A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells

Abstract

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies, we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system, which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor, yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures, revealing that the reaction engenders both MBC subsets with different immune effector function and, ultimately, LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity, for vaccine development, and for understanding long-term pathogen resistance.

Figure 1. Kinetics of the formation of long lived immune effector cells in a synchronized response

(A) Experimental outline and (B) possible labeling scenarios. (C, E, G, I, J, K, L) Transfer recipients were injected with BrdU at indicated times post NP-CGG immunization, as depicted in (A), and the frequencies of BrdU⁺ of live (=EMA⁻) IgM⁺ MBC (C), IgG1⁺ MBC (E), and BM IgG1⁺ LLPC (G) were analyzed 8 weeks post-immunization by flow cytometry (Fig. S2A, S2B). Each dot represents one mouse; combined data from 3–6 experiments. (I) Flow cytometric analysis of CD80 and PD-L2 distribution among EMA⁻ BrdU⁺ NP⁺ CD19⁺ splenic MBC at week 8 (n=6–19, symbols are mean +/− SEM; combined data of 3 experiments; Fig. 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. (K, L) n=6–14, symbols are mean +/− SEM. (D, F, H) Recipients were treated as depicted in (A) and sacrificed 1h after the last BrdU injection at the indicated day. Symbols are mean +/− SEM of 3–4 mice per group (combined data of 2 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⁺ AFC from spleen (circles) and BM (squares) of mice depicted in (C) and (E) were measured by ELISpot assay.

Figure 2. Validation of experimental design

(A, 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 (Fig. S1) three times a day at d6–8 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 panel) to further analyze their BrdU and VPD450 fluorescence at d4 post in vitro activation (right panel). (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 post in vitro activation). (C, D, E) The majority of GCBC 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, E). BrdU was i.p. injected three times into transfer recipients exclusively at d7 after NP-CGG immunization. Groups of 5 mice received 1 injection of 1mg EdU i.p. one, two, three or four days later to mark previously BrdU-labeled cells that were still in S-phase of the cell cycle. (D) Validation of EdU and BrdU double detection assay. Mice were sacrificed 30min 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 panel) to assess their BrdU and EdU fluorescence (middle panel). 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 panel) and is quantified in (E). Arrows indicate subsequent gating of populations and numbers next to outlined areas indicate percent gated population.

Figure 3. Immunohistological analysis of early MBC formation

(A, B) Transfer recipients were injected three times with EdU at d2 (A) and d6 (B) post NP-CGG immunization to label proliferating cells. Spleens were harvested 30min 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 panels) and are magnified (right panels) without depicting B220 and CD4 staining. Micro-anatomic location of proliferating Igλ⁺ B cells is quantified in (C) from 3 individual whole reconstructed splenic sections of 2 mice per time point (Fig. S3A). Error bars represent +/− SEM. GC phenotype was confirmed by PNA positivity on consecutive sections (Fig. 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 (Fig. S3C). Scale bars are 200µm.

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 Fig. 1A and the frequencies of BrdU⁺ of live splenic NP⁺ CD38⁺ CD95⁻ IgM⁺ MBC (blue dashed line, squares) or splenic NP⁺ CD38⁺ CD95⁻ IgG1⁺ MBC (red solid line, circles) and BM B220^low CD138⁺ NP^surface− NP^{intracellular+} IgG1⁺ LLPC (black solid line, triangles) were analyzed 8 weeks later by flow cytometry (Fig. S4). Symbols are mean +/− SEM of 15–21 mice per group. (B) Accumulation of long-lived MBC and BM LLPC over time. Plotted values are based on experimental data presented in (A) while missing data (*) were interpolated using the spline function in the R statistical package. Dotted ledger lines indicate the time post-immunization when 40% of each immune compartment is formed.

Figure 5. Disruption of peak GC reaction diminishes LLPC but not MBC

Transfer recipients were injected i.p. with 350µg 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- (NP₁₆-BSA; A) and high-affinity (NP₂-BSA, B) BM AFC 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 1 out of 2 independent experiments.

Figure 6. Mutational content of MBC matches overall mutational content of early GC whereas mutational content of LLPC is reflected in late GC

(A) Experimental outline. B1–8 mice were injected with 0.75mg EdU three times a day at indicated time points after NP-CGG immunization, rested for 8 weeks, after which EdU⁺ or total NP⁺ IgG1⁺ splenic MBC and BM LLPC 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 200–5000 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.

Figure 7. Gene expression profiling of early and late GCBC

On d8 and d18 post NP-CGG immunization of transfer recipients 3×10⁵ live CD19⁺ NP+ kappa light chain⁻ CD38⁻ CD95⁺ splenic GCBC 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 GCBC. 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.