Peptide profiles of plasma membrane and membrane rafts.

<p>Peptide profiles of plasma membrane and membrane rafts of RAW264.7 cultured for 72h in basic medium. Peptidome separation was done using the CLINPROT profiling purification kit from Bruker Daltonics (Billerica, USA) by means of magnetic particles with magnetic bead-hydrophobic interaction (MB-HIC C8).</p

Fatty acid patterns [nmol/mg protein] and MBI in membrane rafts of PUFA supplemented cells.

<p>Fatty acid patterns [nmol/mg protein] and Methylene Bridge Index (MBI) in membrane rafts of RAW264.7 cultured for 72h in basic medium as well as in medium supplemented with 15 µmol/l alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), gamma-linolenic acid (γ-LNA) and arachidonic acid (AA) respectively. Data are mean±S.D. (n = 6). Superscript letters across a row denote significant differences. n.d.  = below detection limit.</p

Fatty acids [nmol/mg protein] dominating in plasma membrane and membrane rafts of cells cultured in basic medium.

<p>Fatty acids [nmol/mg protein] dominating in plasma membrane and membrane rafts of RAW264.7 cultured for 72h in basic medium. Data are mean±S.D. (n = 6). Superscript letters across a row denote significant differences. C18∶1n9: oleic acid; C18∶1n7: vaccenic acid; C16∶1n7: palmitoleic acid; C18∶0: stearic acid; C16∶0: palmitic acid; C14∶0: myristic acid.</p

Fatty acid patterns [nmol/mg protein] and MBI in plasma membrane of PUFA supplemented cells.

<p>Fatty acid patterns [nmol/mg protein] and Methylene Bridge Index (MBI) in plasma membrane of RAW264.7 cultured for 72h in basic medium as well as in medium supplemented with 15 µmol/l alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), gamma-linolenic acid (γ-LNA) and arachidonic acid (AA) respectively. Data are mean±S.D. (n = 6). Superscript letters across a row denote significant differences.</p

Differences in the CD44 expression level is strengthened by polarization.

<p>(A) Representative dot plot of IL-17 and IFN-γ production by CD4^<b>+</b> T cells in non-polarized, Th1- and Th17-polarized allogeneic co-cultures for either total CD4^<b>+</b> T cells or for naïve-sorted (CD45RB^<b>high</b>) CD4^<b>+</b> T cells. Percentage of cells is indicated in the plot. Analysis was performed on day four after co-culture. (B) Representative dot plot of the CD44 surface level expression of CD4^<b>+</b> T cells in non-polarized, Th1- and Th17-polarized allogeneic co-cultures for either total CD4^<b>+</b> T cells or for naïve-sorted (CD45RB^<b>high</b>) CD4^<b>+</b> T cells. Percentage of cells within different subpopulations is indicated in the plot. Analysis of total CD4^<b>+</b> T cells was performed on day four and of naïve-sorted on day three after co-culture. (C) Percentage of CD44^<b>+++</b>CD4^<b>+</b> T cells (total CD4^<b>+</b> T cells) under different polarization conditions (n = 6–9, <i>M</i> ± SEM, Kruskal-Wallis test and <i>post-hoc</i> Dunn’s comparison, not significant). (D) Histogram overlay of geometric mean fluorescence intensity (gMFI) for CD44 of IFN-γ producers of Th1-polarized co-cultures (grey) and IL-17 producers of Th17-polarized co-cultures (black line). (E and F) GMFI for IFN-γ and IL-17 producers of different generations is shown as histogram overlay (E) and bar chart (F; n = 6, <i>M</i> ± SEM; 2-way ANOVA and <i>post-hoc</i> Sidak`s multiple comparison, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001).</p

CD44 expression level discriminates allo-reactive IL-17⁺ and IFN-γ⁺ CD4⁺ T cells.

