- S. Crotty, Follicular helper CD4 T cells (TFH).Annu. Rev.
Immunol. 29 , 621–663 (2011). doi:10.1146/annurev-immunol-
031210-101400; pmid: 21314428 - C. G. Vinuesa, M. A. Linterman, D. Yu, I. C. MacLennan,
Follicular helper T cells.Annu. Rev. Immunol. 34 , 335– 368
(2016). doi:10.1146/annurev-immunol-041015-055605;
pmid: 26907215 - S. Z. Josefowicz, L. F. Lu, A. Y. Rudensky, Regulatory T cells:
Mechanisms of differentiation and function.Annu. Rev.
Immunol. 30 , 531–564 (2012). doi:10.1146/annurev.
immunol.25.022106.141623; pmid: 22224781 - M. A. Lintermanet al., Foxp3+follicular regulatory T cells
control the germinal center response.Nat. Med. 17 , 975– 982
(2011). doi:10.1038/nm.2425; pmid: 21785433 - I. Wollenberget al., Regulation of the germinal center reaction
by Foxp3+follicular regulatory T cells.J. Immunol. 187 ,
4553 – 4560 (2011). doi:10.4049/jimmunol.1101328;
pmid: 21984700 - Y. Chunget al., Follicular regulatory T cells expressing Foxp3
and Bcl-6 suppress germinal center reactions.Nat. Med. 17 ,
983 – 988 (2011). doi:10.1038/nm.2426; pmid: 21785430 - P. T. Sage, A. H. Sharpe, T follicular regulatory cells in the
regulation of B cell responses.Trends Immunol. 36 , 410– 418
(2015). doi:10.1016/j.it.2015.05.005; pmid: 26091728 - V. R. Fonseca, F. Ribeiro, L. Graca, T follicular regulatory (Tfr)
cells: Dissecting the complexity of Tfr-cell compartments.
Immunol. Rev. 288 , 112–127 (2019). doi:10.1111/imr.12739;
pmid: 30874344 - P. Gonzalez-Figueroaet al., Follicular regulatory T cells
produce neuritin to regulate B cells.Cell 184 , 1775–1789.e19
(2021). doi:10.1016/j.cell.2021.02.027; pmid: 33711260 - T. Kornet al., Myelin-specific regulatory T cells accumulate
in the CNS but fail to control autoimmune inflammation.
Nat. Med. 13 , 423–431 (2007). doi:10.1038/nm1564;
pmid: 17384649 - Z. Shulmanet al., T follicular helper cell dynamics in germinal
centers.Science 341 , 673–677 (2013). doi:10.1126/
science.1241680; pmid: 23887872 - Y. Y. Wan, R. A. Flavell, Identifying Foxp3-expressing
suppressor T cells with a bicistronic reporter.Proc. Natl. Acad.
Sci. U.S.A. 102 , 5126–5131 (2005). doi:10.1073/
pnas.0501701102; pmid: 15795373 - D. J. Firl, S. E. Degn, T. Padera, M. C. Carroll, Capturing change
in clonal composition amongst single mouse germinal centers.
eLife 7 , e33051 (2018). doi:10.7554/eLife.33051; pmid: 30066671 - Z. L. Benetet al., CCL3 promotes germinal center B cells
sampling by follicular regulatory T cells in murine lymph nodes.
Front. Immunol. 9 , 2044 (2018). doi:10.3389/
fimmu.2018.02044; pmid: 30271404 - T. Okadaet al., Antigen-engaged B cells undergo chemotaxis
toward the T zone and form motile conjugates with helper
T cells.PLOS Biol. 3 , e150 (2005). doi:10.1371/journal.
pbio.0030150; pmid: 15857154 - T. A. Schwickertet al., A dynamic T cell-limited checkpoint
regulates affinity-dependent B cell entry into the germinal
center.J. Exp. Med. 208 , 1243–1252 (2011). doi:10.1084/
jem.20102477; pmid: 21576382 - D. Liuet al., T-B-cell entanglement and ICOSL-driven feed-
forward regulation of germinal centre reaction.Nature 517 ,
214 – 218 (2015). doi:10.1038/nature13803; pmid: 25317561 - Z. Shulmanet al., Dynamic signaling by T follicular helper cells
during germinal center B cell selection.Science 345 ,
1058 – 1062 (2014). doi:10.1126/science.1257861;
pmid: 25170154 - C. D. Allen, T. Okada, H. L. Tang, J. G. Cyster, Imaging of
germinal center selection events during affinity maturation.
