Fig 1.
Cutaneous immunity against Malassezia depends on IL-17A and IL-17F cytokines.
The ear skin of wild type (WT) and Il17af-/- mice was associated with M. pachydermatis or treated with olive oil (vehicle control) and analysed at the indicated time points (days post infection, dpi). A. Il17a and Il17f transcript levels in the M. pachydermatis-associated and control ear skin of WT mice. B. Increase of ear thickness in WT and Il17af-/- mice. C. Hematoxylin and eosin-stained ear tissue sections. D. Skin neutrophil numbers. E. Skin fungal load (CFU). DL, detection limit. Data in A-D are from two independent experiments with 2–4 mice per group. Data in E are compiled from five independent experiments with at least 3 mice per group and time point (except at 60 dpi, which is 2 mice per group). In A, the median of each group is indicated. In B, D and E, the mean +/- SEM of each group is indicated. Statistical significance was determined using two-way ANOVA. **p<0.01, ****p<0.0001. See also S1 Fig.
Fig 2.
Vγ4+ γδ T cells are the main IL-17A producers in Malassezia-associated skin.
The ear skin of WT mice was associated with M. pachydermatis or treated with olive oil (vehicle control) and IL-17 production in skin and draining lymph nodes was analysed by flow cytometry at the indicated time points (dpi) or on day 7 (if not specified otherwise). A. Gating strategy for identifying the IL-17+ cellular subsets among viable CD45+ CD11b- single cells in the skin. B.-C. Quantification of IL-17A+ cells among overall skin CD90+ cells (B) or among Vγ4+ TCRγδ+ CD90+ T cells (C) after ex vivo re-stimulation with PMA and ionomycin. D. Quantification of non-restimulated Vγ4+ γδ T cells in the ear skin. E.-G. Proportion (E), total numbers (F), and IL-17A median fluorescence intensity (G) of the indicated IL-17A producing cell populations in the ear skin after ex vivo re-stimulation with PMA and ionomycin. H.-I. Proportion (H) and total numbers (I) of the indicated IL-17A-producing CD44+ cell populations in the ear-draining lymph nodes (dLN) after re-stimulation with PMA and ionomycin or with heat-killed M. pachydermatis (hkM.pach.)-pulsed DCs, as indicated. Data in A-I are from one representative of two independent experiments with 3–5 mice per group. In B, C, D, G and I, each symbol represents one animal. In B, C, D and I, the median of each group is shown. In F and G, the mean +/- SD of each group is shown. K.-L. IL-22, IFN-γ or TNF co-production with IL-17 by Vγ4+ (K) and CD4+ T cells (L) in the dLN after re-stimulation with hkM.pach. pulsed DCs in M. pachydermatis-associated (dark blue) or vehicle-treated control mice (light blue). Four or two concatenated samples from associated or control mice, respectively, are shown with numbers in quadrants indicating percentages of CD44+ TCRγδ+ (K) or CD4+ (L). Data for IL-22 and IFN-γ are from day 7 in one of two representative experiments while TNF is from one experiment on day 5. Statistical significance was determined using one-way ANOVA (B, C, F, G), two-way ANOVA (D) or unpaired Student’s t test (I). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also S2 Fig.
Fig 3.
γδ T cells are key mediators of antifungal immunity to Malassezia.
The ear skin of WT and Tcrd-/- mice was associated with M. pachydermatis or treated with olive oil (vehicle control) and analysed at the indicated time points (dpi). A. IL-17A transcript levels in the ear skin. B. Quantification of IL-17A+ cells among overall CD44+ CD90+ draining lymph node (dLN) cells at 7 dpi after re-stimulation with heat-killed M. pachydermatis (hkM.pach.)-pulsed DCs. C. Increase in ear thickness. D. Hematoxylin and eosin-stained ear tissue sections. E. Skin neutrophil numbers. F. Skin fungal load (CFU). Data in A, C and E are pooled from, and data in B and D are from one representative of two independent experiments with 3–6 mice per group. Data in F are compiled from five independent experiments with 2–6 mice per group resulting in 4–9 mice per time point except for the 30 and 120 dpi time points which are 2 and 3 mice per group, respectively. The mean +/- SEM of each group is shown in A, C, E and F. The median of each group is shown in B. DL, detection limit. Statistical significance was determined using two-way ANOVA (A, C, E and F) or unpaired Student’s t test (B). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 4.
A pool of γδ T cells in lymph nodes supports long-term protection against Malassezia.