<p>(A) Cytokine production in CD4^<b>+</b> T cells four days after allogeneic co-culture with bone-marrow derived dendritic cells. Percentage of cells is indicated in the dot plot. (B) CD44 subpopulations of CD4^<b>+</b> T cells and cytokine production within these subpopulations four days after allogeneic co-culture. (C) CD44 expression level of <i>in vitro</i> generated IFN-γ^<b>+</b>CD4^<b>+</b> (grey) and CD4^<b>+</b>IL17^<b>+</b> T cells (black line). Values for the geometric mean fluorescence intensity (gMFI) for CD44 are indicated in the histogram. (D) CD44 expression level (gMFI) of <i>in vitro</i> generated IL-17^<b>+</b>CD4^<b>+</b> and IFN-γ^<b>+</b>CD4^<b>+</b> T cells (n = 13, <i>M</i> ± SEM, Wilcoxon rank sum test). (E) IL-17 and IFN-γ production prior to as well as seven, 14 and 28 days after allogeneic skin transplantation (n = 3–4, <i>M</i> ± SEM, 2-way ANOVA and <i>post-hoc</i> Sidak`s multiple comparison). (F) CD44 expression level of <i>in vivo</i> formed IFN-γ^<b>+</b>CD4^<b>+</b> (grey) and CD4^<b>+</b>IL17^<b>+</b> T cells (black line) from lymph nodes after transplantation (skin-tx) and without transplantation (w/o). GMFI for CD44 is indicated in the histograms. (G) CD44 expression level (gMFI) of <i>in vivo</i> formed IL-17^<b>+</b>CD4^<b>+</b> and IFN-γ^<b>+</b>CD4^<b>+</b> T cells from spleen prior to as well as seven, 14 and 28 days after transplantation (n = 3–4, <i>M</i> ± SEM, 2-way ANOVA and <i>post-hoc</i> Sidak’s multiple comparison. (H) CD44 expression level (gMFI) of <i>in vivo</i> formed IL-17^<b>+</b>CD4^<b>+</b> and IFN-γ^<b>+</b>CD4^<b>+</b> T cells from lymph nodes after transplantation (n = 3–4 <i>M</i> ± SEM, 2-way ANOVA and <i>post-hoc</i> Sidak’s multiple comparison). Statistical significance (<i>p</i>) is within the groups is indicated in the figures (*<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001).</p

CD44 levels are independent of TNF-α expression and influences differentiation of IL-17⁺ cells.

<p>(A) Overlay of IFN-γ^<b>+</b> (grey) and IL-17^<b>+</b> (black), IFN-γ^<b>+</b> (grey) and IFN-γ^<b>+</b>TNF-α^<b>+</b> (black), IL-17^<b>+</b> (grey) and IL-17^<b>+</b>TNF-α^<b>+</b> (black) CD4^<b>+</b> T cells regarding their CD44 expression levels. Geometric mean fluorescence intensity (gMFI) for CD44 is indicated. (B) GMFI of CD44 for IL-17 and IFN-γ single producers, as well as for IL-17/TNF-α and IFN-γ/TNF-α double producing CD4^<b>+</b> T cells (n = 5, <i>M</i> ± SEM, Friedman test and post-hoc Dunn`s comparison). (C) Ratio of IL-17^<b>+</b> or IFN-γ^<b>+</b>CD4^<b>+</b> T cells, respectively, in αCD44-treated versus untreated co-cultures (n = 5; <i>M</i> ± SEM; Wilcoxon rank sum test). (D) Living cells in allogeneic co-cultures treated with or without (w/o) αCD44 (n = 5; <i>M</i> ± SEM; Wilcoxon rank sum test, not significant). (E) Proliferation of CD4^<b>+</b> T cells in allogeneic co-cultures treated with (grey) or without (w/o, black line) αCD44. Statistical significance (<i>p</i>) is within the groups is indicated in the figures (*<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001).</p

(A) IL-17 production (white bars, left Y-axis) and CD44 expression (grey bars, right Y-axis) of naïve-sorted CD4+ T cells before, one, two, three and four days after allogeneic stimulation with BMDCs (n = 4, ± SEM).

