CD44^act expression on CD4CD25⁺ Treg cells is associated with elevated expression of relevant cytokine mRNA. (A) CD4CD25⁺ T cells were activated for 16 to 18 hours with plate-bound anti-CD3/anti-CD28 then sorted immediately on the basis of CD44^act expression (gates indicated by boxes). (B) Sorted activated CD4CD25⁺ cells and freshly isolated, unactivated (Unact) CD4CD25⁺ cells were immediately lysed for multiplex RT-PCR to assess cytokine mRNA levels. Results shown are representative of 4 independent experiments. Cycles for each cytokine: GMCSF, TNFα, 25 cycles; IL-13, IFNγ, TGFβ, 30 cycles; IL-4, -5, -10, 40 cycles. Internal control GAPDH was amplified for 25 cycles. (C) Relative cytokine mRNA levels as measured by real-time quantitative PCR analysis in CD44^act+ and CD44^act– fractions of CD4CD25⁺ cells activated for 24 hours and separated as in panel A. Cytokine levels in unactivated CD4CD25⁺ T cells are shown for comparison. Data shown are the mean ± SD of 3 experiments. *P ≤ .001 and **P ≤ .005 for comparisons of CD44^act+ to CD44^act– mRNAs.