1. Volume 47, Issue 6, Pages 1142-1153.e4 (December 2017)
CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity 
Dan Suan, Nike J. Kräutler, Jesper L.V. Maag, Danyal Butt, Katherine Bourne, Jana R. Hermes, Danielle T. Avery, Clara Young, Aaron Statham, Michael Elliott, Marcel E. Dinger, Antony Basten, Stuart G. Tangye, Robert Brink 
Immunity 
Volume 47, Issue 6, Pages e4 (December 2017)
DOI: /j.immuni
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2. Immunity 2017 47, 1142-1153.e4DOI: (10.1016/j.immuni.2017.11.022)
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3. Figure 1
CCR6-Expressing Cells Are Enriched in the GC LZ and Exhibit a Phenotype Transitional to That of MBCs
All data are from day 9 of the C57BL/6 splenic response to SRBC immunization.
(A) Flow-cytometry plots illustrating the gating strategy for identifying the analyzed splenic B cell subpopulations, including the expression of CCR6.
(B) The proportion of CCR6+ cells in the LZ and DZ compartments of the IgG1+ GC.
(C) Surface expression of IgG1, CD38, CCR6, CD31, and EBI2 on naive B, GC, and MBC subpopulations as measured by flow cytometry.
(D) Flow-cytometry plots comparing CD23 with CCR6 on GC and MBC subpopulations.
Each data point represents an individual spleen. Data represent 3–14 independent experiments of 4–10 mice per experiment. Error bars indicate the mean. Statistics were calculated with a paired t test (B) or a matched one-way ANOVA (C).
Immunity  , e4DOI: ( /j.immuni )
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4. Figure 2
CCR6+ LZ B Cells Exit the Cell Cycle but Do Not Preferentially Undergo Apoptosis
All data are from day 9 of the C57BL/6 splenic response to SRBC immunization.
(A) Flow-cytometry plots from mice treated with BrdU drinking water for 24 or 48 hr; splenic B cell subpopulations were analyzed for BrdU incorporation and CCR6 expression.
(B) The proportion of BrdU+ cells in naive B, GC, and MBC subpopulations at 24 and 48 hr of treatment with BrdU drinking water.
(C) Expression of the proliferation marker Ki67 in B cell subpopulations as detected by flow cytometry.
(D) Forward-scatter characteristics of B cell subpopulations as determined by flow cytometry.
(E) Flow-cytometry plots showing intracellular active caspase-3 and surface CCR6 expression in B cell subpopulations.
(F) The proportion of active caspase-3+ cells in B cell subpopulations as detected by flow cytometry.
Each data point represents an individual spleen. Data represent three independent experiments of five mice per group. Error bars indicate the mean. Statistics were calculated with a matched one-way ANOVA.
Immunity  , e4DOI: ( /j.immuni )
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5. Figure 3
CCR6+ GC B Cells Are Enriched in the Low-Affinity Compartment of the LZ
All data are from day 9 of the SWHEL B cell response to HEL3X-SRBC immunization in C57BL/6 congenic recipients.
(A) Flow-cytometry plots illustrating the gating strategy for identifying the analyzed splenic B cell subpopulations, including high- and low-affinity SWHEL B cells, and their expression of CCR6.
(B) The proportion of CCR6+ cells in the LZhi, LZlo, DZhi, and DZlo compartments of the SWHEL-derived IgG1+ GC B cells.
(C) Surface expression of IgG1, CD38, CCR6, CD31, and EBI2 on GC and MBC subpopulations as measured by flow cytometry.
(D) Flow-cytometry plots comparing CD23 with CCR6 on GC and MBC subpopulations.
Each data point represents an individual spleen. Data represent 12 independent experiments of 4–15 mice per experiment. Error bars indicate the mean. Statistics were calculated with a matched one-way ANOVA.
Immunity  , e4DOI: ( /j.immuni )
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6. Figure 4
CCR6+ LZlo B Cells Are the Most Quiescent in the GC and Exhibit a SHM Pattern Consistent with Direct Transition into the MBC Compartment
All data are from day 9 of the SWHEL B cell response to HEL3X-SRBC immunization in C57BL/6 congenic recipients.
