1. Volume 44, Issue 1, Pages 116-130 (January 2016)
A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells 
Florian J. Weisel, Griselda V. Zuccarino-Catania, Maria Chikina, Mark J. Shlomchik 
Immunity 
Volume 44, Issue 1, Pages (January 2016)
DOI: /j.immuni
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2. Figure 1
Kinetics of the Formation of Long-Lived Immune Effector Cells in a Synchronized Response
(A and B) Experimental outline (A) and possible labeling scenarios (B).
(C, E, G, and I–L) Transfer recipients were injected with BrdU at indicated times after NP-CGG immunization, as depicted in (A).
(C, E, and G) The frequencies of BrdU+ of live (=EMA−) IgM+ MBCs (C), IgG1+ MBCs (E), and BM IgG1+ LLPCs (G) were analyzed 8 weeks after immunization by flow cytometry (Figures S2A and S2B). Each dot represents one mouse; combined data are from 3–6 experiments.
(I) Flow cytometric analysis of CD80 and PD-L2 distribution among EMA−BrdU+NP+CD19+ splenic MBCs at week 8 (n = 6–19, symbols are mean ± SEM; combined data of three experiments; Figure S2C).
(J) Frequency of CD73+ of EMA−NP+CD19+ cells in transfer recipients 8 weeks after immunization.
(K) Frequency of CD73+ of EMA−BrdU+NP+CD19+IgG1+ cells for each BrdU labeling window.
(L) Frequency of BrdU+ (IgM+ squares, IgG1+ circles) of EMA−CD73+NP+CD19+ cells for each BrdU labeling window.
n = 6–14, symbols are mean ± SEM in (K) and (L).
(D, F, and H) Recipients were treated as depicted in (A) and sacrificed 1 hr after the last BrdU injection at the indicated day. Symbols are mean ± SEM of 3–4 mice per group (combined data of two experiments are shown).
(D) Phenotype of B cells labeled during each BrdU labeling window.
(F) Frequencies of EMA−NP+CD19+ cells with GC (CD38−CD95+, circles) or non-GC (CD38+CD95−, squares; CD38+CD95−IgG1+, triangles) phenotype over the time course of the immune response.
(H) Kinetics of the AFC response. IgG1+ AFCs from spleen (circles) and BM (squares) of mice depicted in (C) and (E) were measured by ELISpot assay.
Immunity  , DOI: ( /j.immuni )
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3. Figure 2 Validation of Experimental Design
(A and B) BrdU positivity is quickly lost in B cells undergoing further cell divisions in the absence of bioavailable BrdU. BrdU or PBS was i.p. injected in transfer recipients (Figure S1) three times a day at d6–d8 after NP-CGG immunization and splenocytes were harvested 6 weeks later. Cells were labeled with the violet proliferation dye (VPD450) and cultured in the presence of CpG ODN1826 to induce polyclonal in vitro proliferation. At d3 and d4, cultured cells were harvested and BrdU fluorescence of EMA−NP+ B cells was correlated with their VPD450 fluorescence indicating distinct cell divisions.
(A) Strategy to gate live B220+NP+ B cells (left) to further analyze their BrdU and VPD450 fluorescence at d4 after in vitro activation (right).
(B) Quantification of flow cytometry data depicted in right panel of (A). The percentage of EMA− NP-reactive B cells, which retained BrdU positivity, is shown for each generation. The results were normalized to 100% for the generation 0 (symbols are mean ± SEM; combined data of d3 and d4 after in vitro activation).
(C–E) The majority of GCBCs do not undergo further cell division from their initial commitment to the MBC pool until reaching a final resting state.
(C) Experimental outline for (D) and (E). BrdU was i.p. injected three times into transfer recipients exclusively at d7 after NP-CGG immunization. Groups of five mice received one injection of 1 mg EdU i.p. 1, 2, 3, or 4 days later to mark previously BrdU-labeled cells that were still in S phase of the cell cycle.
(D and E) Validation and analysis of EdU and BrdU double detection assay (D). Mice were sacrificed 30 min after EdU injection on d+1 and single-cell suspensions of red-blood-cell-depleted splenocytes were subjected to flow-cytometric double detection of EdU and BrdU. EMA− NP-reactive GC (CD38−CD95+) and non-GC (CD38+CD95−) B cells were identified (top) to assess their BrdU and EdU fluorescence (middle). Mice given PBS instead of BrdU and/or EdU served as controls. The frequency of EdU+ of BrdU+NP+IgG1+B220+CD38+CD95− cells was assessed (bottom) and is quantified in (E). Arrows indicate subsequent gating of populations and numbers next to outlined areas indicate percent gated population.
