IL-17+ γδ T cells are neurotoxic <i>in vitro</i>.

<p><b>(A-E)</b> γδ T cells were isolated from C57BL/6J mice and cultured with IL-1β (10 ng/ml), IL-23 (10 ng/ml), anti-CD3 (1μg/ml) and anti-CD28 (10μg/ml) to induce IL-17+ γδ T cells. After 3 days, polarized IL-17+ γδ T cells with their culture media or supernatants only were co-cultured with cortical neurons for 72 h. Neuronal cultures without the addition of γδ T cells served as a control. To evaluate neuronal viability cultures were subsequently immunostained with antibodies against neuronal nuclei (NeuN), neurofilament (NF) to mark neurons (both in red), CD3 to mark γδ T cells (green) and DAPI (blue). Representative images are shown for co-culture with 1x10⁵ polarized γδ T cells after 72 h, in <b>(A)</b> magnification 20x, scalebar 100μm, in <b>(D)</b> magnification 100x, scalebar 50μm, white arrowheads indicate apoptotic nuclei. In <b>(B, C)</b> γδ T cells were isolated, polarized, and co-cultured as described above at indicated concentrations for 72 h or 1x10⁵ polarized γδ T cells were co-cultured for up to 4 days. (<b>E)</b> Quantification of DAPI+ nuclei displaying apoptotic hallmarks. <b>(F, G)</b> Primary cortical neurons were incubated with recombinant IL-17 for 72 h at indicated concentrations or with 50 ng/ml for indicated time points. Neurons treated with imiquimod (10 μg/ml) or LPS (100 ng/ml) served as a positive and negative control, respectively. Cultures were then stained with NeuN Ab and DAPI. Each condition was performed in duplicate and averaged. NeuN-positive cells were quantified and expressed as relative neuronal viability. Mean ± SEM of 3–5 individual experiments, ANOVA with Dunnett´s multiple comparison post test of each time point/condition <i>vs</i>. control, (B) <i>p</i><0.0001, (C) <i>p</i> = 0.0032, (E) <i>p</i> = 0.0151, (F) <i>p</i> = 0.7851, (G) <i>p</i> = 0.0064.</p

Supernatants derived from microglia stimulated through TLRs activate naïve γδ T cells and induce expression of IL-17, but not IFN-γ.

<p><b>(A)</b> Microglia were stimulated with the TLR ligands Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), imiquimod (10 μg/ml), or CpG (1 μM) for 24 h. Microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells, or γδ T cells were directly stimulated with the TLR ligands. Unstimulated cells served as a control. After 2 days, γδ T cells were collected and analyzed by flow cytometry regarding CD3, CD25, CD69, and CD62L expression. Each condition was performed in duplicate and averaged. Mean ± SEM of 3 to 9 individual experiments. <b>(B, C)</b> Microglia were stimulated for 24 h with TLR ligands as described in <b>(A)</b> for 24 h. Subsequently, either the microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells or γδ T cells were co-cultured with both microglia and their supernatant. After 3 days, γδ T cells were harvested, restimulated with PMA/ionomycin, and analyzed by flow cytometry for intracellular IFN-γ and IL-17 expression. <b>(C)</b> Each condition was performed in duplicates and averaged. Mean ± SEM of 4 individual experiments. <b>(D)</b> γδ T cells were cultured with microglia-conditioned supernatant, as described in <b>(A).</b> After indicated time points supernatants were analyzed by ELISA regarding IL-17 production. Each condition was performed in duplicates and averaged. Mean ± SEM of 3 to 7 experiments. <b>(E)</b> Overview of Vγ-chain usage (Vγ1.1, Vγ2, Vγ3 and Vγ5) found on IL-17+ γδ T cells activated by supernatants derived from TLR-stimulated microglia. Mean ± SEM of 3 individual experiments. <b>(F)</b> Bone marrow-derived macrophages (BMDMs) were stimulated for 24 h with various TLR ligands as named in <b>(A)</b>. BMDM-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. γδ T cells were collected after two days and analyzed by flow cytometry regarding CD3, CD25, CD69, and CD62L expression, and supernatants were collected after one, 2 and 3 days, and analyzed regarding the presence of IL-17 by ELISA <b>(G)</b>. Each condition was performed in duplicate and averaged. Mean ± SEM of 4 to 5 individual experiments. <b>(A)</b>, <b>(C)</b> and <b>(F)</b> ANOVA with Dunnett´s multiple comparison post test of each ligand <i>vs</i>. unstimulated control, (A) <i>p</i> = 0.7198, <i>p</i><0.0001, <i>p</i> = 0.9415, <i>p</i><0.0001, <i>p</i> = 0.9707, <i>p</i> = 0.0001, (C) <i>p</i> = 0.0061, <i>p</i> = 0.9883, <i>p</i> = 0.2590, <i>p</i> = 0.1599, (E) <i>p</i><0.0001, <i>p</i> = 0.0004, <i>p</i> = 0.0521. <b>(D)</b> and <b>(G)</b> 2-way ANOVA with Bonferroni post test compared to unstimulated control; <i>p</i>*<0.05, <i>p</i>***<0.001.</p

Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation

<div><p>Background</p><p>Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.</p><p>Objective</p><p>In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.</p><p>Results</p><p>We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability <i>in vitro</i>. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity <i>in vitro</i>. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell contact between T cells and neurons.</p><p>Conclusion</p><p>Taken together, these results point to a crucial role for microglia activated through TLRs in polarization of γδ T cells towards neurotoxic IL-17+ γδ T cells.</p></div

Supernatants from microglia stimulated via TLRs induce activation patterns alike to IL-17+ γδ T cells dependent on MyD88 expressed in microglia and in γδ T cells.

<p><b>(A)</b> Wild-type (WT) or MyD88KO microglia were stimulated for 24 h with the TLR ligands Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), imiquimod (10 μg/ml) or CpG (1 μM). Microglia-conditioned supernatants were transferred to freshly isolated naïve WT, TLR2KO, TLR7KO, or MyD88KO γδ T cells. After 3 days, supernatants were collected and analyzed regarding IL-17 by ELISA. Mean ± SEM of 3–4 individual experiments. <b>(B, C)</b> WT microglia were stimulated as in <b>(A)</b> and microglia-conditioned supernatants were either used for cytokine analysis or transferred to freshly isolated naïve γδ T cells. Also, naïve γδ T cells were directly stimulated with TLR ligands as indicated or IL-1β, IL-23, anti-CD3, and anti-CD28. After 3 days, supernatants were analyzed by bead based multiplex assay or ELISA for indicated cytokines. Mean ± SEM of 3 individual experiments. Amounts of IL-1β and IL-18 were analyzed by ELISA for n = 6 individual experiments. <b>(D)</b> Naïve γδ T cells were directly analyzed. <i>In vitro</i> polarized IL-17+ γδ T cells were harvested after 3 days in culture with IL-1β, IL-23, anti-CD3, and anti-CD28, re-stimulated with PMA/ionomycin, and analyzed by flow cytometry for intracellular granzyme B expression. Representative FACS plots of n = 3 individual experiments are shown. <b>(E, F)</b> WT microglia were stimulated as in <b>(A)</b> and microglia-conditioned supernatants were preincubated with 10 μg/ml anti-IL-1β, anti-IL-23 or respective isotype controls before transfer to naïve γδ T cells. After 2 days, γδ T cells were collected and analyzed by flow cytometry regarding CD3, CD25, CD69, and CD62L expression <b>(E)</b>. After 3 days supernatants were analyzed by ELISA regarding IL-17 secretion <b>(F)</b>. Each condition was performed in duplicate and averaged. Mean ± SEM of 4 individual experiments. <b>(A, B, C, E, F)</b> ANOVA followed by Bonferroni multiple comparison post test, (A) TLR2KO <i>p</i> = 0.3340, TLR7KO <i>p</i> = 0.0989, (B) IL-6 <i>p</i> = 0.0705, IL-23 <i>p</i> = 0.5709, IL-1β <i>p</i> = 0.0011, IL-18 <i>p</i> = 0.7380, (C) IL-13 i = 0.0148, IL-17 <i>p</i><0.0001, IL-22 <i>p</i><0.0001, IFN-γ <i>p</i> = 0.0001, granzyme B <i>p</i> = 0.4176, (E) <i>p</i><0.0001.</p

Supernatants from microglia stimulated via TLR2, TLR4, or TLR9 induce neurotoxic γδ T cells.

