Telomere lengthening and telomerase activation during human B cell differentiation

Abstract

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.

Figure 1

Telomere length in tonsil B cell subsets. (A) TRF blot of naive (N), GC (G), and memory (M) B cells from three tonsils. One microgram of digested genomic DNA from each sample was loaded in a 0.6% agarose gel and separated by electrophoresis. Hybridization was carried out in the dried gel with a ³²P-labeled (CCCTAA)₃ oligonucleotide at 43°C overnight. (B) Comparison of mean TRF length of naive, GC, and memory B cells from five patients. The mean TRF was calculated by converting the intensity of signals to molecular size based on DNA molecular weight markers, using ImageQuant (software of PhosphorImager). Telomere size effect on the signal intensity was taken into consideration in calculation as previously described (17).

Figure 2

Telomerase activity in tonsil B cell subsets. (A) Representative results of telomerase activity in B cell subsets. Seven subsets of B cells [Bm1–5 and plasma cell (PC)] were isolated from two donors by a combination of immunomagnetic cell separation and fluorescent cell sorting, and three subsets (naive, GC, and memory) were isolated from five donors by immunomagnetic cell separation alone. The relative activity of telomerase was consistent and reproducible among subsets of B cells from these different donors and cell separations. A transformed cell line, 293, was used as a positive control and standard for quantitation of relative levels of telomerase activity. (B) Comparison of the levels of telomerase activity in tonsil B cell subsets. The relative levels of telomerase activity were determined by serial dilution and are minimal estimates, compared with the levels of telomerase activity of Bm1 B cell subsets. The internal standard was used to monitor the reproducibility of the assay and served as a reference for comparing results among experiments.

Figure 3

In vitro activation induces telomerase in naive and memory tonsil B cells. (A) The results shown are representative of telomerase induction in naive and memory tonsil B cells with in vitro activation. Similar results were obtained in three independent experiments. (B) Relative levels of telomerase activity in naive and memory tonsil B cells induced by different stimulation conditions. The relative levels of telomerase activity were determined as described in the text and in the legend of Fig. 2.

Figure 4

Telomerase RNA template expression in B cell subsets. (A) Representative Northern blot of hTR expression in naive (N), GC (G), and memory (M) B cells. (B) Abundance of hTR expression in tonsil B cell subsets. The abundance of hTR was analyzed by normalization to 7SK level, presented as in arbitrary units (a.u.), and the results were summarized from B cells of three tonsils.

Figure 5

Model of coordinated regulation of telomere length and telomerase activity in peripheral B cell differentiation. Arrows indicate telomeres of chromosomes and the shaded end reflects the length of telomeres.