Age-related functional decline of human B cells

Abstract

This study aimed to investigate the changes in B cell functional decline and antigen sensitization with aging using two Epstein Barr virus (EBV)-immortalized human B cell lines, one from a 22-year-old man (EBV-B young) and the other from a 65-year-old man (EBV-B old). The activity of senescence-associated β-galactosidase, a marker of cellular senescence, was enhanced in the EBV-B old cells compared with EBV-B young cells. Moreover, the levels of p16, p21, IL-6, TNF-α, and TGF-β1, which are senescence-associated secretary phenotypes, were also increased in EBV-B old cells. In vitro immunization of EBV-B cells with β-lactoglobulin further showed that EBV-B old cells had a reduced cell population of naïve B cells than that of EBV-B young cells. Furthermore, HLA-DR expression, which is important for antigen presentation, was decreased in the EBV-B old cells. Comparative microarray analysis between EBV-B young and old cells also showed decreased expression of antibody genes, such as those of the heavy chain and light chain (κ chain). These results suggest that cellular senescence and decreased gene expression are responsible, at least in part, for the decline in B cell function and antigen sensitization capacity with aging, which ultimately impairs the function of the acquired immune system.

Keywords: Antigen sensitization; Epstein Barr virus-immortalized B cell; Immune senescence; Senescence-associated secretory phenotype.

Fig. 1

Epstein Barr virus-immortalized human B (EBV-B) old cells show a cellular senescence phenotype. a Activity of senescence-associated β-galactosidase (SA-β-gal) in EBV-B cells derived from a 22-year-old male (EBV-B young: HEV174) and a 65-year-old male (EBV-B old: HEV039) was evaluated by flow cytometry. b–f Expression of SASP genes (p16, p21, IL-6, TNF-α, and TGF-β1) in EBV-B young and old cells was analyzed by quantitative real-time polymerase chain reaction. The samples were analyzed in triplicate, and gene expression levels were normalized to those of GAPDH (b, c) and β-actin (d–f). *P < 0.05, **P < 0.01 by two-sided Student’s t-tests. g Frequency of IgM-producing cells was analyzed flow cytometry. This experiment was repeated three times, and similar results were obtained

Fig. 1

Epstein Barr virus-immortalized human B (EBV-B) old cells show a cellular senescence phenotype. a Activity of senescence-associated β-galactosidase (SA-β-gal) in EBV-B cells derived from a 22-year-old male (EBV-B young: HEV174) and a 65-year-old male (EBV-B old: HEV039) was evaluated by flow cytometry. b–f Expression of SASP genes (p16, p21, IL-6, TNF-α, and TGF-β1) in EBV-B young and old cells was analyzed by quantitative real-time polymerase chain reaction. The samples were analyzed in triplicate, and gene expression levels were normalized to those of GAPDH (b, c) and β-actin (d–f). *P < 0.05, **P < 0.01 by two-sided Student’s t-tests. g Frequency of IgM-producing cells was analyzed flow cytometry. This experiment was repeated three times, and similar results were obtained

Fig. 2

Antigen sensitization (specific antibody production) to β-lactoglobulin (β-LG) is decreased in EBV-B old cells. EBV-B cells were sensitized with β-LG antigen (10 μg/mL) and interleukin (IL)-4 (5 μg/mL). Cells producing antigen-specific antibodies were detected using the enzyme-linked immunospot assay. The number of spots was counted using the ImageJ software (https://imagej.nih.gov/ij/index.html), and is indicated in the upper right corner of the well

Fig. 3

Expression of HLA-DR is decreased in EBV-B old cells. Expression of HLA-DR was detected by flow cytometry using an anti-HLA-DR-PE antibody in EBV-B young and old cells

Fig. 4

Gene expression related to B cell function is decreased in EBV-B old cells. a IL-12A and b AICDA levels were assessed in EBV-B young and old cells by qPCR in triplicate and normalized to those of β-actin. *P < 0.05, **P < 0.01 determined by two-sided Student’s t-tests