DOI: 10.5061/dryad.v15dv4215
title: Data from: Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells

description: Follicular regulatory T cells (Tfr) can play opposite roles in the
 regulation of germinal center (GC) responses. Depending on the studies,
 Tfr suppress or support GCs and B cell affinity maturation. However, which
 factors determine positive versus negative effects of Tfr on the GC B
 cells is unclear. In this study we show that GC centrocytes that express
 MYC upregulate expression of CCL3 chemokine that is essential for both the
 positive and negative regulation of GC B cells by Tfr. B cell intrinsic
 expression of CCL3 contributes to Tfr-dependent positive selection of
 foreign Ag-specific GC B cells. At the same time, expression of CCL3 is
 critical for direct Tfr-mediated suppression of GC B cells that acquire
 cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1
 receptors promote Tfr migration to CCL3 and
 highlights Ccr5 expression on Tfr subset that
 expresses Il10. Based on our findings and previous studies, we
 suggest a model of chemotactically-targeted checkpoint control of B cells
 undergoing positive selection in GCs by Tfr, where Tfr directly probe and
 license foreign antigen-specific B cells to complete their positive
 selection in GCs but, at the same time, suppress GC B cells that present
 self-antigens cognate to Tfr.
description: <strong>All flow data were collect based on the following
 procedures.</strong> Single-cell suspensions from
 draining lymph nodes and spleens were prepared and filtered through a
 70-µm nylon cell strainer (BD). Red blood cells were lysed. Cells were
 washed in FACS buffer (2% FBS, 1mM EDTA, 0.1% NaN<sub>3</sub>
 in PBS) and followed by surface staining for the indicated markers for 20
 min at 4℃.  For intracellular staining, after surface markers were
 stained, cells were fixed and stained by using regulatory T cell Staining
 Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s
 instructions. All samples were acquired on a BD FACSCanto flow cytometer.
 For cell sorting, enriched B cells and T cells were incubated with
 antibodies in Sorting buffer (0.5% FBS and 2 mM EDTA in PBS) and were
 performed on a BD FACSAria III cell sorter. 
 <strong>All flow data were analyzed with FlowJo (version
 10.6.0) software.</strong> <strong>VH186.2
 sequence analysis of GC B cells based on the following
 protocol.</strong> Genomic DNA was extracted
 using QIAamp DNA Micro Kit (Qiagen) from sorted NP-specific GC B cells
 (B220<sup>+</sup>
 Fas<sup>+</sup>GL7<sup>+</sup>IgD<sup>lo</sup>NP<sup>+</sup>). The isolated genomic DNA was used for PCR amplification of variable heavy chain region 186.2-joining heavy chain region 2 segments (VH186.2-JH2). PCR was performed using 25 ng of genomic DNA with a previously described semi-nested PCR protocol (refer paper Germinal Center Dynamics Revealed by Multiphoton Microscopy with a Photoactivatable Fluorescent Reporter). Briefly, two sequential rounds of PCR were performed using high-fidelity DNA polymerase (TAKARA) with the outer forward primers 5’-CATGGGATGGAGCTGTATCATGC-3’ and outer reverse primer 5’-CTCACAAGAGTCCGATAGACCCTG-3’ for 30 cycles 98 °C for 10 s, 55 °C for 15 s and 68 °C for 120 s, followed by the inner forward primers 5’-GGTGACAATGACATCCACTTTGC-3’ and inner reverse primer 5’-GACTGTGAGAGTGGTGCCTTG-3’ with the same conditions. Final blunt-ended PCR products were added an A-tailing and then subcloned into pGEM-T Easy vector (Promega). Single clones were sequenced using T7 primers. <strong>Sequencing results were analyzed with IgBLAST (NCBI). Only the unique sequences (clones) that matched to VH186.2 gene (IgHV1-72*01) were counted.</strong>
description: # Targeted checkpoint control of B cells undergoing positive selection in
 germinal centers by follicular regulatory T cells
 This dataset includes all
 the flow cytometry and VH186.2 sequence analysis of GC B cells raw data.
 These original data are the sources of the results of our paper. Our
 results show that GC centrocytes that express MYC upregulate expression of
 CCL3 chemokine that is essential for both the positive and negative
 regulation of GC B cells by Tfr. B cell intrinsic expression of CCL3
 contributes to Tregs-dependent positive selection of foreign Ag-specific
 GC B cells. At the same time, expression of CCL3 is critical for direct
 Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear
 proteins. # **Description of the Data and file structure** This dataset is
 organized by different experimental contexts. Original Data |\_ Flow
 Cytometry Data/ \| |\_ Bone Marrow Chimeras immunized with SA-NucPr and
 SA/ \| |\_ CCL3 WT and CCL3 KO Bone Marrow chimeras data/ \| |\_
 Follicular T cells sorting for CCR1 and CCR5 qPCR analysis/ \| |\_
 Foxp3-Cre Bcl6+/+ and Bcl6 fl/fl mice data/ \| |\_ Foxp3-DTR mice data/ \|
 |\_ Hy10 CCL3 WT CFP and Hy10 CCL3KO GFP competition at day 6, 8, 12, 15
 data/ \| |\_ Mb1Cre+ IL-10R ++ and IL-10R flfl Bone Marrow Chimeras/ |\_
 VH186.2 sequence analysis of GC B cells/ \| |\_ CCL3 WT/ \| |\_ CCL3KO/
 File Details Details for Bone Marrow Chimeras immunized with SA-NucPr and
 SA: We generated mixed 1:1 CCL3-/- CD45.2 : CCL3+/+ CD45.1 and control
 CCL3+/+ CD45.2 : CCL3+/+ CD45.1 bone marrow chimeras (BMChs). After BM
 reconstitution, mixed BMChs were immunized with SA-DEL and boosted with
 SA-NucPr or SA for control. Tfr, Tfh, and SA-specific GC B cells were
 analyzed. All flow raw files are included. About the file naming, for
 example, Specimen_001_* SA-NucPr_0##.fcs, where * is WT (Wild type) or KO
 mouse number (#2, 3 and 4) and ## denotes the order of the flow cytometry
 machine recording data. Details for CCL3 WT and CCL3 KO Bone Marrow
 Chimeras: We generated mixed 1:1 CCL3-/- CD45.2 : CCL3+/+ CD45.1 and
 control CCL3+/+ CD45.2 : CCL3+/+ CD45.1 bone marrow chimeras (BMChs).
