, fractional abundance of
EPHB6- but not EPHB4-suppressed OT-II TFH cells
was increased after the formation of wild-type
MD4 GCs (fig. S6, A and B). However, CXCR5
overexpression on OT-II T cells did not enhance
Lu et al., Science 356, eaai9264 (2017) 19 May 2017 3 of 10
Fig. 2. EFNB1 repulsively regulates TFH
positioning and trafficking in GCs. (A to C) TFH
distribution in follicles and GCs, 7 days after
HEL-OVA immunization of wild-type mice receiving
GFP-expressing OT-II cells and nonfluorescent
MD4 B cells of the indicated Efnb1 genotypes.
(A) Representative images (>50 GC-containing
follicles from more than 10 mice from more than
three independent experiments); (B) TFH densities
in individual GCs and follicles of indicated types;
(C) GC-FM distribution ratios (GFDRs), defined as
the ratio between the TFH density in a GC normalized
against the TFH density in the corresponding FM that
encompasses the GC. Each circle represents one
follicle per GC; data pooled from four independent
experiments, in each of which tissue sections from
four or five mice per condition were analyzed; lines
denote the mean values; *P < 0.05, ***P < 0.001.
(D to G) TFH dynamics around EFNB1-sufficient and
-deficient GCs, as visualized using dsRed-expressing
OT-II cells and CFP-expressing MD4 B cells of the
indicated genotypes in B6 recipients 7 days after
immunization. (D) Typical distribution patterns of
OT-II cells as intravitally visualized in the two types of
GCs. (E) TFH migratory tracks overlaid on the two
types of GC volumes; incoming cells that migrated
from the follicle into the GC are highlighted in red, and
those that migrated back to the follicle are in green
(upper row); outgoing cells that reached the GC
border from within but then migrated back into the
GC are highlighted in red, and those that went further
out to the follicle are highlighted in green (bottom
row). (F) Frequencies of incoming migratory tracks
that ended in the follicle or the GC or the 10-mm buffer
zone in-between (P < 0.05, c2 test); (G) Frequencies
of outgoing migratory tracks that ended in the
follicle or the GC or the 10-mm buffer zone in-between
(P < 0.0001, c2 test); n denotes the total number
of incoming or outgoing tracks; data are pooled
from two independent experiments that each
involved at least three separate mice per condition.
(H and I) Conjugation between OT-II Tcells and
EFNB1- or control vector–transduced B cells that
were pulsed with the indicated concentrations of the
OVA323-339 peptide. (H) Representative fluorescence-activated cell sorting (FACS) plots. (I) Mean ± SEM
of conjugate frequencies from three independent
experiments. *P < 0.05. (J and K) Interactions
between dsRed-expressing OT-II TFH cells and
CFP-expressing Cd19+/creEfnb1fl/y or Cd19+/creEfnb1+/y
MD4 GC B cells in GCs that contained ~95%
nonfluorescent MD4 B cells. (J) Time-lapse images of
two OT-II TFH cells (red) differentially interacting with
a wild-type (green) and a Cd19+/creEfnb1fl/y (blue)
MD4 GC B cell, with the two pairs highlighted by
white arrowheads, as in corresponding Movie 3.
(K) The surface engagement index of OT-II TFH cells
interacting with GC B cells of the indicated
genotypes; data were pooled from three independent
experiments, with each involving at least three mice.
Red lines denote means; ns, not significant.
