Science - USA (2019-08-30)

Cd4CreBcl6fl/flandIl13−/−mice as donors. TFH
cells in these chimeras did not produce IL-13
but still made IL-4 to Alt+OVA immunization
(Fig.7C).Like13CreBcl6fl/flmice, TFH-Il13−/−
chimeric mice produced comparable titers of
total and low-affinity IgE to that of controls (Fig.
7, D and E) but had impaired high-affinity IgE
(Fig. 7F). The residual high-affinity IgE induced
in TFH-Il13−/−mice was unable to elicit a PCA re-
sponse (Fig. 7G). Thus, two complementary ex-

perimental approaches revealed that TFH13 cells

and the IL-13 they produce are both required for

the induction of anaphylactic IgE to allergens.

Discussion

The signals that instruct B cells to make high-

affinity IgE remain unresolved. Our work dem-

onstrated that a rare TFH13 cell population

coexpressing IL-4, IL-5, IL-13, BCL6, and GATA3

was responsible for theproduction of high-

affinity anaphylactic IgE. Eliminating TFH 13

cells or TFHcell–derived IL-13 during allergen

immunization resulted in the abrogation of

high-affinity anaphylactic IgE while leaving low-

affinity IgE intact. Using scRNA-seq, we confirmed

that TFH13 cells are transcriptionally distinct

from TFH2andTH2cells.Previousworkdem-

onstrates that TH2andTFHcells can interconvert

during type 2 responses ( 45 , 46 ). A transitional

stage during TFHdevelopment may exist between

Gowthamanet al.,Science 365 , eaaw6433 (2019) 30 August 2019 9of14

Fig. 7. TFHcell–derived IL-13 is required for anaphylactic IgE
production.(A) Expression of IL-13Ra1 on MedLN GC B cells at day 9
after immunization withAlternariaand NP-OVA is shown as histogram
overlay (left) and summary bar graphs (right). Gating strategy as in
fig. S15B. (B) Day 8 MedLN lymphocytes fromAlternariaand NP-OVA
immunized WT mice were cultured witha-CD40 and IL-4 and/or IL-13 for
3 days, and IgE plasma cells were quantified by staining the cells for
intracellular IgE and CD138. (CtoG) Mixed bone marrow chimeric mice
were generated fromIl13−/−andCd4CreBcl6fl/fldonor bone marrow
(TFH-Il13−/−chimeric mice) or WT andCd4CreBcl6fl/fldonor bone marrow
(control chimeric mice). Chimeric mice were immunized and boosted
withAlternariaand NP20-OVA. (C) IL-4 and IL-13 expression by day 8 TFH
cells after boost from TFH-Il13−/−or control chimeras is shown as flow

cytometry plots (left) or summary bar graphs (right). (D to F) Day 8 post-

boost sera from TFH-Il13−/−or control chimeras were analyzed by ELISA

for (D) total IgE, (E) low-affinity IgE using NP-40 BSA, and (F) high-affinity

IgE using NP7-BSA. (G) Evans blue dye quantification after PCA assay

with day 9 post-boost sera from TFH-Il13−/−or control chimeras after

i.v. challenge with NP7-BSA. Each symbol indicates an individual mouse.

Error bars indicate SEM. Dotted lines in bar graphs represent (A)

IL-13Ra1 expression on naïve B cells or (E to G) background readings

of sera from naïve mice. Statistical tests: ANOVA (A and B); Student’s

ttest (C and G); Mann-WhitneyUtest (D to F). *P< 0.05, **P< 0.01,

***P< 0.001, ****P< 0.0001. Data are either from two pooled

experiments (D and F) or are representative of at least two independent

experiments with three to seven mice per group.

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