DIO reduces thymopoiesis and restricts TCR diversity. (A) The splenic CD4⁺ T cells were isolated to prepare the DNA, and signal-joint TREC levels were analyzed by using quantitative PCR analysis. A total of 6 to 10 mice per group were used for sjTREC assay, and the data are expressed as mean ± SEM. The obesity in mice significantly reduced TRECs at all ages examined. (B) Real-time PCR analysis of cytokine mRNA expression in purified CD4 cells from 13-month-old control and DIO mice (n = 6/group). (C) The TCR spectratyping analyses of CD4 T cells from 3- and 9-month-old mice on chow and high-fat diets are shown (n = 5). A polyclonal profile is Gaussian with 6 to 8 peaks, whereas alterations from Gaussian distributions are measure of oligoclonality. The Gaussian distribution profiles were translated into probability distributions as functions of the AUC for each CDR3 length. The average distribution of the CD4⁺ repertoire from 3- and 9-month-old ad libitum–fed chow diet controls is compared with the DIO mice. The statistical quantitation of the CDR3 size of all the TCR Vβ between control and DIO mice was performed by using CDR3QAssay software. The extent of the change in the CDR3 size distribution is defined as the percentage of improvement (distance from the mean value). (The percentage of improvement greater than 3 SDs in the fragment length of each family indicates that there are significant changes in the Vβ family, based on Gorochov et al.⁴⁷ ) These improvements in TCR diversity are represented as landscape surfaces, in which smooth (blue) landscapes represent an unchanged TCR repertoire (diversity). The mountain (in green, yellow, and orange) depicts perturbation in amplified peaks of CDR3 lengths compared with control mice. Each line crossing on the y-axis of the landscape denotes perturbation for a specific CDR3 length or size (x-axis) of a particular Vβ family (z-axis).