The effect of aging on the frequency, phenotype and cytokine production of human blood CD4 + CXCR5 + T follicular helper cells: comparison of aged and young subjects

Maohua Zhou, Ruqiong Zou, Huiquan Gan, Zhimei Liang, Fujun Li, Ting Lin, Yanfei Luo, Xiaoming Cai, Fang He, Erxia Shen, Maohua Zhou, Ruqiong Zou, Huiquan Gan, Zhimei Liang, Fujun Li, Ting Lin, Yanfei Luo, Xiaoming Cai, Fang He, Erxia Shen

Abstract

Background: T cell-dependent B-cell responses decline with age, indicating declined cognate helper activity of aged CD4 + T cells for B cells. However, the mechanisms remain unclear. T follicular helper (Tfh) cells, a novel T helper subset, play an essential role in helping B cells differentiation into long-lived plasma cells in germinal center (GC) or short-lived plasma cells. In the present study, we proposed that there might existe changes of proportion, phenotype or cytokine production of blood Tfh cells in healthy elderly individuals compared with healthy young individuals.

Results: The results showed that frequencies of aged blood CXCR5 + CD4 + Tfh cells increased compared with young subjects. Both aged and young blood CXCR5 + CD4 + Tfh cells constitutively expressed CD45RO, CCR7 and CD28, and few of these cells expressed CD69 or HLA-DR, which indicated that they were resting memory cells. There was no significant difference of IL-21 frequency production by aged blood CXCR5 + CD4 + Tfh determined by FACS compared with young individuals, however, aged PBMCs produced significantly higher levels of IL-21 evaluated by ELISA. Furthermore, there were no significant differences of percentages of IFN-γ, IL-4, IL-17 or IL-22 production by aged Tfh cells compared with their counterparts of young individuals respectively. However, frequencies of IL-17+ cells within aged CD4 + CXCR5-T cells were markedly lower than in the young individuals. Furthermore we observed different frequencies of IFN-γ, IL-17, IL-4 or IL-22 production by Tfh or by CD4 + CXCR5- cells in aged and young subjects respectively.

Conclusions: Our data demonstrated that the frequencies of blood memory CXCR5 + CD4 + Tfh cells increased in the elderly population. There were similar frequencies of Th characterized cytokine production such as IL-21, IFN-γ, IL-4, IL-17 or IL-22 in aged and young Tfh cells. However, aged PBMCs produced a significantly higher amount of IL-21 compare to young subjects.

Keywords: Aging; IL-21; Tfh cells.

