Influence of AHSCT and cGVHD episodes on peripheral blood naive T-cell frequencies and de novo T-cell production. (A) Naive T-cell frequencies, as defined by CD45RA and CD27 dual expression on CD3⁺ cells, were quantified in PBMCs by multiparameter flow cytometry. A minimum of 3 × 10⁵ events in the live cell gate, as defined by forward and side scatter, was accumulated for each sample. (B) Age-related decrease of sjTREC content normalized for CD3⁺ T-cell frequencies. Peripheral blood sjTREC frequencies were obtained using real-time quantitative PCR and normalized to CD3⁺ T-cell frequencies as estimated by FACS analysis. Healthy adult controls (n = 37) are represented with empty triangles (▵), while the solid line (—) indicates the negative linear regression trend from control individuals' data (y = 31 581e^-0,1012x). AHSCT patients exempt of cGVHD (n = 22) at sampling time are shown as empty circles (▴), whereas patients who underwent transplantation currently undergoing cGVHD (n = 10) are illustrated with filled circles (•). (C) Histogram representation of average peripheral sjTREC frequencies in these cohorts. Statistical significance (P ≤ .05) was calculated by the 2-tailed Student t test and is shown on top of graphics. The vertical lines indicate the group's standard deviation. N.S. indicates not significant.