IL-21 preferentially enhances IL-15-mediated homeostatic proliferation of human CD28+ CD8 memory T cells throughout the adult age span

Abstract

An age-related decline in human immune response is marked by the accumulation of CD28(-) CD8 T cells, which is attributed to repeated antigenic stimulation and to homeostatic proliferation mediated by cytokines such as IL-15. However, the identity of the cytokines that are responsible for the maintenance of CD28 expression is less known. Here, we report the role of IL-21 in the regulation of IL-15-mediated growth and CD28 expression of CD8 memory T cells of young and old donors. We showed that IL-21 drives more IL-15-stimulated cells to enter cell division and to undergo apoptosis. Furthermore, IL-21 preferentially enhanced IL-15-induced proliferation of CD28(+) CD8 memory T cells over their CD28(-) counterparts, as CD28(+) cells expressed higher levels of IL-15R and IL-21R and greater pSTAT5 upon IL-15 and IL-21 stimulation. In addition, IL-21 reduced IL-15-induced CD28 down-regulation in CD8 memory T cells. Finally, the ability of proliferation and survival in response to homeostatic cytokines IL-15 and IL-21 of CD28(+) CD8 memory T cells was well-maintained with age. Together, these findings suggest that IL-21 enhances IL-15-mediated proliferation of CD8 memory T cells, particularly CD28(+) memory T cells, and also serves as an antagonist to the IL-15-induced increase of CD28(-) CD8 T cells.

Figure 1.

IL-21 enhances IL-15-mediated CD8 memory T cell growth in a dose-dependent manner. (A) Growth of CD8 memory T cells cultured with IL-15 and IL-21 alone or in combination. CD8 memory T cells isolated from PBMCs of normal adult donors were cultured with IL-15 and IL-21 at concentrations indicated (ng/mL). Live cell numbers were counted by a standard trypan blue staining method after 7 days, and cell expansion (Fold Change) was calculated by dividing the live cell number at Day 7 over the initial seeding cell number at Day 0. Values represent the mean value ± sem obtained from independent experiments of five to 15 different donors. (B) Growth curves of CD8 memory T cells stimulated with IL-15 alone or IL-15 + IL-21. CD8 memory T cells isolated from PBMCs were stimulated with IL-15 alone or with either two concentrations of IL-21. Cells were counted at days indicated, and cell expansion (Fold Change) was calculated. Values represent the mean value ± sem obtained from independent experiments of nine different donors. Differences in cell count between IL-15 alone and IL-15 + IL-21 (low) have P< 0.05 for Days 7 and 28 and P < 0.01 for Days 14 and 21.

Figure 2.

IL-21 induces pSTAT3 and promotes more CD8 memory T cells to enter cell division. (A) pSTAT3 and pSTAT5 in CD8 memory T cells upon stimulation with IL-15 (50 ng/mL) in the absence or presence of IL-21 (25 ng/mL). pSTAT3 and pSTAT5 were determined by specific antibodies against pSTAT3 and pSTAT5 and represented as the MFI. Values represent the average MFI ± sem, obtained from experiments for cells isolated from eight adult donors; **, P < 0.01; ***, P < 0.001. (B) Cell division profiles of CD8 memory T cells cultured with IL-15 alone or IL-15 + IL-21. Isolated CD8 memory T cells were labeled with CFSE and cultured with IL-15 alone or with IL-15 + IL-21. Representative histograms of CFSE profiles of cultured CD8 memory T cells from one of eight adult donors are presented. Values represent the mean percentage of undivided cells of eight donors with P = 0.0002.

Figure 3.

IL-21 induces more cell death at a late stage of IL-15-mediated culture. Isolated CD8 memory T cells were cultured with IL-15 (50 ng/ml) alone or with IL-15 (50 ng/ml) plus IL-21 (25 ng/ml). A representative dot-plot of cells stained for Annexin-V (ANXA-V) and 7-AAD after culture from one of six different adult donors presented. Values represent the mean percentage, P≤ 0.05 for Days 14 and 21.

Figure 4.

