CD8+ T cells from PCC immunized mice protect naïve mice from EAE.

<p>A. Naïve C57BL/6 mice were immunized with PBS or 100µg of 50V PCC in CFA. Three weeks later, CD8+ T cells purified from the draining lymph nodes and spleen of the PBS immunized (CD8 PBS, squares) or PCC immunized (CD8 PCC, triangles) mice were adoptively transferred into naïve C57BL/6 recipients (CD8 PBS n = 10, CD8 PCC n = 10). Sixteen hours later, EAE was induced in the recipients upon subcutaneous immunization with MOG peptide accompanied by intravenous pertussis toxin on the day of EAE induction and 2 days later. B. Similar protocol was carried out with a second transfer of CD8+ T cells 9 days after EAE induction. The clinical course of EAE was analyzed using a paralysis grading score. Mean ± SEM. * indicates that the p-value<0.05 as assessed using ANOVA Fisher's PLSD. Arrows indicate the time points of CD8+ T cell transfer.</p

Qa-1-binding molecular pattern.

<p>A. The alignment of previously described 9 amino acid-long Qa-1-binding peptides (Qdm, pre-proinsulin, HSP60 signal sequence derived peptide) using the Geneious software (ClustalW analysis). B. A consensus sequence with conserved amino acids in position (P) 1, 2, 5, 7 and 9 was revealed from this alignement. C. The search of such a consensus sequence in mouse Vß chains derived from the IMGT database revealed the presence of consenting 9 amino acid long peptides in the leader sequences of all mouse Vß chains. * Described as being a Qa-1-binding peptide in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021628#pone.0021628-Panoutsakopoulou1" target="_blank">[12]</a>.</p

Regulatory CD8+ T cells are restricted to Qa-1.

<p>Naïve C57BL/6 mice were immunized with PBS or 100µg of 50V PCC in CFA. Three weeks later, CD8+ T cells purified from the draining lymph nodes and spleen of PBS-immunized (CD8 PBS) and PCC-immunized (CD8 PCC) mice were adoptively transferred into either wildtype (WT, n = 7 for ‘CD8 PBS in WT’ and ‘CD8 PCC in WT’) or Qa-1-deficient mice (Qa-1°, n = 5 for ‘CD8 PBS in Qa-1°’ and n = 6 for ‘CD8 PCC in Qa-1°’). EAE was induced in the recipients 16 hours later. * indicates that the p-value<0.05 as assessed using ANOVA Fisher's PLSD.</p

Regulatory CD8+ T cells prevent lymphocyte infiltration and CNS inflammation.

<p>Naïve mice were adoptively transferred with CD8+ T cells from PBS-immunized (CD8 PBS, n = 4) or PCC-immunized (CD8 PCC, n = 4) mice followed by EAE induction 16 hours later. Thirteen days after EAE induction, the mice were sacrificed and the lymphocyte populations in the brain and spinal cord were examined by flow cytometry. A. Absolute numbers of lymphocytes in the CNS gated according to their forward and side-scatter characteristics. B. MOG-specific CD4+ T cells were stained using MOG_38–49-I-Ab tetramers. Represented is the percentage of tetramer-positive cells among total CD4+ T cells in the CNS. C. Absolute numbers of CD4+ T cells that produce IL-17 in the CNS. D. Absolute numbers of IFNγ-producing CD4+ T cells in the CNS. Mean ± SEM. * indicates that the p-value<0.05 as assessed using Mann-Whitney non-parametric analysis.</p

Phenotypic and functional features of M1 and M2 macrophages.

<p><b>A</b>: The effect of an overnight IFNγ priming step was tested on C57Bl/6 mouse bone marrow-derived MØ subjected to M1- or M2-polarizing conditions. The expression of iNOS, ArgI, ArgII, and Ym1/2 were determined by real time RT-PCR on RNA extracted 10 hours after the induction of polarization. Data were calculated using the 2^−ΔΔCt Pfaffl formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008852#pone.0008852-Pfaffl1" target="_blank">[30]</a> in which experimental conditions (M1 and M2) are compared to Ct values obtained in M0 MØ and normalized to the Ct values of the HPRT house-keeping gene. <b>B</b>: Primary aortic vascular smooth muscle cells (VSMCs) from C57Bl/6 mice were cultured for 48 hours in the presence of media conditioned by C57Bl/6 M1 or M2 MØ which were polarized in the presence of the PPARγ agonist pioglitazone (Pio), the PPARγ antagonist GW9662 (GW), or the arginase inhibitor Nor-NOHA. As controls, VSMCs were also cultured with the same concentration of the polarizing agents, of the PPARγ agonists and antagonists, or of the arginase inhibitor. At the end of the assay, the number of viable cells in each condition was evaluated by the MTT assay and by using a standard curve established with known numbers of cells. *; **: p<0.05; p<0.01 vs matched medium conditioned by MØ polarized in the standard way (−). Note that the number of cells obtained with the M2-conditioned medium were significantly greater than with M1-conditioned medium (p<0.01 vs matched condition).</p