<p>(B) IFN-γ production (white bars, left Y-axis) and CD44 expression (grey bars, right Y-axis) of naïve-sorted CD4^<b>+</b> T cells before, one, two, three and four days after allogeneic stimulation with BMDCs (n = 4, ± SEM). (C) Magnetically sorted CD4^<b>+</b> T cells were stimulated four days with allogeneic BMDCs. Co-cultures were left untreated or were treated with 10 μg/ml αCD44 directly before PMA/ionomycin restimulation (n = 5, <i>M</i> ± SEM, Wilcoxon rank sum test, <i>p</i> = 0.06).</p

Low TCR and CD28 stimulation supports T_H17 development.

<p>(A) IL-17 and IFN-γ production of CD4^<b>+</b> T cells stimulated with different amounts of αCD3. Percentage of positive cells is indicated in the dot plot. (B) Depicted is the IL-17 and IFN-γ production of CD4^<b>+</b> T cells stimulated with 0.01 or 1 μg/ml αCD3 (n = 6, <i>M</i> ± SEM, Wilcoxon rank sum test, between IL-17^<b>+</b>CD4^<b>+</b> cells: not significant, between IFN-γ^<b>+</b>CD4^<b>+</b> cells: <i>p</i> = 0.06). (C) Ratio of αCD44-treated and untreated IL-17^<b>+</b>CD4^<b>+</b> or IFN-γ^<b>+</b>CD4^<b>+</b> T cells stimulated with 0.01 or 1 μg/ml αCD3 (n = 3, <i>M</i> ± SEM, paired t test, between IL-17^<b>+</b>CD4^<b>+</b> cells: *<i>p</i> ≤ 0.05, between IFN-γ^<b>+</b>CD4^<b>+</b> cells: not significant). (D and E) Magnetically sorted CD4^<b>+</b> T cells were stimulated four days with allogeneic BMDCs. Co-cultures were left untreated or 1 μg/ml CTLA-4-Ig was added. Percentage of IL-17^<b>+</b>CD4^<b>+</b> T cells of differently treated co-cultures is shown as a representative dot plot (D) and in a bar chart (E: n = 7, <i>M</i> ± SEM; Wilcoxon rank sum test, *<i>p</i> ≤ 0.05). (F) Magnetically sorted CD4^<b>+</b> T cells were stimulated four days with allogeneic BMDCs. Co-cultures were left untreated or were treated with 1 μg/ml CTLA-4-Ig or 10 μg/ml αCD44 or both. IL-17 and IFN-γ production of CD4^<b>+</b> T cells of the differently treated co-cultures is shown as a representative dot plot out of two independent experiments.</p

CD44⁺⁺⁺CD4⁺ T cells have a high cellular activation status.

<p>(A) CD45RB expression level of <i>in vitro</i> generated IFN-γ^<b>+</b>CD4^<b>+</b> (grey) and CD4^<b>+</b>IL-17^<b>+</b> (black line) T cells. GMFI for CD45RB is indicated. (B) CD45RB geometric mean fluorescence intensity (gMFI) of IL-17^<b>+</b>CD4^<b>+</b> and IFN-γ^<b>+</b>CD4^<b>+</b> T cells (n = 5, <i>M</i> ± SEM, Wilcoxon rank sum test, <i>p</i> = 0.06). (C and D) Representative histogram (C) and summary (D) of the ZAP-70 phosphorylation for CD4^<b>+</b>CD44^<b>+</b> (grey solid), CD44^<b>++</b> (grey dotted) and CD44^<b>+++</b> (black line) T cell populations. After four days of allogeneic co-culture cells have been rested for one day. Values for the gMFI of phospho-ZAP-70 are indicated. (E) GMFI of phospho-ZAP-70 of CD4^<b>+</b>CD44^<b>+</b> (grey solid), CD44^<b>++</b> (grey dotted) and CD44^<b>+++</b> T cells (black line) with or without (w/o) αCD44 treatment. A representative histogram overlay out of four independent experiments is shown.</p