Growth of GP33-expressing tumor after a single injection of BM-DC.

<p>Time axis of experimental setup (<i>a</i>). 3×10⁶ BM-DC either loaded with peptide (<i>c</i>) or left unpulsed (<i>b</i>) were injected once (i) i.c. (<i>cycles</i>), (ii) i.p. (<i>triangles</i>) or (iii) i.v. (<i>diamonds</i>). Two days later 1×10⁶ cells of the tumor cell line B16.F10_GP33 was inoculated s.c. and tumor sizes were measured every second day for up to 56 days. Diameters of tumors are depicted. Figure shown for i.c. and i.p. injection route of BM-DC included 13 animals in total, with n = 5 animals per group in two experiments and n = 3 animals in one experiment. Figure including the i.v. vaccination route include five animals in total from one experiment. Error bars: SEM.</p

Proliferation and homing receptor expression on tumor specific T cells after DC vaccination via different injection routes.

<p>Time axis for experimental set up (<i>a</i>). 1.5×10⁶ Thy1.1⁺ P14 splenocytes were adoptively transferred i.v. into C57BL/6 (Thy1.2⁺) mice. One day later 3×10⁶ DC-GP33 or DC-Av were injected (i) i.c. or (ii) i.p. On day 8 after P14 transfer mice were sacrificed and cells from the spleen (<i>b & d</i>) and blood (<i>c</i>) were analyzed. Mean population size of CD3⁺CD8⁺Thy1.1⁺ cells from spleen are depicted (<i>b</i>) significant difference was calculated using an unpaired <i>t</i>-Test after i.c. or i.p. DC-GP33 vaccination route. N = 6 mice, error bars: SEM. One representative analysis of homing receptor expression on Thy1.1⁺ T cells in the blood after DC-GP33 vaccination via the i.c. or the i.p. route is shown. Homing receptors are specified below each panel (<i>c</i>). Numbers in dot blots indicate % of positive cells. Skin homing receptor E-lig expression was measured via FACS analysis in the spleen after DC-GP33 vaccination via the i.c. or the i.p. route (<i>d</i>). Mean E-lig expression level of CD3⁺CD8⁺Thy1.1⁺ cells are depicted from n = 3 mice. Three mice were included in every group per experiment, two (<i>b & d</i>) and three (<i>c</i>) independent experiments were performed.</p

Tumor growth after T cell transfer and repeated DC vaccination via different injection routes.

<p>Time axis of experimental set up (<i>a</i>). 1.5×10⁶ Thy1.1⁺ P14 splenocytes were adoptively transferred i.v. into C57BL/6 (Thy1.2⁺) mice. One and three days later 3×10⁶ BM-DC, either pulsed (<i>c & e</i>) or left unpulsed (<i>b & d</i>) were injected (i) intracutaneously (i.c.) (<i>grey cycles</i>), (ii) intraperitoneally (i.p.) (<i>black triangles</i>) or (iii) intravenously (i.v.) (<i>grey diamonds</i>). On day seven after P14 transfer 1×10⁶ cells of the melanoma cell lines B16.F10 (<i>b & c</i>) and B16.F10_GP33 (<i>d & e</i>) were inoculated subcutaneously (s.c.) into the left and the right flank, respectively. Every second day tumor sizes were measured. Tumor diameters are depicted. Three independent experiments were performed with 4 mice in each group. Error bars: SEM.</p

Growth of GP33-expressing tumor only after repeated DC vaccination.

<p>Time axis of experimental set up (<i>a</i>). BM-DC (3×10⁶) were injected twice into C57BL/6 recipient mice, either peptide-pulsed (<i>open symbols</i>) or unpulsed (<i>filled symbols</i>) via the (i) i.c. (<i>cycles</i>) or (ii) i.p. (<i>triangles</i>) route on day 0 and day 2. Two days later 1×10⁶ cells of the GP-33 expressing tumor cell line were injected s.c. into the left flank (<i>b</i>). Tumor sizes were measured every second day. Diameters of tumors are shown. Four independent experiments were performed with n = 5 mice per group in three experiments and n = 3 in one experiment. Error bars: SEM.</p

Tumor growth after repeated DC vaccination without T cell transfer.

