In vivo T cell proliferation and
TFHdifferentiation
To assess proliferation, CD4+T cells from OT-II
Dock8−/−(CD45.2+)orOT-IIWT(CD45.1.2+)
mice were prepared from the spleen and lymph
nodes by negative selection using the EasySep
CD4 T Cell Isolation kit (STEMCELL Technolo-
gies) according to the manufacturer’s instruc-
tions. For T cell proliferation assays, OTII cells
were labeled with 1mMcarboxyfluoresceindi-
acetate succinimidyl ester (CFSE) before transfer
in to CD45.1+recipient mice and CFSE dilution
was used to assess proliferation. For TFHcell
identification, OT-II cells were not labeled with
CFSE and were gated using CD44+CXCR5+PD1+.
A total of 2 × 10^5 purified OTII cells were
transferred into mice by retro-orbital injection.
Mice were intranasally immunized 24 hours
later with 10mg of NP-OVA (Biosearch) and 2mg
of LPS (Sigma). MedLNs were harvested 3 days
(for T cell proliferation) or 6 days after immu-
nization (for TFHcell analyses). Single–cell sus-
pensions were prepared, stained, and then
analyzed on an LSRII flow cytometer (BD
Biosciences).
Statistical analysis
ANOVA or Student’sttest were performed on
normally distributed data (analyzed by Shapiro-
Wilk test). Mann-WhitneyUor Kruskal-Wallis
Htests were used otherwise. AP-value of <0.05
was considered significant. Data were analyzed
with Prism 7 (GraphPad Software).
REFERENCES AND NOTES
- H. C. Oettgen, R. S. Geha, IgE regulation and roles in asthma
pathogenesis.J. Allergy Clin. Immunol. 107 , 429–441 (2001).
doi:10.1067/mai.2001.113759; pmid: 11240941 - H. Mita, H. Yasueda, K. Akiyama, Affinity of IgE antibody to
antigen influences allergen-induced histamine release.
Clin. Exp. Allergy 30 , 1583–1589 (2000). doi:10.1046/
j.1365-2222.2000.00921.x; pmid: 11069567 - R. Suzukiet al., Molecular editing of cellular responses by the
high-affinity receptor for IgE.Science 343 , 1021–1025 (2014).
doi:10.1126/science.1246976; pmid: 24505132 - J. Wanget al., Correlation of IgE/IgG4 milk epitopes and
affinity of milk-specific IgE antibodies with different
phenotypes of clinical milk allergy.J. Allergy Clin. Immunol.
125 , 695–702.e6 (2010). doi:10.1016/j.jaci.2009.12.017;
pmid: 20226304 - J. S. Heet al., IgG1 memory B cells keep the memory of IgE
responses.Nat. Commun. 8 , 641 (2017). doi:10.1038/
s41467-017-00723-0; pmid: 28935935 - H. Xiong, J. Dolpady, M. Wabl, M. A. Curotto de Lafaille,
J. J. Lafaille, Sequential class switching is required for the
generation of high affinity IgE antibodies.J. Exp. Med. 209 ,
353 – 364 (2012). doi:10.1084/jem.20111941; pmid: 22249450 - K. Shimodaet al., Lack of IL-4-induced Th2 response and IgE
class switching in mice with disrupted Stat6 gene.Nature 380 ,
630 – 633 (1996). doi:10.1038/380630a0; pmid: 8602264 - K. Takedaet al., Essential role of Stat6 in IL-4 signalling.
Nature 380 , 627–630 (1996). doi:10.1038/380627a0;
pmid: 8602263 - J. Zhuet al., Conditional deletion of Gata3 shows its essential
function in TH1-TH2 responses.Nat. Immunol. 5 , 1157– 1165
(2004). doi:10.1038/ni1128; pmid: 15475959 - T. Kobayashi, K. Iijima, A. L. Dent, H. Kita, Follicular helper
T cells mediate IgE antibody response to airborne allergens.
