Change of naïve and MP T cell populations in <i>Hoxb4</i> transgenic and wt mice with age.

<p>Scatter plots showing the percentage of (<b>A</b>) CD4 and (<b>B</b>) CD8 T cells that are naïve (CD44^lo/CD62L^hi), MP (CD44^hi) or are a subpopulation of MP T cells (CD44^hi/Ly6C^hi) for LN, Spl and BM derived from individual <i>Hoxb4</i> transgenic and wt (n = 6–8) age matched mice. Young mice are between 3–4 months and old mice are all older than 28 months. Naïve and MP populations change significantly with age, but not between <i>Hoxb4</i> and wt mice. *P<0.05, 2-tailed Student ttest. MP = memory phenotype, Wt = wild type, LN = lymph node, Spl = spleen and BM = bone marrow.</p

Competitive short-term homeostatic proliferations (7 days) of <i>Hoxb4</i> transgenic and wt CD4 MP T cells.

<p>(<b>A</b>) Scheme of the experimental approach. CD4 MP T cells are sorted from CellTrace™ Violet (CTV) labelled cells isolated from LN and Spl of <i>Hoxb4</i> (CD45.1/2) and congenic wt (CD45.1) mice. Cells of both genotypes are transplanted in a 1∶1 ratio in CD3ε^−/− (CD45.2) mice. (<b>B</b>) FACS profiles showing <i>Hoxb4</i> and wt fractions to donor derived CD4 MP T population (CD45.1) in LN (left panel). Representative FACS profiles for CD62L and CTV on <i>Hoxb4</i> and wt populations. Loss of CTV tracer indicates that most cells are dividing rapidly (right panels). (<b>C</b>) Average contribution (%) of <i>Hoxb4</i> and wt cells to donor derived MP T cells in LN, Spl and BM (n = 3). (<b>D</b>) Percentage of CD62L^hi MP T cells in <i>Hoxb4</i> and wt population found in lymphoid organs. *P<0.05; paired 2-tailed Student ttest. Wt = wild type, MP = memory phenotype, LN = lymph node, Spl = spleen and BM = bone marrow.</p

Percentage of T cell populations in hematopoietic organs of young adult mice.

<p>Note that no significant differences were observed between T cell populations of <i>Hoxb4</i> and wild type mice. 1-tailed Student ttest, comparing <i>Hoxb4</i> vs. wild type mice. LN = Lymph node; BM = bone marrow.</p

Total donor cells contribution to hematopoietic organs and the proportion of <i>Hoxb4</i> versus wild type cells.

<p>LN = Lymph node; BM = bone marrow, wt = wild type.</p

Medium-term competitive homeostatic proliferations (60 days) of <i>Hoxb4</i> transgenic and wt CD4 MP T cells.

<p>(<b>A</b>) FACS profile showing fractions of <i>Hoxb4</i> and wt cells to donor derived MP T population in lymph node (LN). (<b>B</b>) Stacked bar graphs indicating the average contributions of <i>Hoxb4</i> and wt cells in LN, Spl and BM measured in three independent experiments; n = 9. (<b>C</b>) FACS profiles for the expression of typical memory T cell surface markers on <i>Hoxb4</i> and wt MP T cells in the BM. (<b>D</b>) Average subpopulations of <i>Hoxb4</i> and wt MP T cells expressing the indicated surface markers in LN (upper panel), Spl (middle panel) and BM (lower panel). (<b>E</b>) Percentage of <i>Hoxb4</i> and wt MP T cells (gated on CD44^hi) positive for indicated cytokines (n = 3–6). *P<0.05, 2-tailed Student ttest. MP = memory phenotype, wt = wild type, LN = lymph node, Spl = spleen and BM = bone marrow, TNF = tumor necrosis factor; IL-2 = interleukine-2; IFN = interferon.</p

Long-term competitive homeostatic proliferations (180 days) of <i>Hoxb4</i> transgenic and wt CD4 cells.

<p>(<b>A</b>) Scheme of serial transplantations. 10×10⁶ cells of the LNs of primary hosts that received a transplant composed of equal doses of <i>Hoxb4</i> and wt MP T cells were serially transplanted into secondary and tertiary hosts with a 60 days interval. (<b>B</b>) Compilation of <i>Hoxb4</i> and wt fractions of donor derived cells in LN, Spl and BM of secondary (n = 6) and tertiary hosts (n = 4) from two independent experiments. (<b>C</b>) Bar graphs showing the average percentage of cells positive for CD62L and Ly6C within the <i>Hoxb4</i> or wt memory T populations. *P<0.05, 2-tailed Student ttest. Wt = wild type, MP = memory phenotype, LN = lymph node, Spl = spleen and BM = bone marrow.</p

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

<div><p>The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following <i>in vitro</i> stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.</p></div

Defective proliferation and cytokine production by ERK3-deficient T cells.