Immunity  , DOI: ( /j.immuni )
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4. Figure 3 Immunohistological Analysis of Early MBC Formation
(A and B) Transfer recipients were injected three times with EdU at d2 (A) and d6 (B) after NP-CGG immunization to label proliferating cells. Spleens were harvested 30 min later and sections were stained for B cells (B220, green), T cells (CD4, light blue), proliferation (EdU uptake, red nuclei), and NP specificity (Igλ, dark blue). Areas of active proliferation of Ag-specific B cells are marked with color-coded rectangles in the overview of representative areas (left) and are magnified (right) without depicting B220 and CD4 staining.
(C) Quantification of micro-anatomic location of proliferating Igλ+ B cells from three individual whole reconstructed splenic sections of two mice per time point (Figure S3A). Error bars represent ± SEM. GC phenotype was confirmed by PNA positivity on consecutive sections (Figure S3B). No significant expansion of EdU+Igλ+ B cells was observed in mice not given EdU injection, NP-CGG immunization, or without B1-8 B cell transfer (Figure S3C). Scale bars represent 200 μm.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
Kinetics of the Formation of Long-Lived Immune Compartments in WT Mice
(A) BALB/cJ WT mice were immunized with NP-CGG and i.p. injected with BrdU or PBS at indicated time points as depicted in Figure 1A. The frequencies of BrdU+ of live splenic NP+CD38+CD95−IgM+ MBCs (blue dashed line, squares) or splenic NP+CD38+CD95−IgG1+ MBCs (red solid line, circles) and BM B220loCD138+NPsurface- NPintracellular+IgG1+ LLPCs (black solid line, triangles) were analyzed 8 weeks later by flow cytometry (Figure S4). Symbols are mean ± SEM of 15–21 mice per group.
(B) Accumulation of long-lived MBCs and BM LLPCs over time. Plotted values are based on experimental data presented in (A) and missing data (∗) were interpolated with the spline function in the R statistical package. Dotted ledger lines indicate the time after immunization when 40% of each immune compartment is formed.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5 Disruption of Peak GC Reaction Diminishes LLPCs but Not MBCs
(A–C) Transfer recipients were injected i.p. with 350 μg anti-CD40L Ab or hamster control IgG at d12, d13, and d14 after NP-CGG immunization. Each symbol represents one mouse and lines are means. Numbers of low-affinity (NP16-BSA) (A) and high-affinity (NP2-BSA) (B) BM AFCs per 106 BM cells were measured by ELISpot and numbers of live NP+CD38+CD95−CD19+ splenic B cells were quantified by flow cytometric analysis (C) 8 weeks later.
(D–F) Frequency distributions of MBC subsets, as distinguished by their expression of CD80 and PD-L2, were analyzed by flow cytometry.
Shown are representative results of one out of two independent experiments.
Immunity  , DOI: ( /j.immuni )
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7. Figure 6
Mutational Content of MBCs Matches Overall Mutational Content of Early GCs whereas Mutational Content of LLPCs Is Reflected in Late GCs
(A) Experimental outline. B1-8 mice were injected with 0.75 mg EdU three times a day at indicated time points after NP-CGG immunization, then rested for 8 weeks, after which EdU+ or total NP+IgG1+ splenic MBCs and BM LLPCs were sorted. Similarly prepared mice were directly sacrificed to sort splenic EMA−NP+IgG1+CD19+ cells of GC (CD38−CD95+) or non-GC (CD38+CD95−) phenotype.
(B) Mutational content analysis. DNA from 200–5,000 target cells was used for Vλ1 gene sequencing. Each dot represents a single in-frame sequence from up to 16 sequences from 3 individual mice per population.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7 Gene Expression Profiling of Early and Late GCBCs
On d8 and d18 after NP-CGG immunization of transfer recipients, 3 × 105 live CD19+NP+ kappa light chain−CD38−CD95+ splenic GCBCs were sorted per sample and subjected to gene expression profiling using Illumina MouseWG-6 v2.0 Expression BeadChip arrays. Heatmap of differentially expressed genes of early (d8) and late (d18) NP reactive late GCBCs. Each column represents an independent replicate. Genes with a statistically significant (false-discovery rate q < 0.05) change in expression of ≥1.7 are displayed and were grouped by function.
Immunity  , DOI: ( /j.immuni )
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