Science 315 , 528–531 (2007). doi:10.1126/science.1136736;
pmid: 17185562 - R. L. Lindquistet al., Visualizing dendritic cell networks in vivo.
Nat. Immunol. 5 , 1243–1250 (2004). doi:10.1038/ni1139;
pmid: 15543150 - M. Aloulouet al., Follicular regulatory T cells can be specific
for the immunizing antigen and derive from naive T cells.
Nat. Commun. 7 , 10579 (2016). doi:10.1038/ncomms10579;
pmid: 26818004 - J. B. Winget al., A distinct subpopulation of CD25–T-follicular
regulatory cells localizes in the germinal centers.Proc. Natl.
Acad. Sci. U.S.A. 114 , E6400–E6409 (2017). doi:10.1073/
pnas.1705551114; pmid: 28698369 - M. Feuereret al., Genomic definition of multiple ex vivo
regulatory T cell subphenotypes.Proc. Natl. Acad. Sci. U.S.A.
107 , 5919–5924 (2010). doi:10.1073/pnas.1002006107;
pmid: 20231436
33. T. Chtanovaet al., T follicular helper cells express a distinctive
transcriptional profile, reflecting their role as non-Th1/Th2
effector cells that provide help for B cells.J. Immunol. 173 ,
68 – 78 (2004). doi:10.4049/jimmunol.173.1.68;
pmid: 15210760
34. Y. J. Liuet al., Mechanism of antigen-driven selection in
germinal centres.Nature 342 , 929–931 (1989). doi:10.1038/
342929a0; pmid: 2594086
35. A. R. Maceiraset al., T follicular helper and T follicular
regulatory cells have different TCR specificity.Nat. Commun. 8 ,
15067 (2017). doi:10.1038/ncomms15067; pmid: 28429709
36. S. Houet al., FoxP3 and Ezh2 regulate Tfr cell suppressive
function and transcriptional program.J. Exp. Med. 216 ,
605 – 620 (2019). doi:10.1084/jem.20181134; pmid: 30705058
37. A. R. Albrightet al., TGFbsignaling in germinal center B cells
promotes the transition from light zone to dark zone.J. Exp.
Med. 216 , 2531–2545 (2019). doi:10.1084/jem.20181868;
pmid: 31506281
38. T. E. Mandels, R. P. Phippsi, A. Abbot, J. G. Tew, The follicular
dendritic cell: Long term antigen retention during immunity.
Immunol. Rev. 53 , 29–59 (1980). doi:10.1111/j.1600-
065X.1980.tb01039.x; pmid: 6162778
39. K. Kretschmeret al., Inducing and expanding regulatory T cell
populations by foreign antigen.Nat. Immunol. 6 , 1219– 1227
(2005). doi:10.1038/ni1265; pmid: 16244650
40. J. B. Wing, W. Ise, T. Kurosaki, S. Sakaguchi, Regulatory T cells
control antigen-specific expansion of TFHcell number and
humoral immune responses via the coreceptor CTLA-4.
Immunity 41 , 1013–1025 (2014). doi:10.1016/j.
immuni.2014.12.006; pmid: 25526312
41. 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
42. M. A. Lintermanet al., IL-21 acts directly on B cells to regulate
Bcl-6 expression and germinal center responses.J. Exp. Med.
207 , 353–363 (2010). doi:10.1084/jem.20091738;
pmid: 20142429
43. S. Hanet al., Cellular interaction in germinal centers. Roles of
CD40 ligand and B7-2 in established germinal centers.
