Increased apoptosis, curtailed expansion and incomplete differentiation of CD8+ T cells combine to decrease clearance of L. monocytogenes in old mice

Abstract

Aging is accompanied by altered immunity, resulting in a variable state of poorly understood immunodeficiency. While both the numbers and the functionality of naïve T cells are decreased by aging, the impact of these changes upon immune defense against bacterial pathogens in vivo remains understudied. Using a model of Listeria monocytogenes (Lm), where the primary CD8(+) T-cell response is critically important for immune defense, we show that C57BL/6 (B6) mice exhibit an age-dependent reduction in survival, with delayed bacterial clearance in old animals. Kinetic analysis of antigen-specific CD8(+) T-cell expansion showed that CD8(+) effectors begin dividing at the same time in old and adult mice, but that the proliferative burst remained incomplete during discrete windows of time and was coupled with increased effector apoptosis in old mice. Further, antilisterial CD8(+) T cells in old mice showed altered expression of key phenotypic and effector molecules and diminished polyfunctionality, measured by the ability to simultaneously produce multiple effector molecules. These results suggest that defects in functional maturation of CD8(+) cells in aged mice, compounded by (or perhaps coupled to) their reduced expansion in response to infection, yield effector CD8(+) T-cell populations insufficient in size and capability to effectively clear newly encountered intracellular pathogens.

Figure 1. Old mice are less resistant to Lm infection

Adult and old B6 mice were infected with (A) 3×10⁶, (B) 1×10⁶, or (C) 3×10⁵ CFU of Lm-OVA, then monitored daily for survival. (D-E) Adult and old mice were infected with 1.4×10⁵ CFU Lm-OVA, and spleens (D) and livers (E) homogenized on days 1, 4, 6, 8, and 10 post-infection to determine the bacterial burden. Dotted line represents the limit of detection for the assay, and data are presented as mean +/- SEM. For all experiments, n=6-8 mice/group/time point. *p < 0.05; ***p < 0.001. Significance was calculated by the logrank test (A-C), or 2-way ANOVA with Bonferroni posttests (D-E). Data shown are representative of 3 independent experiments.

Figure 2. The antigen-specific CD8 response to Lm is reduced in old mice

Adult and old B6 mice were infected with 1×10³ CFU Lm-OVA and then alternate cohorts were bled every other day throughout the primary CD8 T-cell response to determine the frequency of CD11a^HI K^b-OVA⁺ CD8 T-cells by flow cytometry. At day 60 post-infection, mice were challenged with 1.1×10⁵ CFU Lm-OVA and the secondary CD8 T-cell response was followed by tetramer staining. (A-B) Representative FACS plots are shown, with the numbers indicating percentages of parental subset, and the graphs below depicting the response of all individual animals assessed at that time point. Significance was determined by unpaired t-test, with horizontal bars indicating which groups were compared for the p value shown. (C) Kinetics of the primary and secondary response in adult and old infected mice, and uninfected adult controls. Significance was determined by 2-way ANOVA with Bonferroni posttests. n=3-8 mice/group/time point. **p < 0.01; ***p < 0.001. Data represent mean +/- SEM of 2 independent experiments.

Figure 3. Old lymphocytes traffic normally to sites of Lm-OVA infection

Adult and old B6 mice were infected with 1×10³ CFU Lm-OVA. On day 8 post-infection, the % and # of (A) total CD4, (B) total CD8, and (C) OVA-specific CD8 T-cells were determined in the blood, spleen and liver by tetramer staining and flow cytometry. The MFI of (D) CCR5 and (E) CXCR3 on K^b-OVA⁺ cells was evaluated in the blood, spleen and liver. Significance was calculated by unpaired t-test, with horizontal bars indicating which groups were compared for the p value shown. n=8 mice/group. *p < 0.05; **p < 0.01 ***p < 0.001. Data represent mean +/- SEM of 2 independent experiments.

Figure 4. OVA-specific CD8 T-cells from old mice show altered effector cytokine capacity and phenotype

Adult and old B6 mice were infected with 1×10³ CFU Lm-OVA, and splenic OVA-specific CD8 T-cell polyfunctionality was evaluated 8 days later. (A, B) Polyfunctionality was characterized as the ability of IFNγ-producing cells to additionally produce TNFα, Granzyme B, or both. (A) A representative distribution of the OVA-specific CD8 population into each effector cytokine profile, and (B) the number of OVA-specific cells recovered from the spleen with each effector profile are shown. (C) The relative mean fluorescent intensity of IFNγ, TNFα, or Granzyme B produced individually by the OVA-specific CD8 T-cell population is shown. (D) The surface expression of the phenotypic markers on K^b-OVA⁺ CD8 T-cells was determined on days 5 (top row) and 8 (bottom row) following infection. Solid black line = adult, shaded grey = old. (E) The functional avidity of OVA-specific CD8 T-cells was determined by intracellular cytokine staining following stimulation with a peptide gradient. (F) The on-rate for IFNγ production, and (G) the frequency of CD8 T-cells producing IFNγ following OVA-8p peptide stimulation relative to the frequency of K_b-OVA tetramer binding cells was calculated. Significance was calculated by unpaired t-test, with horizontal bars indicating which groups were compared for the p value shown. n=8 mice/group. *p<0.05; **p<0.01. Data represent mean +/ SEM of 2-4 independent experiments.

Figure 5. Aged CD8 T-cells show reduced cytotoxicity

(A) Direct ex-vivo lysis of peptide pulsed target cells was performed. Significance was calculated by 2-way ANOVA with Bonferroni posttest. Data represent mean +/- SEM of 2 independent experiments. (B) Linear regression was performed to determine the relationship between the number of K^b-OVA⁺ cells and percent specific target cell lysis (A) for each age group. The r² values and deviation from zero (p) for each line are shown, while ** denotes the significant difference between the slopes of the two lines. n=8 mice/group. *p<0.05; **p<0.01; ***p < 0.001.

Figure 6. Old CD8 T-cells show reduced in vivo proliferation following Lm-OVA infection

Adult and old B6 mice were infected with 1×10³ CFU Lm-OVA, then either given 1 mg BrdU IP 24 hours prior to lymphocyte analysis (A-C), or placed on BrdU-containing water until analysis at day 8 post-infection (D). (A) The % of total CD8 T-cells incorporating BrdU and relative mean fluorescent intensity (rMFI) of α-BrdU staining was evaluated for each of the 24 hour periods between days 1-5 following Lm-OVA infection. n=4/group/time point. (B, C) The % of BrdU⁺ K^b-OVA⁺ CD8 T-cells and the intensity of α-BrdU staining within the K^b-OVA⁺ subset was determined at (B) days 4-5, (C) 5-6 post-infection, or (D) from day 0-8 following infection. (E) Annexin V and 7- AAD staining of total CD8 and K^b-OVA⁺ CD8 splenic T-cells were evaluated on days 5 and 7 after infection. Significance was calculated by unpaired t-test, with horizontal bars indicating which groups were compared for the p value shown. n=8/group/time point. * p<0.05; **p<0.01; ***p < 0.001. Data represent mean +/- SEM of 3 independent experiments.