Prolonged exposure of naïve CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length

Abstract

Interleukin (IL)-7 and IL-15 are cytokines implicated in homeostatic control of the peripheral CD8 T-cell pool. We compared the effects of IL-7 and IL-15 on survival and proliferation of purified human CD8+ T-cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl-2 up-regulation. Surprisingly, although minimal proliferation of naïve CD8+ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL-15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8+ T cells through several cell divisions resulted in up-regulation of telomerase and the maintenance of telomere length. These data show that IL-7 and IL-15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset-dependent manner, and have implications for the homeostatic expansion of the naïve CD8+ T-cell pool.

Figure 1

Expression of CD132 (common γ-chain), CD122 (IL-2/15R β-chain) and CD127 (IL-7R α-chain) on subsets of CD8⁺ T cells. PBMC were stained as described in materials and methods. One representative experiment of three is shown.

Figure 2

Induction of proliferation and prevention of apoptosis by IL-7 and IL-15. (a) Purified CFSE-labelled CD8⁺ T cells were incubated with graded doses of recombinant human IL-7 or IL-15 for 6 days and the percentage of cells that had undergone division measured (one representative experiment of three). (b) Purified CD8⁺ T-cell subsets were stimulated with high or low doses of IL-7 or IL-15 and proliferation measured by [³H]-thymidine incorporation on day 6. (c) Apoptosis of purified subsets in the presence or absence of IL-7 or IL-15 was measured by annexin V–FITC staining on day 6 (mean ± SE of data from six individuals). *Indicates P ≤ 0·05 compared to the same cells cultured in medium alone.

Figure 3

Induction of Bcl-2 by IL-7 and IL-15. Purified subsets of CD8⁺ T cells were cultured for 4 days with non-proliferative doses (1 ng/ml) of IL-15 or IL-7 and up-regulation of Bcl-2 measured by flow cytometry using a live cell gate. Solid fill, Bcl-2 on day 4; solid line, Bcl-2 on day 0; dotted line, isotype control for day 0 and; dashed line, isotype control for day 4.

Figure 4

Delayed proliferation of CD45RA⁺ CCR7⁺ CD8⁺ T cells in response to IL-7 or IL-15. (a, b) Purified cells cultured with 50 ng/ml of IL-7 or IL-15 for 34 days (representative data shown for one experiment of four). Spontaneous apoptosis of cells in medium alone was ≥90% by day 7 (data not shown). (a) Viable cell numbers were determined at different time points by sampling bulk cultures repeatedly (trypan blue exclusion). (b) Proliferation was measured by [³H]-thymidine incorporation at the indicated time points. (c–e) CFSE-labelled cells were cultured as indicated and cell division measured by flow cytometry. (d, e) dotted line day 3, solid line day 13, solid fill day 21.

Figure 5

Phenotype of purified CD45RA⁺ CCR7⁺ CD8⁺ T cells cultured for a prolonged period in IL-7 or IL-15. (a) Flow cytometric analysis of cell surface markers on purified cells cultured for 15 days with 50 ng/ml of IL-7 or IL-15. Filled histogram represents expression on day 0, dotted line cells cultured with IL-7, and solid line cells cultured in the presence of IL-15. (b) CD69 expression measured after 6 hr culture with CD3/CD28 expander beads/cytokines, filled histogram represents CD69 expression on day 0, dotted line and dashed line represent culture with IL-15 and IL-7, respectively, solid line represents culture with CD3/CD28 expander beads. (c) CD69 expression during 34 days culture with 50 ng/ml of IL-7 or IL-15. (d) CD95 expression was measured on cells at day 0 (dotted line) or cultured for 16 days (solid line) with 50 ng/ml IL-15. Solid fill represents isotype day 0, dashed line isotype day 16. (e) CD95 expression measured after culture with IL-15 for 25 days (solid line), or after 16 days culture with cytokines followed by washing and culture in medium alone for 9 days (dotted line). Isotype control for day 25 represented by dashed line, isotype for washed and recultured cells represented by solid fill histogram.

Figure 6

Analysis of effector function in purified subsets of CD8⁺ T cells stimulated with 50 ng/ml of IL-7 or IL-15. Expression of granzyme A (a), granzyme B (b), or perforin (c) measured by intracellular staining of purified subsets on day 0 or after culture with IL-15 or IL-7 for 7, 15 or 20 days (CD45RA⁺ CCR7⁺ cells only) as indicated. (d, e) Expression of IFN-γ and TNF-α, on purified subsets cultured for 7, 15 or 20 days (CD45RA⁺ CCR7⁺ cells only) with cytokines, incubated for the last 5 hr with PMA + ionomycin + GolgiPlug. Cytokines were detected by intracellular staining. All results are representative of three independent experiments.

Figure 7

Induction of telomerase activity and maintenance of telomere length by CD45RA⁺ CCR7⁺ CD8⁺ T cells cultured with 50 ng/ml of IL-15 or IL-7. (a) Purified cells (from three individuals A, B, C) analysed on day 0 or after 14 days of culture with cytokines. Telomerase activity in cell lysates was measured by Southern blot analysis. TSR8 denotes the internal quantitative control. (b) Telomere length analysis of purified cells on day 0 or after 19 days of culture with 50 ng/ml of IL-7 or IL-15. 1 µg of genomic DNA was digested with HinfI and RsaI and probed with ³²P-labelled (CCCTAA) to detect repeat sequences.