Low responsiveness of ICL CD4⁺ T cells to TCR stimulation. (A and B) PBMCs from healthy (n = 28), elderly (n = 6), SARC (n = 8), and ICL (n = 20) individuals were CFSE-labeled and stimulated on anti-CD3/anti-CD28 Ab-coated plates or left untreated (basal) for 5 days. Fluorescence intensity loss resulting from division cycles was determined by flow cytometry. Representative plots of the mean fluorescence intensity (MFI) of CFSE in CD4⁺-gated T cells are shown (A). Results (mean ± standard deviation [SD] of 10 independent experiments) are expressed as percentages of proliferating CFSE^low (>1 cell division) CD4⁺ T cells (B). (C and D) PBMCs were stimulated for 5 min by CD3 cross-linking or left untreated and immediately analyzed by PhosphoFlow for intracellular phospho-ERK-1/2 (pERK) content. Representative plots of the MFI of pERK in CD4⁺-gated T cells are shown. Background fluorescence (shaded area) was assessed using an isotype control Ab (C). The bar graphs summarize the results (expressed as mean ± SD) from all analyzed subjects (D). Kruskal-Wallis H test and associated P values are indicated. *P < 0.05 and ***P < 0.0005 compared with healthy CD4⁺ T cells (as determined using the Mann-Whitney U test).