Increased TCR dependent MAPK activation and enhanced AICD in CD5-CK2BD deficient mice is independent of peripheral T cells activation and thymocyte FasL expression.

<p>(A) Total cell numbers (left panel) of CD5-WT vs CD5-ΔCK2BD newborn mice after injection of either PBS (control) or α-CD3 (20 μg) for 24h. To evaluate the effect of anti-CD3 stimulation, both CD5-ΔCK2BD or CD5-WT were compared with their respective unstimulated controls. <b>S</b>ubpopulation analysis show DP (middle panel) or CD4+ SP thymocytes (right panel) (B) Scatter plots showing Bcl-2 levels in DP and CD4+ SP thymocytes. (C) Bar graphs of FasL expression in PBS injected and anti-CD3 injected mice. Adult thymocytes were used as positive control. (D) Scatter plots show pERK expression 24h after α-CD3 injection or PBS as in DP and CD4+ SP cells from CD5-WT and CD5-ΔCK2BD newborn mice. (E) Graphs showing expression of pJNK (F) pP38 and (G) pZap70. Each graph is representative of 1 independent experiment (n = 5–6 each group). *p<0.05, **p<0.01, ***p<0.001, unpaired two-tailed Student-t test.</p

CD5-CK2 Signaling Modulates Erk Activation and Thymocyte Survival

<div><p>CD5 is well recognized for its importance in thymic selection. Although this property of CD5 has been attributed to its ITIM-domain dependent regulation of TCR-signal strength, the mechanism has not been established. A second major signaling domain within the cytoplasmic tail of CD5 is a CK2 binding/activation domain (CD5-CK2BD). Using a gene-targeted mouse in which the CD5-CK2BD is selectively ablated (CD5-ΔCK2BD), we determined that loss of function of CD5-CK2 signaling in a MHC-II selecting TCR transgenic (OT-II) mouse resulted in decrease in double positive (DP) thymocytes, which correlated with enhanced apoptosis. Remarkably, DP cells expressing high levels of CD5 and CD69 and single positive (CD4+SP) thymocytes were increased in CD5-ΔCK2BD mice indicating that CD5-CK2 signaling regulates positive selection and promotes survival. Consistent with this possibility, we determined that the activation and nuclear localization of ERK as well as apoptosis was greater in thymic populations from OTII CD5-ΔCK2BD mice than OTII CD5-WT mice following injection of OVA_323-339-peptide. The mobilization of Ca²⁺, an early event of TCR activation, was not altered by the loss of CD5-CK2 signaling. Collectively, these data demonstrate that the CD5-CK2 signaling axis regulates positive selection by modulating activation of ERK and promoting survival independent of proximal TCR signals.</p></div

Nuclear translocation pERK is independent of CD5-CK2 signaling.

<p>(A) Images of DP and CD4+SP thymocytes obtained from PBS or OVAp injected CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice and analyzed by imaging flow cytometry. The images show each channel individually represented as well as an overlay of all channels. The data are representative of at least 900 images each from 2 independent experiments (n = 2 mice each group). (B) Quantitative representation of all events represented in (A). The graphs reflect mean±SEM pERK levels inside the nucleus (left panels) or ratio of pERK in nucleus to whole cell (right panels) within DP (upper panels) or CD4+SP (lower). (C) Images of pERK in DP and CD4+SP thymocytes obtained from non-TCR transgenic CD5-WT and CD5-ΔCK2BD mice. The images are representative of 900–1500 images from 3 independent experiments (n = 3 mice each group). (D) Quantitative representation analyzed similar to (B). **p<0.01 ****p<0.0001, unpaired two-tailed Student-t test.</p

Ablation of CD5-CK2 binding domain alters thymic T cell development.

<p>Thymic DN, DP (CD4+CD8+) and SP (CD4+ or CD8+) populations (dot plots), proportions and absolute numbers (graphs) in CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice (A) and CD5-WT and CD5-ΔCK2BD mice (B). Ratio of CD4:CD8 SP cells in CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice (C) and CD5-WT and CD5-ΔCK2BD non-TCR Tg mice (D). The flow cytometric dot plots are representative of one of five mice from each strain. The graphs represent data from 4 independent experiments (n = 4–7 each group) *p<0.05, **p<0.01, ***p<0.001, unpaired two-tailed Student-t test.</p

Enhanced <i>in vitro</i> and <i>in vivo</i> thymocyte apoptosis in the absence of the CD5-CK2BD.

