Data from: Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells
Cite this dataset
Grigorova, Irina; Ke, Fang (2024). Data from: Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells [Dataset]. Dryad. https://doi.org/10.5061/dryad.v15dv4215
Abstract
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.
README: Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells
https://doi.org/10.5061/dryad.v15dv4215
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.
Methods
All flow data were collect based on the following procedures.
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% NaN3 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.
All flow data were analyzed with FlowJo (version 10.6.0) software.
VH186.2 sequence analysis of GC B cells based on the following protocol.
Genomic DNA was extracted using QIAamp DNA Micro Kit (Qiagen) from sorted NP-specific GC B cells (B220+ Fas+GL7+IgDloNP+). 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.
Sequencing results were analyzed with IgBLAST (NCBI). Only the unique sequences (clones) that matched to VH186.2 gene (IgHV1-72*01) were counted.
Funding
National Institute of Allergy and Infectious Diseases, Award: AI106806
National Institute of Allergy and Infectious Diseases, Award: AI142032