TSC subjects with germline mutation of <i>TSC1</i> reveal proper representation of CD4 and CD8 mature T cell subsets.

<p>PBMC from healthy donors and TSC subjects were isolated by density gradient centrifugation, counted and surface stained with anti-CD3, CD4, CD8, CD45RA and CD27 mAb and analyzed by FACS. A–B) Viable cell counts per ml of blood and the frequency of CD4⁺ and CD8⁺ cells within CD3⁺ lymphocytes is depicted for individual subjects. Open squares refers to Pt1 and 2 carrying the Leu129Pro mutation, black squares refers to Pt3 and 4 carrying a stop codon mutation in the position of Arg692. C–E) Distribution of naïve (Naïve, CD45RA^highCD27^high), central memory (CM, CD45RA^lowCD27^high), effector memory (EM, CD45RA^lowCD27^low) and terminally differentiated effectors (EF, CD45RA^highCD27^low) within CD3⁺CD4⁺ (C, D) or CD3⁺CD8⁺ (E) cells is depicted. F–G) Freshly isolated PBMC were stimulated for 4 h with PMA and Ionomycin. Expression of IL-2 and IFN-γ was determined by intracellular cytokine staining. Results depict the frequency of cytokine secreting cells after gating on CD3⁺CD4⁺ (F) or CD3⁺CD8⁺ (G) cells (2 HD and Pt3, 4). Cytokine production was also analyzed after a 5d CD3/CD28-driven culture for 8 HD and the 4 TSC1 Pts with no differences (not shown). H) The frequency of CD3⁺CD4⁺CD25⁺FoxP3⁺ cells is shown. Data depicted in D, E, H reflect the mean ± SEM of 8 HD and 4 TSC1 Pts. Statistical significance was evaluated by unpaired two-tailed Student t-Test (A–B, F–H) or Mann-Whitney test (D–E).