Daan Pieren

35 Novel features of ageing in murine T cells RESULTS Proportion of splenic regulatory T cells increases with progressing age We assessed the composition of the total splenic CD3 + T-cell pool of young (n=6 per experiment, 2 months old) and aged mice of various ages (n=4-6 per experiment, 17 to 18 months, 22 to 24 months, and 28 months old). Using flow cytometry, significantly lower frequencies of CD3 + T cells were detected in the spleens of aged mice compared to young mice, except for the oldest group of mice ( Figure 1A, Supplementary Figure 1 ). Within this T-cell pool, we found decreased proportions of Th cells (CD3 + CD4 + FoxP3 - ) ( Figure 1B ), comparable proportions of Tc cells (CD3 + CD4 - FoxP3 - ) ( Figure 1C ), and increased proportions of Treg cells (CD3 + CD4 + FoxP3 + ) ( Figure 1D ) with progressing age. Defining Tc cells as CD3 + CD4 - FoxP3 - may also include small proportions of non-Tc-cell subsets, such as CD4 - CD8 - T cells or γδ T cells. However, an additional data set showed that the proportion of these cells did not differ between young and aged mice ( Supplementary Figure 1 ). T cells Helper T cells Regulatory T cells Cytotoxic T cells A B C D Young (2) Aged (17-18) Aged (22-24) Aged (28) 0 10 20 30 40 50 % CD3 + of viable lymphocytes *** p =0.11 *** Young (2) Aged (17-18) Aged (22-24) Aged (28) 0 20 40 60 80 % Foxp3 - CD4 + of CD3 + ** ** ** Young (2) Aged (17-18) Aged (22-24) Aged (28) 0 20 40 60 80 % Foxp3 - CD4 - of CD3 + Young (2) Aged (17-18) Aged (22-24) Aged (28) 0 10 20 30 % Foxp3 + CD4 + of CD3 + *** *** *** Figure 1. Proportion of regulatory T cells increases with progressing age. The frequencies of T cells (CD3 + of viable lymphocytes) ( A ), Th cells (FoxP3 - CD4 + of CD3 + ) ( B ), Tc cells (FoxP3 - CD4 - of CD3 + ) ( C ), and Treg cells (Foxp3 + CD4 + of CD3 + ) ( D ) were determined in spleens of young (2 months old, n=36) and aged mice of three age categories (17-18 months old, n=11; 22-24 months old, n=11; 28 months old, n=9). Numbers between brackets indicate the age in months. Mean ± SD; ** p < 0.01, *** p < 0.001 for difference between young and each group of aged mice using Kruskal-Wallis test. Proliferative T-cell responses decline with progressing age Next, we exposed splenic single-cell suspensions of young and aged mice to a low (low anti-CD3), intermediate (low anti-CD3 + anti-CD28), or high (high anti-CD3 + anti-CD28) strength of stimulation to investigate the proliferative capacity of Th, Tc, and Treg cell subsets. Proliferation of Th, Tc, and Treg cells was measured by flow cytometry up to four days after stimulation ( Figure 2 ). Proliferative capacity of all three T-cell subsets showed a gradual decline with 2