ApoE modulates the expression of PPARγ.

<p><b>A</b>: Expression of IL-4Rα1 and of IL-13Rα1 in non-polarized MØ^B6 and MØ^ApoE determined by real time PCR and normalized by HPRT (au: arbitrary unit). <b>B</b>: Expression of PPARγ in non-polarized MØ from MØ^B6 (black) and MØ^ApoE (white) mice with increasing doses of recombinant ApoE (rApoE) determined by real time PCR and normalized by HPRT (au: arbitrary unit). **: p<0.001; ***: p<0.0001 vs MØ^B6. <b>C</b>: Expression of ArgI in M1 and M2 MØ^B6 (black) and MØ^ApoE (white) mice polarized with or without (−) pioglitazone (Pio) or GW9662 (GW) determined by real time PCR. The Results are expressed as 2^−ΔΔCt<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008852#pone.0008852-Pfaffl1" target="_blank">[30]</a>. *: p<0.05; **: p<0.001 vs matched MØ^B6 condition; †: p<0.05; ††: p<0.01 vs matched M1 condition.</p

Preserved plasticity of fully polarized macrophages.

<p>MØ^ApoE (white) and MØ^B6 (black) were primed and subjected to a first M1 or M2 polarization. After ten hours, the culture conditions were switched during 10 additional hours in order to induce the opposite phenotype. Expression of iNOS, Arg I and ArgII was evaluated by real time PCR and normalized by HPRT (au: arbitrary unit). Results are representative of three distinct experiments.</p

Macrophages of early atherosclerotic lesions in ApoE KO mice express Arg I while Arg II predominates in late stages.

<p><b>A</b>: Immunofluorescence of atherosclerotic lesions from 20 and 55 week-old ApoE KO mice. MØ were identified as Mac3⁺ (red) cells. Co-expression of Mac3 and Arg I (violet) and/or Arg II (green) were identified by image overlay. Merge: overlay of bright field, DAPI, Mac3, Arg I and Arg II stainings. Inset: magnification of the zone delimited in the “Merge” frame. <b>B</b>: Arg I, Arg II and Arg I⁺ Arg II⁺ double positive surface areas within 3 random fields/plaque of 20 (white) and 55 (grey) week-old mice. <b>C, D</b>: Regression analysis between the surface area of plaques (X-axis) and total MØ area (Mac3, C) or the Arg I⁺/Arg II⁺ ratio (D). R²: regression correlation coefficient. <b>E</b>: Expression of IL-4 and IFNγ transcripts on microdissected atherosclerotic lesions from 20 and 55 week-old ApoE KO mice determined by real time PCR and normalized by HPRT (au: arbitrary unit). **; ***: p<0.001; p<0.0001 vs 20 w. <b>F–I</b>: Microdissection of aortic atherosclerotic lesions. Lesions can be detected (delimited by black dashes) through the vascular wall in the dissected aortic root (white dashes). The vascular wall was opened longitudinally to expose the luminal side of the vessel (G). The aortic cusps are readily identified. Arrow heads indicate the plane of fracture at the lesion/media interface, which allows the separation of the lesion from the media. The arrows in H indicate the movement performed with tweezers to detach the lesion. Appearance of the vessel after the dissection of the lesion is shown in I.</p

Kinetic expression of M1 and M2 markers.

<p>Bone marrow-derived MØ from C57BL/6 mice (MØ^B6, closed circles) and ApoE KO mice (MØ^ApoE, open circles) were differentiated, primed with IFNγ and polarized or not (No polarization) towards the M1 (M1 polarization) or the M2 phenotype (M2 polarization). Expression of Arg I, iNOS, Arg II were determined by real time PCR and normalized by HPRT (au: arbitrary unit). IL-6 was monitored in cell culture supernatant by ELISA. Results are representative of three independent experiments.</p