TurboGFP impairs T cell clustering after CD3/CD28 activation.

<p>T cells were transfected with either the Scr-Vec-GFP(+) or the Scr-Vec-GFP(−) vectors, then activated by plate-bound anti-CD3 and soluble CD28 for 20 hours. A) GFP-expressing T cells exibit impaired clustering after CD3/CD28 activation. B) Normal cluster formation of CD3/CD28-activated T cells in sample transfected with the Scr-Vec-GFP(−) are indicated by black arrows. A representation of 3 independent experiments is shown. C & D) Activated cell clusters were quantified within one representative field of view at 100x for the Scr-Vec-GFP(−) vector and the Scr-Vec-GFP(+) vector transfections to obtain the total number of clusters (C), or the numbers of clusters by size range (<25 μm, 25–50 μm, and >50 μm) (D) where the black bars indicate the Scr-Vec-GFP(−) transfections and the white bars indicate the Scr-Vec-GFP(+) transfections. Mean and SEM are shown.</p

CD25 expression levels by RT-PCR.

<p>RNA was isolated from CD4⁺ (A) and CD8⁺ (B) populations from six donors following 20 hours activation with anti-CD3/anti-CD28. Relative CD25 expression levels were normalized to beta-actin, calculated using the 2-(delta-delta(CT)) method and compared by Student's paired t-test. Mean and SEM are shown and <i>P</i>≤0.05 was considered to be significant.</p

Analysis of CD25 and CD69 surface expression by flow cytometry.

<p>T cells from seven donors were transfected with the vectors Scr-Vec-GFP(−) (black bars) or Scr-Vec-GFP(+) (white bars). Following 20 hours of anti-CD3/anti-CD28 activation, CD4⁺ and CD8⁺ populations were analyzed for CD25⁺ expression and compared by Student's paired t-test in the CD4⁺ (A) and CD8⁺ (B) populations (<i>P</i> = 0.006 and <i>P</i> = 0.04, respectively). Mean Fluorescence Intensity (MFI) for CD25 expression in CD4⁺ (C) and CD8⁺ (D) populations indicate significant differences for CD4⁺ populations (<i>P</i> = 0.02) and CD8⁺ populations (<i>P</i> = 0.003). E–H) CD69 expression analysis indicates no significant differences in either proportion of CD4⁺ or CD8⁺ CD69-expressing cells or in the MFIs. Mean and SEM are shown and <i>P</i>≤0.05 was considered to be significant.</p