CD40-B cell immunization generates effectors expressing similar level of T-bet and Blimp-1, higher level of Eomes and lower amount of Bcl-6.

<p>A. Expression of T-bet, Eomes and Bcl-6 by CD8⁺ Te cells generated following CD40-B cell and DC immunizations. Four days post-immunization with 2×10⁶ CD40-B cells or DCs matured with LPS and loaded with the OVA peptide, Te cells were stained intracellularily with antibodies against T-bet, Eomes and Bcl-6 transcription factors. The representative overlay histogram shows expression of the transcription factor by endogenous T cells (CD8⁺CD45.2⁻) and OVA-specific Te cells (CD8⁺CD45.2⁺). The MFI is shown on each overlay, the upper bold number indicates the MFI of OVA-specific effectors (CD8⁺CD45.2⁺) while the lower number is for the endogenous population (CD8⁺CD45.2⁻). B. Quantification of the level of expression of T-bet, Eomes and Bcl-6. The histograms shows the MFI of expression for T-bet, Eomes and Bcl-6 by OVA-specific CD8⁺ Te cells (CD8⁺CD45.2⁺) normalized to the MFI of endogenous CD8⁺ T cells (CD8⁺CD45.2⁻). The results are from at least 2 independent experiments. C. Similar expression of Blimp-1 by OVA-specific Te cells following CD40-B cell or DC immunization. At the peak of the response (d4), Te cells were sorted (CD8⁺CD45.2⁺) from spleen to extract RNA. The relative expression of Blimp-1 was determined by quantitative RT-PCR. Expression relative to a reference sample is shown. Results are from 4 independent experiments. * p<0.05 and *** p<0.001.</p

CD40-B cells have an activated phenotype.

<p>After 3 d of culture on murine 3T3-CD40L fibroblasts, CD40-B cells were matured or not with LPS (1 µg/mL) or CpG (2 mM) for 24 h (CD40-B LPS and CD40-B CpG). Freshly isolated splenocytes were used as a naïve B cell control. The histogram bars show the mean of fluorescence intensity (MFI) +/− standard deviation of the mean (SEM) for the expression of CD86, CD80, CD62L, CD40, K^b and I-A^b gated on the CD19⁺ population. The results are pooled from at least three independent experiments except for CD40 expression on B cells (n = 2). * p<0.05, ** p<0.01 and *** p<0.001.</p

Effectors generated with CD40-B cell immunization contract more rapidly than the one obtained with DC immunization.

<p>A. Contraction of the OVA-specific CD8⁺ T cell response. Mice were immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030139#pone-0030139-g002" target="_blank">Figure 2</a>. Lymph nodes were surgically removed at 4, 7, 10 and >30 days post-immunization. Cells were stained to determine the percentage of Te cells generated. The graph shows the percentage of remaining Te cells (CD8⁺CD45.2⁺) over time relative to the peak of the response (d4). B. Effectors generated with CD40-B cell and DC immunization express similar amount of Bcl-2 during the course of the CD8⁺ T cell response. The MFI of Bcl-2 for OVA-specific CD8⁺ Te cells was normalized to the MFI of endogenous CD8⁺ T cells to obtain a MFI ratio. 3 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001.</p

Immunization with CD40-B cells induces an <i>in vivo</i> CD8⁺ T cell response.

<p>A. CD40-B cell vaccination generates CD8⁺ Te cells but not Tm cells. 10⁶ female OT-1 T cells (CD8⁺CD45.2⁺) were adoptively transferred into congenic B6SJL female mice (CD45.1⁺) followed by immunization two days later with 2×10⁶ CD40-B cells, matured or not with LPS (1 µg/mL) or CpG (2 mM) and loaded with 4 µg/mL OVA or with an irrelevant peptide (IRR). As a reference recipients were immunized with 2×10⁶ DCs matured with LPS and loaded with OVA peptide. OVA-specific T cells (CD8⁺CD45.2⁺) were analyzed in the same mouse by surgical removal of superficial lymph nodes at d4 (effector) and d30–45 (memory) post-immunization. Te and Tm cells were identified as CD8⁺CD45.2⁺ by flow cytometry. The percentage of Te and Tm cells generated are indicated on each dot plot. B. Percentage of CD8⁺ Te (left panel) and Tm (right panel) cells recovered at d4 (Te) and d>30 (Tm) in one lymph node is shown. C. Yield of CD8⁺ Tm cell generation. The yield of Tm cell formation was calculated as the percentage of Te cells that develop into Tm cells. D. The percentage of mice that generates more then 5% of CD8⁺ Tm cells is shown for the different immunization conditions. E and F. Lm challenges. 30 d post immunization, mice were challenged with a lethal dose of Lm-OVA (10⁵ CFU). 3 d post challenged, CFU were determined in the spleen (E) and liver (F) for each mouse. A–D are from at least four independent experiments with at least two mice per group while E and F are from one independent experiment with three mice per group. * p<0.05, ** p<0.01 and *** p<0.001.</p

CD40-B cell vaccination generates functional effector.

<p>A. <i>In vivo</i> killing. Mice were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030139#pone-0030139-g002" target="_blank">Figure 2</a>. Four days post-immunization, CFSE-labeled splenocytes pulsed or not with OVA were injected as target cells. After 4 h, the percentage of CFSE^hi (OVA-pulsed; gate labeled OVA on the histogram) and CFSE^lo (unpulsed; gate labeled neg on the histogram) cells were analyzed in the spleen. Percentage of specific lysis is indicated on the histogram and was calculated using the indicated gate and as described in Material and Methods. B. Percentage of specific killing by OVA CD8⁺ Te cells. Mean +/− SEM of specific lysis are shown for the different immunization conditions. 2 mice per conditions, 3 independent experiments. C and D. Lm challenge. Four days post-immunization, mice were challenged with a lethal dose of Lm-OVA (10⁵ CFU). 3 d post-infection (peak of bacterial load), mice were killed and CFU were determined in the spleen (C) and the liver (D). Mean +/− standard (SD) are shown. 2–4 mice per conditions, 3 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001.</p

Phenotype of the CD8⁺ Te cells generated after CD40-B cell immunization.

<p>A. Phenotype of effectors at d4 post-immunization with CD40-B cells treated or not with TLR ligands. Immunizations were realized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030139#pone-0030139-g002" target="_blank">Figure 2</a> with CD40-B cells (2×10⁶) treated or not with CpG or LPS and with DCs (2×10⁶) matured with LPS. The bar chart shows the MFI of expression for CD44, 1B11, CD127, CD122, CD27 by CD8⁺ Te cells (CD8⁺CD45.2⁺) normalized to the MFI of endogenous CD8⁺ T cells (CD8⁺CD45.2⁻). For CD62L, the percentage of CD8⁺ Te cells expressing high level of CD62L is shown. B. No correlation between CD127 expression level and memory generation. The results are from 2 independent experiments for CD27 and CD122 and from at least 3 independent experiments for the other cell surface molecules. * p<0.05, ** p<0.01 and *** p<0.001.</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

<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

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