J. Immunol. 155 , 556–567 (1995). pmid: 7541819
44. A. K. Hadjantonakis, S. Macmaster, A. Nagy, Embryonic stem
cells and mice expressing different GFP variants for multiple
non-invasive reporter usage within a single animal.BMC
Biotechnol. 2 , 11 (2002). doi:10.1186/1472-6750-2-11;
pmid: 12079497
45. K. Vinterstenet al., Mouse in red: Red fluorescent protein
expression in mouse ES cells, embryos, and adult animals.
Genesis 40 , 241–246 (2004). doi:10.1002/gene.20095;
pmid: 15593332
46. A. J. Wolfet al., Initiation of the adaptive immune response
toMycobacterium tuberculosisdepends on antigen
production in the local lymph node, not the lungs.J. Exp.
Med. 205 , 105–115 (2008). doi:10.1084/jem.20071367;
pmid: 18158321
47. T. A. Shih, M. Roederer, M. C. Nussenzweig, Role of antigen
receptor affinity in T cell-independent antibody responses
in vivo.Nat. Immunol. 3 , 399–406 (2002). doi:10.1038/ni776;
pmid: 11896394
48. M. J. Barnden, J. Allison, W. R. Heath, F. R. Carbone, Defective
TCR expression in transgenic mice constructed using cDNA-
baseda- andb-chain genes under the control of heterologous
regulatory elements.Immunol. Cell Biol. 76 , 34–40 (1998).
doi:10.1046/j.1440-1711.1998.00709.x; pmid: 9553774
49. H. A. Zariwalaet al., A Cre-dependent GCaMP3 reporter
mouse for neuronal imaging in vivo.J. Neurosci. 32 ,
3131 – 3141 (2012). doi:10.1523/JNEUROSCI.4469-11.2012;
pmid: 22378886
50. E. Bettelliet al., Reciprocal developmental pathways for the
generation of pathogenic effector TH17 and regulatory T cells.
Nature 441 , 235–238 (2006). doi:10.1038/nature04753;
pmid: 16648838
51. T. Chinenet al., An essential role for the IL-2 receptor in Treg
cell function.Nat. Immunol. 17 , 1322–1333 (2016).
doi:10.1038/ni.3540; pmid: 27595233
52. R. Roozendaalet al., Conduits mediate transport of low-
molecular-weight antigen to lymph node follicles.Immunity 30 ,
264 – 276 (2009). doi:10.1016/j.immuni.2008.12.014;
pmid: 19185517
53. E. F. J. Meijeret al., Murine chronic lymph node window for
longitudinal intravital lymph node imaging.Nat. Protoc. 12 ,
1513 – 1520 (2017). doi:10.1038/nprot.2017.045;
pmid: 28683064
54. J. T. Jacobsen, G. D. Victora, Microanatomical labeling of
germinal center structures for flow cytometry using
photoactivation.Methods Mol. Biol. 1623 , 51–58 (2017).
doi:10.1007/978-1-4939-7095-7_4; pmid: 28589346
55. J. M. Taset al., Visualizing antibody affinity maturation in
germinal centers.Science 351 , 1048–1054 (2016).
doi:10.1126/science.aad3439; pmid: 26912368
56. P. Dashet al., Paired analysis of TCRaand TCRbchains at the
single-cell level in mice.J. Clin. Invest. 121 , 288–295 (2011).
doi:10.1172/JCI44752; pmid: 21135507
57. A. Han, J. Glanville, L. Hansmann, M. M. Davis, Linking T-cell
receptor sequence to functional phenotype at the single-cell
level.Nat. Biotechnol. 32 , 684–692 (2014). doi:10.1038/
nbt.2938; pmid: 24952902
58. A. P. Masella, A. K. Bartram, J. M. Truszkowski, D. G. Brown,
J. D. Neufeld, PANDAseq: Paired-end assembler for illumina
sequences.BMC Bioinformatics 13 , 31 (2012). doi:10.1186/
1471-2105-13-31; pmid: 22333067
59. M. P. Lefrancet al., IMGT, the international ImMunoGeneTics
information system.Nucleic Acids Res. 37 , D1006–D1012
(2009). doi:10.1093/nar/gkn838; pmid: 18978023
60. J. J. Trombettaet al., Preparation of single-cell RNA-Seq
libraries for next generation sequencing.Curr. Protoc. Mol. Biol.