 After BM reconstitution the BMChs were immunized with NP-KLH to track the
 NP-specific B cell response over time. This fold including two operated
 the experiments on separately date. The folder has all raw flow files.
 About the file naming, for example, BM chimeric_1 CCL3+,2f,+ d15, where
 \_1 is mouse number, CCL3+,2f,+ means CCL3+/+, and d15 means harvest the
 mouse at day 15 post immunization. Details for Follicular T cells sorting
 for CCR1 and CCR5 qPCR analysis: Sorting Follicular regulatory T cells,
 CXCR5 int PD1 int and CXCR5 low PD1 low Foxp3+ Tregs, and follicular help
 T cells from immunized Foxp3-GFP mice and CD4+CD25+ cells from CCR5 KO
 mice for qPCR analysis of CCR5 and CCR1. This folder has all raw flow
 files. Details for Foxp3-Cre Bcl6+/+ and Bcl6 fl/fl mice data: CCL3 WT CFP
 and CCL3 KO GFP Hy10 B cells were co-transferred into Tfr deficient Bcl6
 fl/fl Foxp3-Cre and control Bcl6 +/+ Foxp3-Cre mice to assess their
 competition in GCs at day 15 after immunization. Tfr, Tfh, and GC B cells
 were analyzed. All flow raw files are included. About the file naming, for
 example, Specimen_001_Cre-1, where Cre-1 means Foxp3-Cre Bcl6+/+ mouse
 number 1. Specimen_001_FC-1, where \_FC-1 means Foxp3-Cre Bcl6 fl/fl mouse
 number 1. Details for Foxp3-DTR mice data: CCL3 WT CFP and CCL3 KO GFP
 Hy10 B cells were co-transferred into the Foxp3-DTR mice, that were then
 immunized with DEL-OVA. At day 12 after immunization the recipient mice
 were injected with diphtheria toxin at concentrations that lead to
 transient depletion of Tregs or with PBS for control. Tfr, Tfh, and GC B
 cells were analyzed. All flow raw files are included. About the file
 naming, for example, day15_DTx-1, where day15 means day 15 after DEL-OVA
 immunization. Where \_DTx-1 means diphtheria toxin treated mouse number 1.
 day15_PBS-1, where \_PBS-1 means PBS treated mouse number 1. Details for
 Hy10 CCL3 WT CFP and Hy10 CCL3KO GFP competition at day 6, 8, 12, 15 data:
 Recipient mice were co-transferred with CCL3 WT CFP amd CCL3 KO GFP Hy10 B
 cells and were immunized with DEL-OVA for time-course of Hy10 B cell
 participation in the GC response in draining LNS. GC B cells were
 analyzed. All flow raw files are included. About the file naming, for
 example, day8_boyJ 503 f, where day8 means day 8 post immunization. boyJ
 503 means the mouse background is BoyJ strain, and mouse number is 503. f
 means this mouse is a female. Details for Mb1Cre+ IL-10R ++ and IL-10R
 flfl Bone Marrow Chimeras: We generated mixed BMChs with irradiated CD45.1
 mice that were co-transferred with 1:1 ratio of BM cells from Mb1Cre+/-
 IL10R fl/fl CD45.2 mice or Mb1Cre+/- IL10R +/+ CD45.2 mice and WT CD45.1
 mice. Reconstituted BMChs were immunized with NP-KLH and draining LNs were
 isolated for analysis at day 10 and day 18 post immunization. The dark
 zone (DZ) and light zone (LZ) GC B cells, and NP-specific GC B cells were
 analyzed. This fold including two operated experiments on separately date.
 The folder has all raw flow files. About the file naming, for example,
 Specimen_001_1 d10 where \_1 d10 is mouse number 1 harvest at day 10 post
 immunization. Details for CCL3 WT and CCL3 KO: We generated mixed 1:1
 CCL3-/- CD45.2 : CCL3+/+ CD45.1 and control CCL3+/+ CD45.2 : CCL3+/+
 CD45.1 bone marrow chimeras (BMChs). After BM reconstitution the BMChs
 were immunized with NP-KLH. NP specific CD45.2 CCL3 WT and CCL3 KO GC B
 cells were sorted for VH186.2 sequence analysis. All sequencing raw files
 are included.