<p>(A) Apoptosis (Annexin V⁺ 7-AAD^-/+) in thymocytes from CD5-WT and CD5-ΔCK2BD mice following culture for 24h in the presence of α-CD3 and/or α-CD5 or medium alone. The graphs show apoptosis in gated DP, CD4+SP and CD8+SP and populations. Data represent mean±SEM from one experiment (n = 3–4 independent mice). (B) Histograms (lower left) show proportion of cells with elevated caspase 3 levels relative to unstained cells within DP and CD4+SP thymocytes obtained from CD5-WT and CD5-ΔCK2BD OTII TCR-Tg mice after i.p injection with either PBS or OVAp. Solid vertical lines are drawn to reflect unstained MFI and to identify the caspase 3 on the highest expressing population. Scatter plots show MFI of caspase 3 levels in DP cells (top), proportion of DP cells expressing high levels of caspase 3 (middle) and MFI of high caspase 3 expressing CD4+ SP cells (bottom). Data represents 4 independent experiments (n = 4 mice each group). Numbers are presented as mean±SEM, *p<0.05, unpaired two-tailed Student-t test.</p

AKT and CDC37 activation is unaffected by loss of CD5-CK2 signaling pathway.

<p>Scatter plot of pAKT(S473) levels (A) and pCDC37 (S13) levels (B) within DP or CD4+SP thymocytes obtained from CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice after injection with PBS (control) or 300 μg of OVAp. Data represents 4 independent experiments (n = 5 mice each group). NS = not significant, unpaired two-tailed Student-t test.</p

Deletion of the CD5-CK2BD results in increased basal and induced ERK phosphorylation in the absence of enhanced TCR proximal signaling.

<p>(A) Histograms (upper left) show pERK levels within DP or CD4+SP thymocytes obtained from CD5-WT OTII and CD5-ΔCK2BD OTII TCR-Tg mice after PBS or OVAp injection. Vertical solid lines are drawn to identify the MFI of unstained cells and the MFI of cells from PBS treated mice. Scatter plots (right) show data from 4 individual mice. (B) Calcium mobilization in DP, CD4+SP and CD8+SP thymocytes from CD5-WT and CD5-ΔCK2BD mice following stimulation with α-CD3. Each graph is representative of 2 independent experiments (n = 2–3 independent mice). Inset arrows denote time point of addition of α-CD3 or ionomycin. *p<0.05, ****p<0.0001, unpaired two-tailed Student-t test.</p

CD5-CK2 signaling regulates positive selection and maturation of post-selected thymocytes.

<p>(A) Dot plots of CD5 and CD69 expression gated on DP cells from CD5-WT and CD5-ΔCK2BD OTII TCR-Tg (upper) and non-transgenic (lower) mice. Inset polygon represents the CD5^hiCD69^hi post-selected thymocytes within the DP population. (B) Scatter plot shows the frequency of DP CD5^hiCD69^hi population from CD5-WT and CD5-ΔCK2BD OTII TCR transgenic (upper) and non-transgenic (lower) mice. Each dot represents an individual mouse. (C and D) Fewer terminally differentiated CD4+ SP (CD62L^hiCD69^lo) cells in CD5-ΔCK2BD mice. (C) Representative dot plot showing expression of CD69 and CD62L in CD4+SP gated cells from CD5-WT and CD5-ΔCK2BD non-TCR transgenic (upper) and OTII TCR transgenic (lower) mice. The inset polygon represents CD62L^hiCD69^lo terminally differentiated cells. (D) Scatter plot of proportion of CD4+ SP cells with CD62L^hiCD69^lo phenotype from different strains as designated. Each dot represents a mouse. Data represents 4 independent experiments (n = 3–5 mice each group). Numbers are presented as mean± SEM. **p<0.01, unpaired two-tailed Student-t test.</p

IFN-γ-signaling is required for attenuation of EAE by lithium.

<p>EAE was induced in (A) WT (B), <i>Stat1</i>^−/−, (C) <i>Ifngr1^−/−</i>, and (D) <i>Ifnar1^−/−</i>as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052658#s2" target="_blank">Material and Methods</a>. Arrows indicate first day of administration of lithium. (Mean ± SEM, <i>n</i> = 10–13 mice/group *<i>p</i><0.05, or NS, not significant, from start of treatment until day 30, as determined by Mann-Whitney test). (E) RNA was isolated from spinal cords of WT and <i>Ifngr1^−/−</i> mice on day 20 post-immunization and evaluated for gene expression by real-time PCR, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052658#s2" target="_blank">Materials and Methods</a>. <i>n</i> = 3 for immunized, <i>n</i> = 1 for unimmunized controls. *<i>p</i><0.05, as determined by one-way ANOVA.</p

Analysis of disease parameters for adoptive transfer of EAE induced by Th1, Th17, and IL-17F-Thy1.1 cells in untreated and lithium-treated animals.

<p>Data are presented as mean ± SEM (<i>n</i> = 8–11 mice).</p>a<p><i>p</i><0.05; Lithium-treated Th1 compared to untreated Th1.</p>b<p><i>p</i><0.05; Lithium-treated Th17 compared to untreated Th17.</p