1. DUSP4-mediated accelerated T-cell senescence in idiopathic CD4 lymphopenia
by Alexandre Bignon, Alexis Régent, Laurence Klipfel, Aude Desnoyer, Pierre de la Grange, Valérie Martinez, Olivier Lortholary, Ali Dalloul, Luc Mouthon, and Karl Balabanian
Blood
Volume 125(16):
April 16, 2015
©2015 by American Society of Hematology

2. Aging gene-expression profiles in CD4+ T cells from patients with ICL
Aging gene-expression profiles in CD4+ T cells from patients with ICL. (A) Regulated genes across the samples were hierarchically clustered according to their mean-normalized intensity across all samples.
Aging gene-expression profiles in CD4+ T cells from patients with ICL. (A) Regulated genes across the samples were hierarchically clustered according to their mean-normalized intensity across all samples. Blue and pink samples correspond to CD4+ T cells from healthy subjects (n = 8) and patients with ICL (n = 9), respectively. (B) Venn diagram of analyzed, expressed, and regulated genes in CD4+ T cells for patients with ICL vs healthy subjects. Upregulated and downregulated genes are indicated by red and green colors, respectively. (C) Results extracted from the Gene Ontology analysis, KEGG pathway, and list of genes regulated in healthy T cells during aging28 were combined to delineate a specific T-cell aging pathway in ICL. 1Genes that are significantly regulated in another hierarchical clustering. (D) Ten representative genes of the upregulated group and 6 of the downregulated group were monitored by quantitative PCR in healthy (n = 12), SARC (n = 8), and ICL (n = 12) CD4+ T cells. Each individual sample was run in triplicate. Results are expressed as mRNA levels after normalization to GAPDH mRNA levels. *P < .05, **P < .005, and ***P < compared with healthy CD4+ T cells (as determined using the Mann-Whitney U test).
Alexandre Bignon et al. Blood 2015;125:
©2015 by American Society of Hematology

3. Low responsiveness of ICL CD4+ T cells to TCR stimulation.
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 CFSElow (>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 < compared with healthy CD4+ T cells (as determined using the Mann-Whitney U test).
Alexandre Bignon et al. Blood 2015;125:
©2015 by American Society of Hematology

4. CD4+ T cells exhibit a senescent profile in ICL
CD4+ T cells exhibit a senescent profile in ICL. (A and B) Flow-cytometric analyses of CD27, CD28, CD57, and KLRG1 expression in CD3+CD4+-gated PBMCs from healthy, elderly, SARC, and ICL subjects.
CD4+ T cells exhibit a senescent profile in ICL. (A and B) Flow-cytometric analyses of CD27, CD28, CD57, and KLRG1 expression in CD3+CD4+-gated PBMCs from healthy, elderly, SARC, and ICL subjects. Quadrants were set on controls stained with the corresponding isotype control Ab. Representative dot plots showing coexpression of CD27 and CD28 or CD57 and KLRG1 are shown (A). Comparison of the frequencies of CD27−, CD28−, HLADR−, CD57−, KLRG1−, and granzyme B-expressing CD4+ T cells in healthy (n = 28), elderly (n = 6), SARC (n = 8), and ICL (n = 20) individuals. Lines indicate the mean ± SD values, and each symbol represents the value from an individual (B). (C and D) Telomerase activity (C) and telomere length (D) were measured in PBMCs from the aforementioned groups by Telomere Repeat Amplification Protocol, followed by enzyme-linked immunosorbent assay detection and quantitative PCR, respectively. Results (mean ± SD) are from 3 independent experiments and show the optical density values (C) or are expressed as the ratio of telomere repeat copy number (T) to albumin copy number (a single-copy gene, S) within the same DNA sample in each group (D). Kruskal-Wallis H test values and associated P values are indicated. *P < 0.05, **P < 0.005, and ***P < compared with healthy leukocytes (as determined using the Mann-Whitney U test).
Alexandre Bignon et al. Blood 2015;125:
©2015 by American Society of Hematology

5. Repeated TCR stimulation leads to defective signaling and DUSP4 overexpression in healthy CD4+ T cells.
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 < compared with healthy (A), untreated (C-F), or stimulated-S1 (B) CD4+ T cells (as determined using the Mann-Whitney U test).
Alexandre Bignon et al. Blood 2015;125:
©2015 by American Society of Hematology

6. DUSP4 inhibition ameliorates TCR signaling in senescent CD4+ T cells.
DUSP4 inhibition ameliorates TCR signaling in senescent CD4+ T cells. (A and B) After 2 rounds of stimulation with anti-CD3/anti-CD28 Abs, activated CD4+ T cells from 3 independent healthy individuals were nucleoporated with 1.5 μg SCR or DUSP4 siRNAs. Twelve hours after transfection, T cells were stimulated on plates coated with anti-CD3/anti-CD28 Abs for 2 days. DUSP4 and DUSP6 transcript or protein levels were then evaluated by quantitative PCR (left and middle) or immunoblot (right). The ratios of DUSP4 over α-tubulin proteins are indicated below the gels. pERK content was determined by PhosphoFlow in siRNA-transfected T cells restimulated by CD3 cross-linking (B). Representative plots of the MFI of pERK in transfected CD4+ T cells are shown (left). Results show the mean pERK content in transfected CD4+ T cells ± SD (right). (C-E) CD4+ T cells from 3 independent elderly or ICL subjects were nucleoporated with 1.5 μg SCR or DUSP4 siRNAs and then stimulated for 2 days, as described earlier. siRNA-transfected cells were recovered and tested for DUSP4 and DUSP6 expression (C) and for their pERK content after CD3 cross-linking (D). On day 3 after stimulation, the frequencies of CD27-expressing cells CD4+ T cells and levels of CD40L expression were determined by flow cytometry and shown as the percentage increase after DUSP4 silencing (E). Results represent the mean ± SD or are representative of 3 independent experiments. *P < 0.05 and **P < compared with SCR siRNA-transfected CD4+ T cells (as determined using the Mann-Whitney U test).
Alexandre Bignon et al. Blood 2015;125:
©2015 by American Society of Hematology