Inflammatory cytokines overcome age-related defects in CD4 T cell responses in vivo

Abstract

Age-related decreases in immune function are thought to contribute to the reduced efficacy of vaccinations seen in elderly populations. Our previous in vitro studies demonstrated that naive CD4 T cells from aged TCR-transgenic mice proliferate less than young cells and generate poorly functioning effectors due to decreased IL-2 production. In this current study, we show that this age-related defect in CD4 T cell response also occurs in vivo and that it is correlated with reduced NF-kappa B activation. After transfer to young hosts, CD4 T cells from aged transgenic mice proliferate less and produce reduced levels of IL-2 upon immunization with Ag and alum. Introducing a combination of the inflammatory cytokines TNF-alpha, IL-1, and IL-6, or the use of an adjuvant such as CFA that induces these cytokines, markedly enhanced responses of these aged CD4 T cells, so that they proliferated and produced IL-2 similar to young cells. This enhancement is correlated with the enhanced activation of the transcription factor NF-kappa B in aged cells. We suggest that induction of inflammatory cytokines via adjuvants may enhance the efficacy of vaccinations in elderly populations.

FIGURE 1

Reduced in vivo expansion of aged CD4 T cells. Naive Tg CD4 T cells from young and aged mice were CFSE labeled and transferred to young syngeneic CD4 KO hosts immunized i.p. with PCC peptide/alum. On days 1–3, spleens and LNs were harvested and CFSE staining was assessed. A, Representative FACS histograms showing CFSE profiles of young (open) and aged (shaded) Tg CD4 cells. As indicated in the FACS dot plots, histograms are gated on Vβ3⁺CD4⁺ cells. B, Fold expansion of donor young and aged Vβ3 CD4 cells from days 1 to 3 after immunization with peptide/alum. Bar graph shows means ± SE of four experiments; *, p < 0.05.

FIGURE 2

Reduced ex vivo IL-2 production by aged CD4 T cells. Young and aged Tg CD4 cells from immunized adoptive hosts described in Fig. 1 were harvested on days 1, 3, and 6 after immunization. Cells (spleen and LN) were stimulated ex vivo with PMA/ionomycin for 4 h and intracellular staining for IL-2 was performed. A, FACS histograms are gated on Vβ3⁺CD4⁺ cells as shown in Fig. 1. G-MFI and percent IL-2 positive staining are indicated for each histogram; dotted lines represent isotype controls. No IL-2-positive cells were observed in control cultures not given PMA/ionomycin. Representative of seven experiments. B, Graph showing the percent IL-2-positive staining and G-MFI of young (solid) and aged (striped) donor populations on day 3 (means ± SE) for seven experiments; *, p < 0.05.

FIGURE 3

T/1/6 and CFA induce increased in vivo expansion and IL-2 production by aged cells. Naive Tg CD4 T cells from young and aged mice were CFSE labeled and transferred to young syngeneic CD4 KO hosts immunized i.p. with peptide in alum, CFA, or plus a combination of TNF-α (250 ng), IL-1 (500 ng), or IL-6 (500 ng). On day 6, spleens and LNs were harvested and CFSE staining was assessed. A, Histograms of young (open) and aged (shaded) cells showing CFSE profiles are gated on Vβ3⁺CD4⁺ cells as described in Fig. 1. B, Bar graph showing the total recovery of young (solid) and aged (striped) donor Tg cells on day 6 after immunization; representative of at least three experiments. C, Graph summarizing IL-2 intracellular staining for young (solid) and aged (striped) donor Tg cells comparing alum, T/1/6, and CFA; graph shows mean ± SE of at least three experiments; *, p < 0.05. D, Graph summarizing G-MFI of IL-2 staining for young (solid) and aged (striped) donor Tg cells comparing alum, T/1/6, and CFA; graph shows mean ± SE of at least three experiments; *, p < 0.05. E, Young nontransgenic B10.Br mice were immunized i.p. with adjuvants alone: alum (striped), CFA (solid) or PBS control (open). At 24 h, PECs were harvested and total RNA was prepared. RNA was assayed by RPA. OD readings of bands for each cytokine were normalized to L32 mRNA to obtain relative mRNA quantities. Other cytokines that were not induced by CFA or alum include IL-12 p35 and p40, IL-10, IL-4, IL-5, IL-13, IL-9, IL-2, and TGFβ. Representative of two experiments.

FIGURE 4

T/1/6 enhance in vitro responses of CD4 T cells from aged mice. Tg CD4 T cells from young and aged mice were stimulated with Ag/APC alone (□) or with T/1/6 (10 ng/ml each; ■). A, IL-2 production by young and aged cells (2 × 10⁵ cells) 24 h after initial stimulation. B, The fold expansion of young and aged Tg cells after 4 days of culture with Ag/APC and cytokines was determined by dividing the total cell recovery on day 4 by the input cell number. C, IL-2 production by 2 × 10⁵ 4 day effector populations after restimulation with Ag/APC. For all graphs, each symbol represents the results of a single experiment; *, p < 0.05; ns, no significant difference.

FIGURE 5

Impaired transcriptional machinery in CD4 T cells from aged mice. Tg CD4 T cells from young and aged mice were stimulated with Ag/APC alone or with T/1/6 (10 ng/ml each). Nuclear extracts from equal numbers of 4-day effectors were prepared: A, before restimulation; B, after restimulation with anti-Vβ3/anti-CD28 for 4 h. EMSAs were performed using [³²P]dCTP-labeled probes for NF-κB (top), NFAT (middle), and AP-1 (bottom) transcription factors. Representative of three experiments.