Transcriptional profiling of antigen-dependent murine B cell differentiation and memory formation

Abstract

Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure.

FIGURE 1

Naive follicular and Ag-specific plasma, germinal center, and memory B cells can be highly purified from wild-type mice. Gating strategies and purities of the starting populations are shown in the left panels. Plasma cells, germinal center cells, and memory B cells were harvested from spleens 7, 14, or >70 days, respectively, after immunization with 100 μg of NP-CGG. Unimmunized mice were injected with only alum. Cells were analyzed after the first sort, shown in the right panels, and sorted again directly into TRIzol to ensure further purity. Plasma and germinal center B cells were enriched using magnetic enrichment of syndecan-1⁺ or PNA-binding cells, respectively, before FACS.

FIGURE 2

Cell-intrinsic differences between naive follicular, plasma, germinal center, and memory B cells. Selected genes with statistically significant transcriptional differences between at least two of the analyzed B cell populations are shown in heatmap form. Functional categories were assigned based on a combination of Gene Ontology categorization and literature searches. Red represents high relative levels of expression, and blue represents low relative transcript levels.

FIGURE 3

B cells use unique mechanisms to survive T-dependent and T-independent responses. A, Retroviral expression of Klf2, Ski, or NF-κB1 provides a competitive advantage to anti-IgM- plus anti-CD40-stimulated B cells. Naive splenic follicular B cells were purified through depletion of non-B lineage and syndecan-1⁺, GL-7⁺, and CD21^high cells; stimulated with anti-IgM and anti-CD40; and transduced with the indicated retrovirus. The percentage of GFP⁺ cells was quantified at 2 and 6 days postinfection. The GFP⁺ advantage was quantified as follows: ((% GFP + day 6)(% GFP − day 2))/((% GFP + day 2)(% GFP − day 6)), and normalized to the control virus for each experiment. Representative plots from one experiment are shown, and the mean ± SEM values are displayed to the right for four independent experiments. B, Retroviral expression of Bcl2, Ski, and NF-κB1, but not Klf2, extends the number of divisions that B cells undergo. Cells were labeled with Far Red DDAO, stimulated, and infected as above, and DDAO expression levels in GFP⁺ cells were monitored at 2 and 5 days postinfection. Gray-shaded histograms in the day 2 plots represent control virus-transduced cells, and overlaid unfilled histograms represent cells transduced by the indicated retrovirus. The percentage of GFP⁺ cells that had undergone 7 or more divisions, calculated by dilution of DDAO signal relative to day 0, is shown in the day 5 plots. In a separate experiment, cells were stained at day 6 postinfection with annexin V and DAPI to quantify apoptotic cells within the GFP⁺ population. C, Retroviral expression of NF-κB heterodimers and Bcl2, but not Klf2 or Ski, protects LPS-stimulated B cells. LPS-stimulated splenic follicular B cells purified as above were transduced with the indicated retroviruses, and GFP⁺ advantage was calculated as above. Mean ± SEM values from three independent experiments are shown.

FIGURE 4

Mist1 expression affects the function, but not the formation of short-lived plasma cells. Mist1^−/− or age-matched wild-type controls were immunized with NP-CGG, and serum (A) and spleens (B) were harvested 7 days later. Serum titers of NP-specific Abs (A) and the total number of splenic B220^low syndecan-1⁺ NP⁺ cells (B) are shown. Mean ± SEM values are shown from a total of 10 Mist1^−/− and 9 Mist^+/+ mice analyzed in two independent experiments.

FIGURE 5

Bmi1 is not required for the generation of memory B cells. Bmi1^−/− or wild-type littermates were immunized with NP-CGG, and spleens were harvested 4 wk after immunization. Plots show only B220⁺ Igλ⁺ cells, and memory cells are identified as IgD⁻ NP⁺ cells.

FIGURE 6

Memory B cells simultaneously express molecules associated with both lymph node and mucosal homing. A, Splenic memory and naive B cells express differential levels of CCR6, CD80, and L-selectin. Naive follicular (B220⁺ IgM^low IgD^high Igλ⁺ NP⁻) and memory B cells (B220⁺ IgM⁻ IgD⁻ Igλ⁺ NP⁺) were analyzed for expression of CD80 and CCR6. Mice were immunized 10 wk before analysis. Gray shaded plots represent fluorescence minus one controls (92), and black unfilled histograms represent cells stained with anti-CCR6, anti-CD80, or anti-L-selectin. B, Differential expression of CCR6 and L-selectin between memory B cells in spleen, Peyer’s patch, and peripheral lymph nodes. Expression of L-selectin and CCR6 was analyzed on B220⁺ IgM⁻ IgD⁻ Igλ⁺ NP⁺ memory B cells isolated from the spleen, Peyer’s patch, and brachial and axillary lymph nodes isolated from mice immunized 12 wk beforehand with NP-CGG.