<p>Time axis of experimental set up (<i>a</i>). 3×10⁶ BM-DC were injected into C57BL/6 recipient mice twice, either peptide pulsed (<i>c & e</i>) or unpulsed (<i>b & d</i>) via the (i) i.c. (<i>grey cycles</i>), (ii) i.p. (<i>black triangles</i>) or (iii) i.v. (<i>grey diamonds</i>) route on day 0 and day 2. Two days later 1×10⁶ cells of both tumor cell lines were injected s.c. into the left and the right flank, respectively. Tumor sizes were measured every second day. Diameters of tumors are shown. N = 6 per group, from two independent experiments. Error bars: SEM.</p

Peripheral leukocytosis after primary percutaneus coronary intervention (PPCI).

<p>Absolute number of total leukocytes, neutrophil granulocytes and monocytes (<b>A</b>), as well as lymphocytes and the main T-cell subsets (<b>B</b>) in peripheral blood. Concentration of leukocyte populations are displayed for patients 24 h following PPCI in STEMI (n = 31), 3 months post PPCI (n = 24), and in age-matched healthy Controls (n = 18). 1-way ANOVA with Tukeys' post-hoc test was performed. *p<0.05, ***p<0.001, ns not significant.</p

Distribution of main CD4 T-cell subsets.

<p>Frequencies of CD4<b>⁺</b> naive (CD45RA<b>⁺</b>CCR7<b>⁺</b>), central memory (CD45RA<b>⁻</b>CCR7<b>⁺</b>), effector memory (CD45RA<b>⁻</b>CCR7<b>⁻</b>) and terminally differentiated effector memory TEMRA cells (CD45RA<b>⁺</b>CCR7<b>⁻</b>) are shown. Groups are identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047155#pone-0047155-g001" target="_blank">Figure 1</a>. 1-way ANOVA with Tukeys' post-hoc test was performed. *p<0.05, ***p<0.001, ns not significant.</p

Clinical patient characteristics of the time course substudy group.

<p>CABG: coronary artery bypass graft; PCI: percutaneous coronary intervention; CHD: coronary heart disease; MI: myocardial infarction; NA: not applicable.</p

Increased expression of CD57 in central memory cells indicates acute proliferation in the CD4 T-cell compartment.

<p>Conventional gating analysis confirmed increased expression of proliferation associated markers CD57 on CM CD4<b>⁺</b> T cells of STEMI pts (MFI – mean fluorescence intensity). This was reflected by significantly increased frequencies of specific KLRG1<b>⁻</b>CD57<b>⁺</b> CD4 CM<b>⁺</b> T cells (A–C). The validity of CD57, PD-1 and KLRG1 as proliferation-associated markers was determined in healthy controls according to Sallusto's model of T-cell differentiation (D). Mean telomere length (mTL) in CD4<b>⁺</b> T cells is reduced in patients with STEMI when compared to age-matched controls (E). *p<0.05, **p<0.01, ***p<0.001, ns not significant.</p

CD4⁺CCR7⁺ T cells are specifically depleted from peripheral blood during myocardial reperfusion.

<p>Time course pilot study showed no significant changes in numbers of monocytes (A) and granulocytes (B) following PPCI. In contrast, T cells decreased by 35% during the first 30 min following reperfusion showing significant relative increase within next 24 hours (C). The absolute cell count analysis revealed a significant decrease in CD4<b>⁺</b> T cells during the first 60 min following PPCI (D). This was mainly due to the specific depletion of circulating CD4<b>⁺</b>CCR7<b>⁺</b> cells within the first 30 min following reperfusion (E). *p<0.05, **p<0.01, ns not significant.</p