Interleukin-12 is sufficient to promote antigen-independent interferon-gamma production by CD8 T cells in old mice

Abstract

Numerous functional defects have been identified in naive T cells from aged mice, including deficiencies in proliferation, cytokine production and signal transduction. It is well documented that the ratio of naïve to memory T cells significantly decreases with age resulting in the majority of T cells from aged hosts expressing activated/memory T-cell markers (CD44(hi)), yet it is unclear whether T cells with a CD44(hi) phenotype in aged hosts are functionally equivalent to T cells with a similar phenotype in young hosts. We have identified a population of CD44(hi) CD8 T cells in old mice that are capable of secreting interferon-gamma (IFN-gamma) in response to interleukin-12 (IL-12) stimulation. This occurred in the absence of T-cell receptor engagement, a function that was not observed in CD8 T cells from young mice. This phenotype was associated with increased IL-12 receptor beta2 gene expression and IL-12 induced signal transducer and activator of transcription 4 (STAT-4) activation, even when CD8 T-cell numbers from young and old mice were normalized for CD44(hi) expression. Furthermore, we demonstrate that IL-12-induced STAT-4 activation was required for T helper type 1 (Th1) cytokine-induced IFN-gamma production in CD8 T cells. These data illustrate that old mice possess a specialized subset of CD44(hi) CD8 T cells with an enhanced responsiveness to IL-12, enabling these cells to produce substantial amounts of IFN-gamma in response to Th1 cytokine stimulation. We have therefore identified a functional difference in the populations of CD44(hi) CD8 T cells from young and old mice, and believe that understanding age-associated immunological changes is essential for helping the elderly combat deadly diseases.

Figure 1

CD8 T cells from old mice have increased phosphorylated signal transducer and activator of transcription 4 (pSTAT-4) levels upon stimulation with interleukin-12 (IL-12). Single-cell suspensions were isolated from the lungs of young (open bars) and old (closed bars) mice and cultured with tissue culture media (TCM) or IL-12 for 4 hr (a–c) or multiple time-points (d,e). Cells were fixed in 1 × lyse/fix buffer, permeabilized with Perm III buffer, and flow cytometry was performed using antibodies against CD3, CD8 and pSTAT-4. Lymphocytes were gated according to their characteristic forward and side scatter and CD8 T cells were identified by CD8⁺/CD3⁺ double staining. (a) Representative histograms illustrating pSTAT-4 expression. (b) Percentage of CD8 T cells expressing pSTAT-4 at 4 hr. (c) Mean fluorescence intensity (MFI) of pSTAT-4 in CD8 T cells at 4 hr. An isotype control antibody for pSTAT-4 was used to set the positive gates. Data represent mean ± SE of four or five mice and are representative of two independent experiments. (d) Percentage of CD8 T cells expressing pSTAT-4 at multiple time-points. (e) MFI of pSTAT-4 in CD8 T cells at multiple time-points. Unstimulated cultures were used to set the positive gates. Data represent the mean ± SE of four or five mice and are representative of two independent experiments. Significance was determined by unpaired Student’s t-test *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

Interleukin-12 (IL-12) stimulation leads to signal transducer and activator of transcription 4 (STAT-4) phosphorylation in purified CD8⁺ cells. CD8⁺ cells were purified from the lungs (a–c) and spleens (d,e) of young (open bars) and old (closed bars) mice using magnetic cell separation. Purified cells were pooled according to age (a–c), or plated individually (d,e) and cultured with tissue culture medium (TCM) or IL-12 for 2 hr (a–c) or 4 hr (d,e). Cells were fixed, permeabilized and stained with antibodies against CD3, CD8 and pSTAT-4. CD8 T cells were identified by CD8⁺/CD3⁺ double staining. (a) Representative dot plots of pSTAT-4 expression in pulmonary CD8 T cells from young and old mice. (b) The percentage of pulmonary CD8 T cells expressing pSTAT-4. (c) Mean fluorescence intensity (MFI) of pSTAT-4 in pulmonary CD8 T cells. Positive gates were set on unstimulated cell cultures. Data represent pools of four or five mice from two independent experiments. Error bars represent the mean ± SE of the two independent experiments. (d) The percentage of splenic CD8 T cells expressing pSTAT-4. (e) MFI of pSTAT-4 in splenic CD8 T cells. Data represent the mean ± SE of four or five mice. Significance was determined by unpaired Student’s t-test. *P < 0·05.