(A) Flow-cytometry plots of BrdU incorporation versus CCR6 expression in mice treated with 1 mg of BrdU i.v. 1, 5, and 10 hr before analysis.
(B) The proportion of BrdU+ cells in GC and MBC subpopulations 1, 5, and 10 hr after i.v. treatment with 1 mg BrdU.
(C) SHM (somatic hypermutation rate; average number of mutations per clone over the whole VDJ coding region) analysis of the CDR2 IgH variable region in the LZhi, LZlo CCR6−, LZlo CCR6+, and MBC subpopulations. Gray columns indicate all detected amino acid substitutions, and red and blue columns indicate Y53D and Y58F substitutions, respectively. n, number of mutated clones analyzed.
Data in (A) and (B) represent three independent experiments of five mice per group. Each data point represents an individual spleen. Error bars represent means. Statistics were calculated with a matched one-way ANOVA. Data in (C) are derived from pooling three independent experiments of four mice per experiment.
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7. Figure 5
CCR6+ MBC Precursors Physically Localize to the LZ of the GC but Do Not Require CCR6 for Their Production
(A) Flow-cytometry plots from day 9 of the SWHEL.Ccr6gfp response to HEL3X-SRBC in C57BL/6 congenic recipients. Wild-type (SWHEL.Ccr6+/+), heterozygous (SWHEL.Ccr6gfp/+), and homozygous (SWHEL.Ccr6gfp/gfp) responses are shown.
(B) The proportion of Ccr6-GFP+ cells in GC and MBC sub-compartments.
(C) Enumeration of cell numbers in the GC and MBC sub-compartments on day 9 of the SWHEL.Ccr6gfp response to HEL3X-SRBC in C57BL/6 congenic recipients.
(D) Histology of Ccr6f+/+ and Ccr6gfp/gfp formalin-fixed spleens on day 9 of the response to SRBC immunization.
(E) Number of Ccr6-GFP+ cells in the LZ and DZ compartments of individual GC sections. Each data point represents a different GC.
Data in (A)–(C) represent two independent experiments of three mice per experiment. Statistics were calculated with a matched one-way ANOVA. Data in (D) and (F) represent three independent experiments of four to five mice per group. Statistics in (E) and (G) were calculated with a paired t test.
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8. Figure 6
CCR6+ B Cells Are Enriched in the LZ of Human Tonsils and Exhibit Phenotypic Characteristics Consistent with Being MBC Precursors
(A) Representative flow-cytometry plots illustrating the gating strategy used for identifying tonsillar B cell subpopulations, including CCR6 expression.
(B) The proportion of CCR6+ B cells in the DZ and LZ of human tonsils.
(C) Forward-scatter, surface, and intracellular expression of selected markers.
Each data point represents an individual tonsil. Error bars represent the mean. Data in (C) represent two to four independent experiments of three to five tonsils per experiment. Statistics were calculated with a paired t test (B) or a matched one-way ANOVA (C).
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9. Figure 7
CCR6+ LZ B Cells from Human Tonsils Exhibit an RNA Expression Profile and Functional Characteristics Consistent with Being MBC Precursors
(A) Heat-map illustrating the top 40 genes most differentially expressed between the LZ CCR6− and MBC populations (p ≤ 0.05; log2FC > 1, where FC stands for fold change).
(B) Heat-map illustrating the genes most differentially expressed between the LZ CCR6− and LZ CCR6+ populations (p ≤ 0.1; log2FC > 1).
(C) Enumeration of live cells per well after B cell subpopulations were sorted and cultured in IL-21 and CD40L for 5 days.
(D) Enumeration of CD20loCD38hiCD27hiCD138+ plasmablasts after B cell subpopulations were sorted and cultured in IL-21 and CD40L for 5 days.
(E) Quantitation of IgM, IgG, and IgA in the supernatants of B cell subpopulations after culture in IL-21 and CD40L for 5 days.
Data in (A) and (B) are from two independent arrays on two different human tonsils. Data in (C)–(E) represent three independent experiments on two different tonsils. Data points in (E) represent the total Ig levels in each well. Statistics were calculated with a one-way ANOVA.
Immunity  , e4DOI: ( /j.immuni )
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