<p><b>(A)</b> Microglia were stimulated with Pam3CysSK4 (100 ng/ml) LPS (100 ng/ml) or CpG (1 μM) for 24 h. Unstimulated cells served as a control. Microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), CpG (1 μM) or imiquimod (10 μg/ml) alone, served as controls. Subsequently, cultures were immunostained with antibodies against CD3 (γδ T cells, green), NeuN, and neurofilament (neurons, red), magnification 100x, scale bar 50 μm. In <b>(C)</b> 1*10⁵ microglia were added to neuronal cultures concurrent with γδ T cells. <b>(D)</b> BMDMs were stimulated with Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml) or CpG (1 μM) for 24 h. Unstimulated cells served as a control. BMDM-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), CpG (1 μM) or imiquimod (10 μg/ml) alone served as controls. Subsequently, cultures were immunostained as in <b>(A)</b>. <b>(B, C, D)</b> NeuN-positive cells were quantified and expressed as relative neuronal viability. Each condition was performed in duplicate and averaged. Mean ± SEM of 3–5 individual experiments with ANOVA followed by Bonferroni multiple comparison post test, (B) <i>p</i><0.0001, (C) <i>p</i><0.0001, (D) <i>p</i><0.0001.</p

Naïve γδ T cells express TLRs but do not secrete IL-17 in response to TLR stimulation.

<p><b>(A, B)</b> γδ T cells were isolated from C57BL/6J mice and stained at their cell surface (TLR1, TLR2, TLR4) or intracellularly (TLR7, TLR9, MyD88) with antibodies directed against the indicated TLR (solid line) and the respective isotype control (dotted line). Data are displayed as delta (Δ) mean fluorescence intensity (MFI) of the specific antibody in relation to the isotype control ± SEM from 3 to 5 individual experiments. <b>(C, D)</b> γδ T cells were isolated from C57BL/6J mice and stimulated with the TLR ligands Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), imiquimod (10 μg/ml), CpG (1 μM), or IL-1β and IL-23 (10 ng/ml each). Unstimulated cells served as a negative control. After 3 days, cells were re-stimulated and analyzed by flow cytometry for intracellular IFN-γ and IL-17 expression. Results in <b>(D)</b> are shown as mean ± SEM of 3 experiments, ANOVA with Dunnett´s multiple comparison post test of each ligand <i>vs</i>. unstimulated control. <b>(E)</b> γδ T cells were stimulated as described in <b>(C)</b>, but supernatants were collected directly after 3 days and analyzed by IL-17 ELISA. Results are shown as mean ± SEM of 3 individual experiments, ANOVA with Dunnett´s multiple comparison post test of each ligand <i>vs</i>. unstimulated control, (D) <i>p</i> = 0.3262, <i>p</i> = 0.0355, (E) <i>p</i> = 0.4647.</p

Regulatory T cells suppress CD8 T cell-mediated hepatitis <i>in vivo</i>.