J. Allergy Clin. Immunol. 139 , 300–313.e7 (2017). doi:10.1016/
j.jaci.2016.04.021; pmid: 27325434 - A. P. Meli, G. Fontés, C. Leung Soo, I. L. King, T follicular helper
cell-derived IL-4 is required for IgE production during intestinal
helminth infection.J. Immunol. 199 , 244–252 (2017).
doi:10.4049/jimmunol.1700141; pmid: 28533444
12. A. Noble, J. Zhao, Follicular helper T cells are responsible for
IgE responses to Der p 1 following house dust mite
sensitization in mice.Clin. Exp. Allergy 46 , 1075–1082 (2016).
doi:10.1111/cea.12750; pmid: 27138589
13. J. J. Dolenceet al., Airway exposure initiates peanut allergy by
involving the IL-1 pathway and T follicular helper cells in mice.
J. Allergy Clin. Immunol. 142 ,1144–1158.e8 (2018).
doi:10.1016/j.jaci.2017.11.020; pmid: 29247716
14. H. E. Lianget al., Divergent expression patterns of IL-4 and
IL-13 define unique functions in allergic immunity.
Nat. Immunol. 13 ,58–66 (2012). doi:10.1038/ni.2182;
pmid: 22138715
15. R. Moritaet al., Human blood CXCR5+CD4+T cells are
counterparts of T follicular cells and contain specific subsets
that differentially support antibody secretion.Immunity 34 ,
108 – 121 (2011). doi:10.1016/j.immuni.2010.12.012;
pmid: 21215658
16. Y. Haradaet al., The 3′enhancer CNS2 is a critical regulator of
interleukin-4-mediated humoral immunity in follicular helper
T cells.Immunity 36 , 188–200 (2012). doi:10.1016/
j.immuni.2012.02.002; pmid: 22365664
17. A. Sahooet al., Batf is important for IL-4 expression in
T follicular helper cells.Nat. Commun. 6 , 7997 (2015).
doi:10.1038/ncomms8997; pmid: 26278622
18. P. Vijayanandet al., Interleukin-4 production by follicular
helper T cells requires the conserved Il4 enhancer
hypersensitivity site V.Immunity 36 , 175–187 (2012).
doi:10.1016/j.immuni.2011.12.014; pmid: 22326582
19. A. Williamset al., Hypersensitive site 6 of the Th2 locus control
region is essential for Th2 cytokine expression.Proc. Natl.
Acad. Sci. U.S.A. 110 , 6955–6960 (2013). doi:10.1073/
pnas.1304720110; pmid: 23569250
20. J. Zhu, T helper 2 (Th2) cell differentiation, type 2 innate
lymphoid cell (ILC2) development and regulation of
interleukin-4 (IL-4) and IL-13 production.Cytokine 75 ,
14 – 24 (2015). doi:10.1016/j.cyto.2015.05.010;
pmid: 26044597
21. J. S. Weinsteinet al., TFH cells progressively differentiate to
regulate the germinal center response.Nat. Immunol. 17 ,
1197 – 1205 (2016). doi:10.1038/ni.3554;pmid: 27573866
22. I. Yusufet al., Germinal center T follicular helper cell IL-4
production is dependent on signaling lymphocytic activation
molecule receptor (CD150).J. Immunol. 185 , 190–202 (2010).
doi: 10 .4049/jimmunol.0903505; pmid: 20525889
23. X. Liuet al., Bcl6 expression specifies the T follicular helper cell
program in vivo.J. Exp. Med. 209 , 1841–1852 (2012).
doi:10.1084/jem.20120219; pmid: 22987803
24. H. C. Su, H. Jing, Q. Zhang, DOCK8 deficiency.Ann.
N. Y. Acad. Sci. 1246 ,26–33 (2011). doi:10.1111/
j.1749-6632.2011.06295.x; pmid: 22236427
25. H. C. Su, H. Jing, P. Angelus, A. F. Freeman, Insights into
immunity from clinical and basic science studies of DOCK8
immunodeficiency syndrome.Immunol. Rev. 287 ,9–19 (2019).