<p><i>A</i>, Defective proliferation of <i>Erk</i>3^−/− T lymphocytes after anti-CD3 stimulation. Splenocytes from <i>Erk3</i>^+/+ or <i>Erk3</i>^−/− hematopoietic chimeras were labeled with CFSE and stimulated with different doses of anti-CD3 Ab for 72 h. CFSE profiles gated on CD4⁺ or CD8⁺ T cells lacking or not ERK3 are shown for the different anti-CD3 Ab concentrations. One representative experiment is shown. <i>B</i>, Quantification of T cell proliferation. T cell proliferation, measured as in A, was quantified by determining the percentage of cells that have divided (one division and more; CFSE^lo). Each dot represents the results from one mouse. Unpaired Student’s t test (two-sided) was used to determine statistical significance. * p<0.05. <i>C</i>, Addition of anti-CD28 Abs does not rescue the proliferation of ERK3-deficient CD4⁺ T cells. Splenocytes were stimulated with a sub-optimal dose of anti-CD3 Ab (0.3 µg/ml) in the presence (bottom) or absence (top) of soluble anti-CD28 Ab (5 µg/ml). CFSE profiles gated on CD4⁺ T cells lacking or not ERK3 are shown. <i>D</i>, Reduced production of IL-2 and IFN-γ by ERK3-deficient T cells after anti-CD3 stimulation. After 72 h of anti-CD3 stimulation, activated T cells were stimulated with PMA and ionomycin for 4 h. Brefeldin A was added for the last 2 h of culture. IL-2 and IFN-γ production was detected using intracellular cytokine staining. CFSE/IL-2 and CFSE/IFN-γ profiles gated on CD4⁺ or CD8⁺ T lymphocytes deficient or not for ERK3 are shown for the different anti-CD3 Ab concentrations. Numbers in parenthesis represent the % of proliferating and cytokine producing cells. The results in this figure are representative of at least three independent experiments with mice from independent hematopoietic chimeras.</p

Normal peripheral compartment in ERK3-deficient hematopoietic chimeras.

<p><i>A</i>, Lymphocyte subsets distribution in the LN and spleen of hematopoietic chimeras deficient or not for ERK3. CD4/CD8 and B220/IgM profiles are shown for mice reconstituted with <i>Erk3</i>^+/+ or <i>Erk</i>3^−/− fetal liver cells. The data are representative of eight independent hematopoietic chimeras. <i>B</i>, Phenotype of ERK3-deficient T lymphocytes in the LN and spleen. CD44/CD62L profiles are shown for CD4⁺ and CD8⁺ T cells recovered from the LN and spleen of hematopoietic chimeras deficient or not for ERK3. The data are representative of eight independepent hematopoietic chimeras. <i>C</i>, CD4⁺ regulatory T cells are produced normally in the absence of ERK3. CD25/FoxP3 profiles gated on CD4⁺ T cells are shown from the spleen of mice reconstituted with <i>Erk3</i>^+/+ or <i>Erk3</i>^−/− fetal liver cells. <i>D</i>, Normal distribution of γδ T cells in the spleen of hematopoietic chimeras deficient or not for ERK3. The histograms show TCRγδ expression gated on CD4⁻CD8⁻CD3⁺ splenocytes. The percentage of TCRγδ⁺ T cells is indicated on the histogram.</p

<i>Erk3</i> transcription is controlled by the classical ERK1/2 MAPK pathway.

<p>Splenocytes from <i>Erk3</i>^+/− mice were stimulated with coated anti-CD3 Abs (1 µg/ml) for 24 h or 48 h in the presence or absence of the selective pharmacologic inhibitor of MEK1/2, U0126. Cells were cell surface stained with anti-CD4 and anti-CD8 Abs followed by FDG staining to measure β-galactosidase activity. The overlays show β-galactosidase activity (FDG) in CD4⁺ (top) and CD8⁺ (bottom) T cells after 24 h (left) or 48 h (right) of stimulation. β-galactosidase activity by unstimulated (NS) T cells is shown as negative control. The data are representative of three independent experiments.</p