<p>4x10⁶ OT-I CD8 T cells were transferred intravenously into TF-OVAxDEREG mice and treated with DT (diphtheria toxin) or PBS as described in methods. (A) Spleens and livers were analyzed for the presence of Foxp3⁺ T cells on day 5. Plots depict data gated on CD4⁺T cells. ALT (B) and bilirubin (C) were measured in serum on days 4 or 5, respectively. Values from individual mice and mean±SEM are depicted. (D) The number of OT-I T cells was estimated by staining for the Vα2 chain, which forms the OT-I TCR, since no clonotypic antibody is available. The number of CD8⁺Vα2⁺ T cells in liver and spleen was determined by flow cytometry. Depicted are mean±SD from 6–7 mice per group. (E) At day 5 after induction of hepatitis, IFN-γ production of CD8 OT-I T cells isolated from liver and spleen was measured after <i>in vitro</i> cultivation in medium or SIINFEKL. Plots depict mean±SD, events are gated on CD8⁺Vα2⁺T cells, data are from 5–7 mice per group. (F) 5 days after the induction of hepatitis, OT-I CD8 Thy1.1⁺ T cells were purified from liver and spleen and specific lysis was analyzed and calculated as described in methods. Plots depict mean±SD from 5 mice per group. (G) Histology was assessed on day 4 using antibodies against CD3, Foxp3 (magnification 100x), and CD8 (magnification 200x). Representative results from 5–6 mice in each group are shown. Statistics in (B-F) were performed using the Mann-Whitney-test; ***p<0.001, **p<0.01, *p<0.05; ns = not significant.</p

Regulatory T cells accumulate in the liver of mice in the course of hepatitis.

<p>4x10⁶ CD8 OT-I T cells were transferred intravenously into TF-OVA mice. Non-parenchymal cells were isolated from liver and spleen at the indicated days and analyzed for the presence of CD4⁺, CD25⁺, and Foxp3⁺ T cells by flow cytometry. Absolute numbers of (A) CD4⁺ T cells and (B) Foxp3⁺CD25⁺CD4⁺ T cells are depicted (mean±SEM from n = 4–25 mice per time point; ***p<0.001, **p<0.01, *p<0.05 by Mann-Whitney test). (C) Immunohistochemistry for CD3 (red membrane staining) and Foxp3 (brown nuclear staining) of livers from mice at the indicated days after transfer of CD8 OT-I T cells (magnification 200x), arrows indicate exemplary CD3⁺Foxp3⁺ cells). Representative images from 3–4 mice per time point are depicted.</p

Hepatic regulatory T cells display effector-Treg phenotype and suppress the proliferation of CD8 OT-I T cells <i>in vitro</i>.

<p>(A) CFSE-labeled CD8 OT-I T cells were stimulated by APCs isolated from spleens of TF-OVA mice for three days alone or in co-culture with CD4⁺CD25^- T cells (naïve CD4 T cells), CD4⁺CD25⁺ Treg isolated from lymph nodes and spleens of wild type-mice (control-Treg) or CD4⁺CD25⁺ Treg isolated from livers of TF-OVA mice suffering from hepatitis (liver-Treg). Depicted are representative histograms showing proliferating CFSE-labeled CD8 OT-I T cells at day three, cultured with different CD4 T-cell types (gated on CD8⁺CFSE⁺). (B) The percentage of proliferating CD8 T cell cultured with control-Treg, liver-Treg, or naïve CD4 T cells was calculated by CFSE-intensity relative to the proliferation of CD8 T cells (100% = dotted line) cultured alone. Box plots depict the percentage of CD8 T-cell proliferation. N = 5–9. p-values were calculated using one-sample t-test against a hypothetical value of 100%. **p<0.01. (C) Liver-Treg and control-Treg were generated and isolated as above. CD4⁺CD25⁺Foxp3⁺ cells were analyzed for expression of various markers by flow cytometry. Depicted are mean ± SD from n = 4 mice per group, ***p<0.001, **p<0.01, *p<0.05 by unpaired t-test.</p

Priming of naive CD4 T-cells by liver-derived antigen <i>in vivo.</i>

<p>OT-II T-cells were purified from the lymph nodes and spleen of OT-II mice and labeled with CFSE. Four million cells were transferred intravenously into splenectomized TF-OVA mice (A), MHC-II^−/− → TF-OVA chimeras (B), or TF-OVA mice (control). Cells from the indicated organs were isolated 44 (A) or 68 hours (B) after cell transfer and analyzed for the presence of proliferating OT-II T-cells by detection of CFSE-dilution. All plots depict data gated on CD4⁺ cells. Events to the right of the vertical line represent the undivided population. Events at the far left of the plot represent unlabeled endogenous cells. Representative results from n = 4 bone-marrow chimeras and n = 4 control mice (A), and n = 6 splenectomized and n = 4 control mice (B) are shown.</p