Figure 3

Phosphorylated signal transducer and activator of transcription 4-positive (pSTAT-4⁺) CD8 T cells are CD44^hi and interleukin-12 receptor β2-positive (IL-12Rβ2⁺). (a,b) Single-cell suspensions were isolated from the lungs of young (open bars) and old (closed bars) mice and stimulated with IL-12 for 4 hr. Cells were fixed, permeabilized and stained with antibodies against CD3, CD8, CD44 and pSTAT-4. Lymphocytes were gated according to their characteristic forward and side scatter and CD8 T cells were identified by CD8⁺/CD3⁺ double staining. (a) Representative dot plots of CD44 and pSTAT-4 expression in pulmonary CD8 T cells from young and old mice. (b) Percentage of CD44^hi and CD44^lo CD8 T cells expressing pSTAT-4. Data represent the mean ± SE of five mice and are representative of two independent experiments. Significance was determined by unpaired Student’s t-test. **P < 0·01, ***P < 0·001. (c). Single-cell suspensions were isolated from the spleens of two young (open bars) and two old (closed bars) mice and pooled together according to age. Cells were stained with antibodies against CD3, CD8 and CD44. Populations of CD44^hi and CD44^lo CD8 T cells were purified using high-speed cell sorting and cultured with IL-12 for 4 hr. Cells were fixed, permeabilized and stained for pSTAT-4, and subsequently analysed for the percentage of CD44^hi and CD44^lo CD8 T cells expressing pSTAT-4. Unstimulated cell cultures were used to set the positive gates. Data represent mean ± SE of the two independent experiments. (d,e) CD8⁺ cells (d) or CD44^hi and CD44^lo CD8 T cells (e) were purified using magnetic cell separation (d) or high-speed cell sorting (e), homogenized in 1 ml Trizol, and frozen at −80°. RNA was isolated and reverse transcription polymerase chain reaction was performed for IL-12Rβ2 messenger RNA (mRNA). (d) IL-12Rβ2 relative units (RU) of mRNA. Data represent the mean ± SE of four mice and are representative of two independent experiments. (e) IL-12Rβ2 mRNA RU. Data represent the mean ± SE of two independent experiments of pooled cells from two mice.

Figure 4

Interleukin-12 (IL-12)-stimulated interferon-γ (IFN-γ) production is increased in CD8 T cells from old mice compared with young mice. (a) CD8⁺ cells were purified from the lungs of young (open bars) and old (closed bars) mice using magnetic cell separation. Cells were plated on an ELISpot plate that had been pre-coated with IFN-γ capture antibody, and were stimulated with tissue culture medium (TCM) or IL-12 for 48 hr at 37°. IFN-γ-producing cells were determined by ELISpot. Data are expressed as pools of two mice and are the combined result of three independent experiments. Significance was determined by unpaired Student’s t-test. *P < 0·05. (b–d) Single-cell suspensions were isolated from the lungs of young (open bars) and old (closed bars) mice and stimulated with IL-2, IL-12 and IL-18 for 30 min, 4 hr or 8 hr at 37°. Cells were fixed, permeabilized and stained with antibodies against CD3, CD8, phosphorylated signal transducer and activator of transcription 4 (pSTAT-4) and IFN-γ. Lymphocytes were gated according to their characteristic forward and side scatter and CD8 T cells were identified by CD8⁺/CD3⁺ double staining. Unstimulated cultures were used to set the positive gates. (b) Percentage of IFN-γ⁺ CD8 T cells. (c) Representative dot plots of pSTAT-4 and IFN-γ expression in pulmonary CD8 T cells. (d) Percentage of pSTAT-4⁺/IFN-γ⁺ CD8 T cells. Data represent the mean ± SE of five mice and are representative of two independent experiments. (e,f) CD8⁺ cells were purified from the spleens of young (open bars) and old (closed bars) mice using magnetic cell separation. Cells were cultured with 10 μm P6 or dimethylsulphoxide in supplemented Dulbecco’s modified Eagle’s minimal essential medium for 30 min, followed by 4 hr of IL-2, IL-12 and IL-18 stimulation at 37°. Cells were fixed, permeabilized and stained with antibodies against CD3, CD8, pSTAT-4 and IFN-γ. Unstimulated cultures were used to set the positive gates. (e) Representative dot plots showing pSTAT-4 and IFN-γ expression in CD8 T cells from old mice. (f) Percentage of IFN-γ⁺ CD8 T cells. Data represent the mean ± SE of four mice and are representative of two independent experiments. Significance was determined by unpaired Student’s t-test. *P < 0·05. **P < 0·01, ***P < 0·001.