Figures

Figure 1
Figure 1
Increased proportion of blood CXCR5 + CD4 + Tfh cells of aged subjects. A. LYM counts: lymphocyte counts of peripheral blood (109/L). B. Percentages of CD4 + T cells (CD3 + CD4+) in lymphocytes from young and aged groups determined by FACS. C. Cell numbers of CD4 + T cells in peripheral blood (109/L). D. Frequencies of Tfh cells (CXCR5 + CD4+) in CD4 + T cells determined by FACS. E. The total cell counts of Tfh cells (CXCR5 + CD4+) in peripheral blood (109/L). F. Correlation of percentages of CXCR5 + CD4 + Tfh cells with age. One open circle or filled square represented one individual. Data were expressed with line at mean with SEM.
Figure 2
Figure 2
CD45RO, CCR7, and CD28 expression on CD4 + CXCR5 + Tfh cells as well as CD4 + T, CD3 + CD4-T and CD4 + CXCR5-T cells. CD45RO (A), CCR7 (B) and CD28 (C) expression on CD4 + T, CD3 + CD4-T, CD4 + CXCR5 + Tfh as well as CD4 + CXCR5-T cells from young and aged subjects. Blood lymphocytes were first gated, and then CD4 + T (CD3 + CD4+), CD3 + CD4-T, CXCR5 + CD4 + Tfh and CXCR5-CD4+ T cells were analyzed by expression of CD45RO, CCR7 or CD28. Representative results were shown. Thin line: isotype (iso) control, heavy line: young individual (young), filled area: elderly individual (aged).
Figure 3
Figure 3
Statistical analysis of CD45RO, CCR7 and CD28 expression on CD4 + CXCR5 + Tfh and other T subsets. Percentages of CD45RO + (A) (young subjects: n = 12, aged subjects: n = 11), CCR7+ (B) (young subjects: n = 6, aged subjects: n = 6) and CD28 + (C) (young subjects: n = 7, aged subjects: n = 9) among CD4 + T, CD3 + CD4-T, CD4 + CXCR5 + Tfh and CD4 + CXCR5-T cells were shown as in histograms. Y: young individuals, A: aged individuals. *: p < 0.05, n.s: no significant difference. All data were expressed with line at mean with SEM.
Figure 4
Figure 4
CD69 and HLA-DR expression on young or aged CD4 + CXCR5 + Tfh cells, CD4 + T, CD3 + CD4-T and CD4 + CXCR5-T cells. CD69 (A) and HLA-DR (B) expression on CD4 + T, CD3 + CD4-T, CD4 + CXCR5 + Tfh and CD4 + CXCR5-T cells from representative results were shown as in histograms. Thin line: isotype control (iso), heavy line: young individual (young), filled area: elderly individual (aged). Statistical results of percentages of CD69 + (C) (young subjects: n = 13, aged subjects: n = 11), and HLA-DR + (D) (young subjects: n = 10, aged subjects: n = 10) among CD4 + T, CD3 + CD4-T, CD4 + CXCR5 + Tfh and CD4 + CXCR5-T cells were shown. Y: young individuals, A: aged individuals. n.s: no significant difference. All data were expressed with line at mean with SEM.
Figure 5
Figure 5
Similar frequency of IL-21 produced by aged and young blood CXCR5 + CD4 + Tfh cells. A. Representative results of IL-21+ cells produced by Tfh or CD4 + CXCR5-cells were shown. B. FACS data of percentages of IL-21+ cells in CD4 + CXCR5 + Tfh cells or CD4 + CXCR5- cells from both aged and young individuals (young subjects: n = 23, aged subjects: n = 23) were shown. C. IL-21 concentration determined by ELISA was shown. PBMCs were prepared and cultured with P + I (PMA and inomycin) or – (medium) for 3 days. Supernatants were collected and IL-21 was evaluated by ELISA. *: p < 0.05. All data were expressed with line at mean with SEM.
Figure 6
Figure 6
Comparison of IFN-γ+, IL-17+, IL-4 + or IL-22+ cells among aged and young CXCR5 + CD4 + Tfh. PBMCs were prepared, stimulated with PMA and inomycin for 4–6 hours, and intracellular staining were done. FACS data were analyzed: lymphocytes were first gated and then CD4 + CXCR5+ or CD4 + CXCR5- cells were gated and cytokines such as IFN-γ, IL-17, IL-4 and IL-22 were analyzed. Statistical data of frequencies of IFN-γ + (A), IL-17 + (B), IL-4 + (C) and IL-22 + (D) among CD4 + CXCR5 + Tfh or CD4 + CXCR5- cells were shown. *: p < 0.05, n.s: no significant difference. All data were expressed with line at mean with SEM.
Figure 7
Figure 7
Different frequencies of IFN-γ+, IL-17+, IL-4+ or IL-22+ cells among Tfh compared with CD4 + CXCR5- cells. Statistical data of frequencies of IFN-γ + (A), IL-17 + (B), IL-4 + (C) and IL-22 + (D) among CD4 + CXCR5 + Tfh or CD4 + CXCR5- cells were shown. All data were expressed with line at mean with SEM. Differences between two groups were assessed by paired t test for two-tails. *: p < 0.05, n.s: no significant difference.

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