Figure 4. AIMT cells in young and aged mice represent identical clones.

(A-F) Lymph node cells from young (7-12 weeks) and aged (65-77 weeks) Vβ5 mice were enriched for CD8⁺ T cells by magnetic beads and stained for CD8, CD44, CD62L, CD49d, TCRVα2 and TCRVα8.3. 2-3 mice were pooled for each sample. n = 3 independent experiments.

(A) The percentage of TCRVα2⁺ and TCRVα8.3⁺ among AIMT and naïve CD8⁺ T cells is shown.

(B-F) AIMT (CD44⁺ CD62L⁺) and naïve (CD44^- CD62L⁺) TCRVα2⁺ CD8⁺ T cells were FACS sorted. Library of TCR sequences was generated from isolated RNA using a two-step PCR and analyzed by deep sequencing.

(B) Frequency of TRAJ52, TRAJ39, and TRAJ43 usage among the analyzed samples. Mean is shown.The statistical significance was tested using one-way repeated measures ANOVA.

(C) PCA analysis of the TCR clonotype (based on CDR3 amino acid sequence) usage in the individual samples. Only clonotypes detected in at least 3 samples were included in the analysis.

(D) A volcano plot showing log2 differential usage between naïve and AIMT cells (both young and aged) and adjusted p-value for each individual TCR clonotype present at least in 3 samples. “AIMT”, “naïve” (2-fold difference in usage, adjusted p < 0.01) and “neutral” (non-significant) clonotypes are indicated in red, blue, and gray respectively.

(E) Pie charts showing the abundance of the “AIMT”, “naïve”, and neutral clonotypes in the T-cell repertoires of young and aged Vβ5 mice.

(F) Relative usage (z-score) of 5 most abundant “AIMT” and 5 most abundant “naïve” clonotypes in the indicated samples. The average frequency of each clone in young naïve, young AIMT, aged naïve, and aged AIMT repertoires is indicated on the right.