107 , 4.22.1–4.22.17 (2014). doi:10.1002/0471142727.
mb0422s107; pmid: 24984854
61. A. Dobinet al., STAR: Ultrafast universal RNA-seq aligner.
Bioinformatics 29 , 15–21 (2013). doi:10.1093/bioinformatics/
bts635; pmid: 23104886
62. B. Li, C. N. Dewey, RSEM: Accurate transcript quantification
from RNA-Seq data with or without a reference genome.
BMC Bioinformatics 12 , 323 (2011). doi:10.1186/1471-2105-12-
323 ; pmid: 21816040
63. T. Stuartet al., Comprehensive integration of single-cell data.
Cell 177 , 1888–1902.e21 (2019). doi:10.1016/
j.cell.2019.05.031; pmid: 31178118
64. M. J. T. Stubbingtonet al., T cell fate and clonality inference
from single-cell transcriptomes.Nat. Methods 13 , 329– 332
(2016). doi:10.1038/nmeth.3800; pmid: 26950746
65. B. J. Haaset al., De novo transcript sequence reconstruction
from RNA-seq using the Trinity platform for reference
generation and analysis.Nat. Protoc. 8 , 1494–1512 (2013).
doi:10.1038/nprot.2013.084; pmid: 23845962
ACKNOWLEDGMENTS
We thank D. Mucida (Rockefeller University) and C. Vinuesa
(Australian National University) for critical reading of our
manuscript; M. Carroll and D. Firl (Harvard University Medical
School) for help with implantation and imaging of iLN lymph node
windows; K. Gordon and K. Chhosphel for cell sorting; P. Strogies,
D. Gross, and J. Petrillo at Rockefeller University’s Precision
Instrumentation Technologies Facility and V. Sherman at
Rockefeller University’s High Energy Physics Instrument Shop for
adapting and producing parts for longitudinal iLN imaging; the
Rockefeller University Genomics Center for RNA sequencing and
Comparative Biosciences Center for animal housing; and
Rockefeller University employees for their continuous assistance.
Funding:This work was supported by NIH grants R21AI138020,
R01AI119006, and R01AI139117; Human Frontier of Science
Program Research Grant RPG003/2015; and a March of Dimes
Basil O’Connor Starting Scholar Award to G.D.V. This work was
also supported by NIH grants DP2GM119419 and RM1HG006193 to
A.K.S. and NCI Cancer Center Support Grant P30 CA008748
and NIH grant R37AI034206 to A.Y.R. Work in the Victora
laboratory is further supported by an NIH Director’s Pioneer Award
(DP1AI144248) and by the Robertson Foundation. J.T.J. was
supported by Fripro mobility grant no. 239757, jointly funded by
the Research Council of Norway and the Co-funding of Regional,
National, and International Programmes (COFUND)–Marie Curie
Actions under the EU Seventh Framework Programme (FP7)
and South-Eastern Norway Regional Health Authority grant no.- W.H. was supported by an Irvington-Cancer Research
Institute Postdoctoral Fellowship. A.Y.R. is a Howard Hughes
Medical Institute (HHMI) Investigator. A.K.S. was supported by a
Sloan Fellowship in Chemistry. A.K.S. and G.D.V. are Pew-Stewart
Scholars. G.D.V. is a Burroughs-Wellcome Investigator in the
Pathogenesis of Infectious Disease, a Searle Scholar, and a
MacArthur Fellow.Author contributions:J.T.J. and G.D.V.
designed and supervised all experiments and wrote the text with
input from all authors. J.T.J. performed most experiments with
help from S.S., A.G., Z.L., S.J.A., L.M., A.M.B., and A.S. W.H. and
A.Y.R. designed and characterized theRosa26Foxp3mouse and
contributed to all experiments involving this strain. T.B.R.C.
designed and executed all computational analyses, with essential
Jacobsenet al.,Science 373 , eabe5146 (2021) 16 July 2021 12 of 13
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