Decreased expression of Kruppel-like factors in memory B cells induces the rapid response typical of secondary antibody responses

Abstract

Secondary antibody responses are characterized by the rapid kinetics of the responding cells, including the production of larger amounts of serum Ig compared with the primary response. Memory B cells, which are responsible for this phenomenon, undergo greater proliferation and differentiation into Ig-secreting plasma cells than naïve B cells. We have found that memory cells rapidly enter cell division, irrespective of extrinsic stimuli. Microarray analysis of human splenic B cells revealed that naïve cells express higher levels than memory B cells of Krüppel-like factor (KLF) 4, KLF9, and promyelocytic leukemia zinc finger (PLZF), transcription factors important in maintaining cellular quiescence. These genes were down-regulated after activation through CD40 and the B cell receptor. Enforced expression of KLF4, KLF9, and PLZF in memory B cells delayed their entry into division and reduced the number of proliferating cells, such that the behavior of transfected memory cells resembled that of naïve B cells. Thus, the accelerated response of memory B cells correlates with reduced expression of KLF4, KLF9, and PLZF and the subsequent regulatory effects they exert on the cell cycle.

Fig. 1.

IgM and switched memory B cells enter division earlier than naïve B cells do regardless of the type or doses of stimulus used. (a–f) Sort-purified naïve (■), IgM memory (▴), and switched memory (●) B cells were cultured (1.5 × 10⁵ per ml) with CD40L alone (a), CD40L plus F(ab′)₂ fragments of anti-Ig (10 μg/ml) (b), CpG (1 μg/ml) (c), CpG plus anti-Ig (d), CD40L and CpG (e), or CD40L, CpG, and anti-Ig (f). Proliferation was assessed every 24 h by determining incorporation of [³H]thymidine during a 4-h pulse. The mitotic inhibitor demecolcine (10 ng/ml) was added at the onset of culture to measure entry of the cells into their first S phase and therefore ttfd. The center of the curve is the mean ttfd; the height is proportional to the number of cells entering division. Values represent the mean ± SEM of triplicate cultures. (g–j) Sort-purified naïve (g) and total memory (h) B cells were cultured (2.5 × 10⁵ per ml) with 2-fold dilutions of CD40L (○, 1/250; □, 1/500; ▵, 1/1,000; ◇, no CD40L). The ttfd (i) and amplitude (j) of the responses of naïve (■) and memory (●) B cells were calculated. These results are representative of two or more independent experiments performed with B cells from different spleens.

Fig. 2.

Differential expression of genes involved in the negative regulation of the cell cycle. (a) Data generated from naïve and memory B cell GeneChips was mined for genes differentially expressed by >2-fold. Genes with distinct roles in cell-cycle regulation and that differ in expression between memory and naïve B cells are shown. The results are presented as fold change for IgM memory (black bars) and switched memory (gray bars) B cells relative to naïve B cells. (b) Differential expression of genes involved in inhibiting the cell cycle, as revealed by microarray analysis, was confirmed by sqPCR using GAPDH as a standardizing control. (c) Naïve B cells were cultured with CD40L and anti-Ig for 72 h. Cells were harvested every 24 h, and expression of KLF4, KLF9, or PLZF was determined by sqPCR.

Fig. 3.

Cell-cycle regulators inhibit B cell blastogenesis. B cells expressing GFP after nucleofection with plasmids encoding KLF4 (a–d), KLF9 (e–h), PLZF (i–l), or the vector alone were cultured in duplicate with CD40L. Responses were assessed after 4–5 days by determining scatter profiles (a, b, e, f, i, and j), cell number (c, g, and k), and [³H]thymidine incorporation during the final 18 h (d, h, and l). Data are the mean ± SEM. and represent two or more experiments.

Fig. 4.

ttfd analysis of subsets of B cells transfected with genes differentially expressed by naïve and memory B cells. (a and b) B cells were transfected with plasmids encoding KLF4-GFP (▾), KLF9-GFP (●), PLZF-GFP (◆), p21-GFP (▴), or GFP alone (■). GFP⁺ cells were isolated and then cultured with CD40L alone (a) or CD40L plus anti-Ig (b) in the presence of demecolcine. ttfd was determined as described for Fig. 1. (c–j) Transfected naïve (■; Left) and memory (■; Right) B cells expressing KLF4-GFP (c and d), KLF9-GFP (e and f), PLZF-GFP (g and h), p21-GFP (i and j), or GFP alone (▴) were isolated and cultured with CD40L and anti-Ig in the presence of demecolcine. ttfd was determined as described for Fig. 1. The values in c–j represent the ttfd for transfected B cells. (k) The relative effect (% change compared with control cultures) of overexpressing KLF4, KLF9, and PLZF on ttfd of naïve (black bars) and memory B cells (gray bars). (l) The fold change in amplitude of the response of naïve (black bars) and memory (gray bars) B cells overexpressing KLF4, KLF9, or PLZF relative to naïve and memory cells transfected with the vector control. Similar results were obtained in at least two independent experiments.