doi:10.1111/imr.12723; pmid: 30565250
26. J. K. Krishnaswamyet al., Migratory CD11b+conventional
dendritic cells induce T follicular helper cell-dependent
antibody responses.Sci. Immunol. 2 , eaam9169 (2017).
doi:10.1126/sciimmunol.aam9169; pmid: 29196450
27. K. L. Randallet al., Dock8 mutations cripple B cell
immunological synapses, germinal centers and long-lived
antibody production.Nat. Immunol. 10 , 1283–1291 (2009).
doi:10.1038/ni.1820; pmid: 19898472
28. F. D. Finkelman, M. E. Rothenberg, E. B. Brandt, S. C. Morris,
R. T. Strait, Molecular mechanisms of anaphylaxis: Lessons
from studies with murine models.J. Allergy Clin. Immunol. 115 ,
449 – 457 (2005). doi:10.1016/j.jaci.2004.12.1125;
pmid: 15753886
29. E. Janssenet al., Dedicator of cytokinesis 8-deficient patients
have a breakdown in peripheral B-cell tolerance and
defective regulatory T cells.J. Allergy Clin. Immunol. 134 ,
1365 – 1374 (2014). doi:10.1016/j.jaci.2014.07.042;
pmid: 25218284
30. A. K. Singhet al., DOCK8 regulates fitness and function of
regulatory T cells through modulation of IL-2 signaling.
JCI Insight 2 , e94275 (2017). doi:10.1172/jci.insight.94275;
pmid: 28978795
31. D. Zotoset al., IL-21 regulates germinal center B cell
differentiation and proliferation through a B cell-intrinsic
mechanism.J. Exp. Med. 207 , 365–378 (2010). doi:10.1084/
jem.20091777; pmid: 20142430
32. K. Ozakiet al., A critical role for IL-21 in regulating
immunoglobulin production.Science 298 , 1630–1634 (2002).
doi:10.1126/science.1077002; pmid: 12446913
33. A. Sutoet al., Interleukin 21 prevents antigen-induced IgE
production by inhibiting germ line Cetranscription of
IL-4-stimulated B cells.Blood 100 , 4565–4573 (2002).
doi:10.1182/blood-2002-04-1115;pmid: 12393685
34. N. Woodet al., IL-21 effects on human IgE production in
response to IL-4 or IL-13.Cell. Immunol. 231 , 133–145 (2004).
doi:10.1016/j.cellimm.2005.01.001; pmid: 15919378
35. K. Takatsu, T. Kouro, Y. Nagai, Interleukin 5 in the link between
the innate and acquired immune response.Adv. Immunol. 101 ,
191 – 236 (2009). doi:10.1016/S0065-2776(08)01006-7;
pmid: 19231596
36. M. Kubo, T follicular helper and TH2 cells in allergic responses.
Allergol. Int. 66 , 377–381 (2017). doi:10.1016/
j.alit.2017.04.006; pmid: 28499720
37. M. Mohrs, K. Shinkai, K. Mohrs, R. M. Locksley, Analysis of type
2 immunity in vivo with a bicistronic IL-4 reporter.Immunity 15 ,
303 – 311 (2001). doi:10.1016/S1074-7613(01)00186-8;
pmid: 11520464
38. N. Debeuf, E. Haspeslagh, M. van Helden, H. Hammad,
B. N. Lambrecht, Mouse models of asthma.Curr. Protoc.
Mouse Biol. 6 , 169–184 (2016). pmid: 27248433
39. X. M. Liet al., A murine model of peanut anaphylaxis: T- and
B-cell responses to a major peanut allergen mimic human
responses.J. Allergy Clin. Immunol. 106 , 150–158 (2000).
