Fig 1.
IL-17+ γδ T cells are neurotoxic in vitro.
(A-E) 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 (A) magnification 20x, scalebar 100μm, in (D) magnification 100x, scalebar 50μm, white arrowheads indicate apoptotic nuclei. In (B, C) 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. (E) Quantification of DAPI+ nuclei displaying apoptotic hallmarks. (F, G) 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 vs. control, (B) p<0.0001, (C) p = 0.0032, (E) p = 0.0151, (F) p = 0.7851, (G) p = 0.0064.
Fig 2.
Naïve γδ T cells express TLRs but do not secrete IL-17 in response to TLR stimulation.
(A, 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. (C, D) 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 (D) are shown as mean ± SEM of 3 experiments, ANOVA with Dunnett´s multiple comparison post test of each ligand vs. unstimulated control. (E) γδ T cells were stimulated as described in (C), 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 vs. unstimulated control, (D) p = 0.3262, p = 0.0355, (E) p = 0.4647.
Fig 3.
Supernatants derived from microglia stimulated through TLRs activate naïve γδ T cells and induce expression of IL-17, but not IFN-γ.
(A) 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, C) Microglia were stimulated for 24 h with TLR ligands as described in (A) 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. (C) Each condition was performed in duplicates and averaged. Mean ± SEM of 4 individual experiments. (D) γδ T cells were cultured with microglia-conditioned supernatant, as described in (A). 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. (E) 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. (F) Bone marrow-derived macrophages (BMDMs) were stimulated for 24 h with various TLR ligands as named in (A). 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 (G). Each condition was performed in duplicate and averaged. Mean ± SEM of 4 to 5 individual experiments. (A), (C) and (F) ANOVA with Dunnett´s multiple comparison post test of each ligand vs. unstimulated control, (A) p = 0.7198, p<0.0001, p = 0.9415, p<0.0001, p = 0.9707, p = 0.0001, (C) p = 0.0061, p = 0.9883, p = 0.2590, p = 0.1599, (E) p<0.0001, p = 0.0004, p = 0.0521. (D) and (G) 2-way ANOVA with Bonferroni post test compared to unstimulated control; p*<0.05, p***<0.001.
Fig 4.
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.
(A) 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, C) WT microglia were stimulated as in (A) 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. (D) Naïve γδ T cells were directly analyzed. In vitro 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. (E, F) WT microglia were stimulated as in (A) 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 (E). After 3 days supernatants were analyzed by ELISA regarding IL-17 secretion (F). Each condition was performed in duplicate and averaged. Mean ± SEM of 4 individual experiments. (A, B, C, E, F) ANOVA followed by Bonferroni multiple comparison post test, (A) TLR2KO p = 0.3340, TLR7KO p = 0.0989, (B) IL-6 p = 0.0705, IL-23 p = 0.5709, IL-1β p = 0.0011, IL-18 p = 0.7380, (C) IL-13 i = 0.0148, IL-17 p<0.0001, IL-22 p<0.0001, IFN-γ p = 0.0001, granzyme B p = 0.4176, (E) p<0.0001.
Fig 5.
Supernatants from microglia stimulated via TLR2, TLR4, or TLR9 induce neurotoxic γδ T cells.
(A) 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 (C) 1*105 microglia were added to neuronal cultures concurrent with γδ T cells. (D) 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 (A). (B, C, D) 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) p<0.0001, (C) p<0.0001, (D) p<0.0001.