American Society of Hematology

Figure 4

$Figure 4. Repeated TCR stimulation leads to defective signaling and DUSP4 overexpression in healthy CD4+ T cells. (A) Steady-state levels of DUSP4 and DUSP6 transcripts in sorted CD4+ T cells from healthy (n = 28), elderly (n = 6), SARC (n = 8), and ICL (n = 20) subjects were assessed by quantitative PCR. Each individual sample was run in triplicate. Results are expressed as DUSP4 or DUSP6/GAPDH ratio. (B) CFSE-loaded CD4+ T cells from 9 independent healthy donors were either left untreated (basal) or successively stimulated once, twice, or 3 times (S1-S3) on anti-CD3/anti-CD28 Ab-coated plates for 5 days. Each round of stimulation was separated by a 2-day culture in complete RPMI medium without any stimulation. Representative plots of the MFI of CFSE in CD4+ T cells (left). Results (mean ± SD of 3 independent experiments) expressed as fractions of proliferating CFSElow CD4+ T cells (right). Cells were gated on forward and side scatter to eliminate debris and on forward scatter-width/area and side scatter-width/area to gate only single and viable cells. Samples contained between 46.5% and 87.3% viable cells. (C) pERK content was determined in CD4+ T cells after restimulation by CD3 cross-linking for 5 minutes. Representative plots of the MFI of pERK in CD4+ T cells (left). Mean pERK content in unstimulated and activated CD4+ T cells ± SD (right). (D) DUSP4 and DUSP6 mRNA levels (mean ± SD, n = 9) were evaluated by quantitative PCR in healthy CD4+ T cells left untreated or repeatedly stimulated with anti-CD3/anti-CD28 Abs. (E and F) Flow-cytometric analyses of CD45RA, HLA-DR, CD27, CD40L, CCR7, and CXCR4 expression on healthy CD4+ T cells left untreated or after 3 rounds of stimulation. Representative dot plots or histograms (left). Background fluorescence is shown as shaded areas in F. Frequencies (mean ± SD, n = 9) of naive (CD45RA+CCR7+) vs memory (CD45RA−CCR7−/+), HLA-DR-expressing, and CD27-expressing CD4+ T cells (E) and levels of CXCR4 and CD40L expression (F; right). Kruskal-Wallis H test values and associated P values are indicated. *P < 0.05, **P < 0.005, and ***P < 0.0005 compared with healthy (A), untreated (C-F), or stimulated-S1 (B) CD4+ T cells (as determined using the Mann-Whitney U test).$

Repeated TCR stimulation leads to defective signaling and DUSP4 overexpression in healthy CD4⁺ T cells. (A) Steady-state levels of DUSP4 and DUSP6 transcripts in sorted CD4⁺ T cells from healthy (n = 28), elderly (n = 6), SARC (n = 8), and ICL (n = 20) subjects were assessed by quantitative PCR. Each individual sample was run in triplicate. Results are expressed as DUSP4 or DUSP6/GAPDH ratio. (B) CFSE-loaded CD4⁺ T cells from 9 independent healthy donors were either left untreated (basal) or successively stimulated once, twice, or 3 times (S1-S3) on anti-CD3/anti-CD28 Ab-coated plates for 5 days. Each round of stimulation was separated by a 2-day culture in complete RPMI medium without any stimulation. Representative plots of the MFI of CFSE in CD4⁺ T cells (left). Results (mean ± SD of 3 independent experiments) expressed as fractions of proliferating CFSE^low CD4⁺ T cells (right). Cells were gated on forward and side scatter to eliminate debris and on forward scatter-width/area and side scatter-width/area to gate only single and viable cells. Samples contained between 46.5% and 87.3% viable cells. (C) pERK content was determined in CD4⁺ T cells after restimulation by CD3 cross-linking for 5 minutes. Representative plots of the MFI of pERK in CD4⁺ T cells (left). Mean pERK content in unstimulated and activated CD4⁺ T cells ± SD (right). (D) DUSP4 and DUSP6 mRNA levels (mean ± SD, n = 9) were evaluated by quantitative PCR in healthy CD4⁺ T cells left untreated or repeatedly stimulated with anti-CD3/anti-CD28 Abs. (E and F) Flow-cytometric analyses of CD45RA, HLA-DR, CD27, CD40L, CCR7, and CXCR4 expression on healthy CD4⁺ T cells left untreated or after 3 rounds of stimulation. Representative dot plots or histograms (left). Background fluorescence is shown as shaded areas in F. Frequencies (mean ± SD, n = 9) of naive (CD45RA⁺CCR7⁺) vs memory (CD45RA⁻CCR7^−/+), HLA-DR-expressing, and CD27-expressing CD4⁺ T cells (E) and levels of CXCR4 and CD40L expression (F; right). Kruskal-Wallis H test values and associated P values are indicated. *P < 0.05, **P < 0.005, and ***P < 0.0005 compared with healthy (A), untreated (C-F), or stimulated-S1 (B) CD4⁺ T cells (as determined using the Mann-Whitney U test).

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