doi:10.1067/mai.2000.107395; pmid: 10887318
40. D. Chianget al., Single-cell profiling of peanut-responsive
T cells in patients with peanut allergy reveals heterogeneous
effector TH2 subsets.J. Allergy Clin. Immunol. 141 , 2107– 2120
(2018). doi:10.1016/j.jaci.2017.11.060; pmid: 29408715
41. Y. Wanget al., IgE sequences in individuals living in an area of
endemic parasitism show little mutational evidence of
antigen selection.Scand. J. Immunol. 73 , 496–504 (2011).
doi:10.1111/j.1365-3083.2011.02525.x; pmid: 21284686
42. G. J. McKenzieet al., Impaired development of Th2 cells in
IL-13-deficient mice.Immunity 9 , 423–432 (1998).
doi:10.1016/S1074-7613(00)80625-1; pmid: 9768762
43. G. J. McKenzie, P. G. Fallon, C. L. Emson, R. K. Grencis,
A. N. McKenzie, Simultaneous disruption of interleukin (IL)-4
and IL-13 defines individual roles in T helper cell type 2-
mediated responses.J. Exp. Med. 189 , 1565–1572 (1999).
doi:10.1084/jem.189.10.1565; pmid: 10330435
44. T. R. Ramalingamet al., Unique functions of the type II
interleukin 4 receptor identified in mice lacking the interleukin
13 receptor alpha1 chain.Nat. Immunol. 9 ,25–33 (2008).
doi:10.1038/ni1544; pmid: 18066066
45. A. Ballesteros-Tatoet al., T follicular helper cell plasticity
shapes pathogenic T helper 2 cell-mediated immunity to
inhaled house dust mite.Immunity 44 , 259–273 (2016).
doi:10.1016/j.immuni.2015.11.017; pmid: 26825674
46. A. Glatman Zaretskyet al., T follicular helper cells differentiate
from Th2 cells in response to helminth antigens.J. Exp. Med.
206 , 991–999 (2009). doi:10.1084/jem.20090303;
pmid: 19380637
47. S. Nakayamadaet al., Early Th1 cell differentiation is marked
by a Tfh cell-like transition.Immunity 35 , 919–931 (2011).
doi:10.1016/j.immuni.2011.11.012; pmid: 22195747
48. S.Keleset al., Dedicator of cytokinesis 8 regulates signal
transducer and activator of transcription 3 activation
and promotes TH17 cell differentiation.J. Allergy Clin. Immunol.
138 , 1384–1394.e2 (2016). doi:10.1016/j.jaci.2016.04.023;
pmid: 27350570
49.C.J.Kearney,K.L.Randall,J.Oliaro,DOCK8regulates
signal transduction events to control immunity.Cell. Mol.
Immunol. 14 ,406–411 (2017). doi:10.1038/cmi.2017.9;
pmid: 28366940
50. H. Wuet al., An inhibitory role for the transcription factor
Stat3 in controlling IL-4 and Bcl6 expression in follicular helper
T cells.J. Immunol. 195 , 2080–2089 (2015). doi:10.4049/
jimmunol.1500335; pmid: 26188063
51. A. Erazoet al., Unique maturation program of the IgE response
in vivo.Immunity 26 , 191–203 (2007). doi:10.1016/
j.immuni.2006.12.006; pmid: 17292640
52. M. Yazdanbakhsh, P. G. Kremsner, R. van Ree, Allergy,
parasites, and the hygiene hypothesis.Science 296 , 490– 494
(2002). doi:10.1126/science.296.5567.490; pmid: 11964470
53. J. S. Heet al., Biology of IgE production: IgE cell differentiation
and the memory of IgE responses.Curr. Top. Microbiol.
Immunol. 388 ,1–19 (2015). doi:10.1007/978-3-319-13725-4_1;
pmid: 25553792
54. C. M. Fitzsimmons, F. H. Falcone, D. W. Dunne, Helminth
allergens, parasite-specific IgE, and its protective role in
human immunity.Front. Immunol. 5 , 61 (2014). doi:10.3389/
fimmu.2014.00061; pmid: 24592267
Gowthamanet al.,Science 365 , eaaw6433 (2019) 30 August 2019 13 of 14
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