IL-21 preferentially enhances IL-15-mediated growth of CD28⁺ CD8 memory T cells. (A) Growth curves of CD28⁺ and CD28^– CD8 memory T cells stimulated with IL-15 alone or IL-15 + IL-21. Total CD8 memory T cells were isolated and cultured. The live cell numbers were determined by trypan blue staining under a regular light microscope. CD28⁺ and CD28^– CD8 memory T cells in the culture were determined by FACS analysis, and the values were then calculated and represent the mean ± sem obtained from results of nine adult donors. Differences in cell count for CD28⁺ cells under each condition have P< 0.05 for Day 7 and P < 0.01 for Days 14, 21, and 28. (B) Comparison of growth of separately cultured CD28⁺ and CD28^– CD8 memory T cells. CD28⁺ and CD28^– CD8 memory T cells were isolated from total CD8 memory T cells by cell sort and cultured separately with IL-15 alone or with IL-21. At the indicated days, the cells were harvested and counted. Values represent the mean value ± sem (n = 13 for Day 7; n = 5 for Day 14); *, P < 0.05; ***, P < 0.001.

Figure 5.

CD28⁺ CD8 memory T cells express higher levels of IL-15R and IL-21R and exhibit stronger STAT5 signaling than their CD28^– counterparts. (A) CD28⁺ and CD28^– CD8 memory (CD45RA^–) T cells were isolated by cell sort and cultured under IL-15 alone or with IL-21. (A) The mRNA levels of the receptors (IL15RA, IL21R, IL2RB, and IL2RG) in freshly isolated CD28⁺ and CD28^– CD8 memory T cells were determined by qRT-PCR and normalized to ACTB. Values represent the mean value ± sem obtained from results of one of six different human donors. (B) Surface protein expression of CD132 (Common γ_c) in CD28⁺ and CD28^– CD8 memory T cells. Values represent the average MFI ± sem obtained from experiments for cells isolated from five different human donors for Day 0 and 14 donors for Day 7. (C) pSTAT5 in CD28⁺ and CD28^– CD8 memory T cells after IL-15 and IL-21 exposure. pSTAT5 was determined using the MFI of fluorescent-labeled antibodies against pSTAT5 molecules and normalized to unstimulated cells. Values represent the average MFI ± sem obtained from experiments for cells isolated from eight different adult donors at 10 and 30 min after treatment. Throughout the figure: *, P < 0.05; ***, P< 0.001.

Figure 6.

IL-21 reduces IL-15-induced down-regulation of CD28 expression in CD8 memory T cells. (A) Analysis of cell division and CD28 expression in cultured CD8 memory T cells under IL-15 alone or IL-15 + IL-21. Isolated CD8 memory T cells were labeled with CFSE and cultured with IL-15 alone or with IL-15 + IL-21 for 14 days. CFSE and CD28 profiles were collected using flow cytometry and analyzed by FlowJo. A representative dot-plot of the CD28 versus CFSE profile was presented from four different donors. Dotted lines indicate cell division. (B) Quantitative presentation of the ratio of CD28⁺ over CD28^– in fast-dividing (≥5 cell divisions) and slow/no-dividing (0–4 cell divisions). (C) MFI of the surface CD28 expression for cells that have undergone little (0–4) or extensive (5 or more) divisions. Values represent the mean value ± sem obtained from experiments for cells isolated from four different human donors. (D) CD28 mRNA levels of CD8 memory T cells cultured with IL-15 alone or IL-15 + IL-21. The data were derived from qRT-PCR analysis and normalized to ACTB and normalized further to a Day 0 value. Values represent the mean value ± sem obtained from experiments for cells isolated from six different human donors for Days 0 and 7 or five donors for Day 14. C and D use the same legend as in B, the hatched bar in D represents freshly isolated cells with no stimulation. Throughout the figure: *, P< 0.05; **, P < 0.01: †, P < 0.06.

Figure 7.

IL-15- and IL-21-induced proliferation of CD28⁺ CD8 memory T cells is stable through adult age. CD28⁺ CD8 memory T cells were isolated from old donors (age ≥70; n = 4) and young donors (age ≤30; n = 6) by cell sort and cultured with IL-15 (50 ng/mL) alone or with IL-21 (25 ng/mL) for 14 days. Live cell numbers were counted with trypan blue staining, and cell expansion (Fold Change) was calculated by dividing the cell count after culture over the initial seeding cell count. Values represent mean ± sem.