A.-E. WT mice were associated with M. pachydermatis (M.pach.) once or twice or treated with olive oil (vehicle control) according to the scheme in (A). Total numbers of overall CD45+ cells (B) and IL-17+ Vγ4+ γδ T cells in ear skin (C) and IL-17A+ Vγ4+ γδ T cells in dLN after re-stimulation with heat-killed M. pachydermatis-pulsed DCs (D). Skin fungal load (E). Data in B, C and E are compiled from two independent experiments, and data in D are from one representative of two independent experiments with 2–4 mice per group. Each symbol represents one mouse. The median of each group is indicated. F.-I. WT mice were associated with M. pachydermatis for 7 days and treated with FTY720 or EtOH (solvent control) in drinking water from day -1 until day 7 of colonisation. Representative flow cytometry plots showing CD44+ TCRγδ+ cells among viable CD45+ CD90+ Ki-67+ dLN cells (F). Total numbers of Ki-67+ γδ T cells in dLN (G) and ear skin (H) and fungal loads (CFU) in the skin (I). Data in G-I are pooled from two independent experiments with 2–4 mice per group. Each symbol represents one mouse. The median of each group is indicated. K.-M. Chemokine receptor expression by IL-17+ and IL-17- Vγ4+ γδ T cells in dLN and skin of M. pachydermatis-associated WT mice after re-stimulation with heat-killed M. pachydermatis-pulsed DCs or PMA and ionomycin, as indicated. Histograms showing three concatenated samples from one representative of of two (dLN) or three (ear) independent experiments (K) including a fluorescence minus one (FMO) control per experiment. Median fluorescence intensity of IL-17A staining in the CCR6- CD103- and CCR6+ CD103+ subsets of Vγ4+ γδ T cells in dLN (L) and ear skin (M) Data are from one representative of three (L) or two (M) independent experiments. Connected data points are from the same mouse. Statistical significance was determined using one-way ANOVA (B-E, G, H) or unpaired (I) or paired (L, M) Student’s t test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also S3 Fig.
Fig 5.
γδT17 cell responses to Malassezia do not depend on CD11c+ cells or T cell receptor signalling.
A.-E. Irradiation chimeras reconstituted with CD11c-DTR transgene positive (TG+) or transgene negative (TG-) bone marrow as a control were treated with diphteria toxin prior to association with M. pachydermatis for 7 days. Total numbers of CD45+ CD11c+ cells in the dLN (A). Total numbers of IL-17A+ γδ T cells (B) and IL-17A+ CD4+ T cells (C) in the ear skin (left) and dLN (right) after re-stimulation with heat-killed M. pachdermatis-pulsed DCs (in case of dLN T cells) or with PMA and ionomycin (in case of skin T cells), respectively. Increase in ear thickness (D). Skin fungal loads (CFU) at 7 dpi (E). Data are pooled from two independent experiments with three mice per group. Each symbol represents one mouse. The median of each group is indicated. DL, detection limit. F.-I. Nur77GFP mice were associated with M. pachydermatis or treated with olive oil (vehicle control) for 7 days and GFP expression by γδ T cells and CD4+ T cells in the dLN or ear was assessed after restimulation with DCs that were or were not pulsed with heat-killed M. pachydermatis (DC +/- hkM.pach.). Histograms are from three (vehicle) or six (M.pachydermatis-associated) concatenated samples (E). GFP median fluorescence intensity (MFI) in CD44+ Vγ4+ γδ T cells and CD4+ T cells (G). GFP MFI of IL-17+ and IL-17- cells after restimulation with M. pachdermatis-pulsed DCs in dLN (H) and ear skin (I). Data are from one representative of two independent experiments with four to six mice per group. Bars in G-I are the mean+SD of each group. Statistical significance was determined using unpaired Student’s t test (A-E) or one-way ANOVA (G-I) **p<0.01, ***p<0.001, ****p<0.0001.
Fig 6.
γδ T cells are activated independently of C-type lectin and Toll-like receptor signalling in response to Malassezia.
A.-I. The ear skin of Card9-/- and Card9+/- littermate control (A-C) and Tlr23479-/- and control (D-F) mice was associated with M. pachydermatis. After 7 days, γδ T cells in skin and dLN were quantified (A, D) and analyzed for IL-17 production (B, E). The fungal load (CFU) was assessed in the skin (C, F). Data are pooled from three (A-C) or two (D-F) independent experiments with 3–5 mice per group. Each symbol represents one mouse. The median of each group is indicated. DL, detection limit. Statistics were calculated using unpaired Student’s t test. See also S4 Fig.
Fig 7.
The antifungal γδ T cell response depends on IL-23 and IL-1 family cytokine signalling.
A.-F. The ear skin of MyD88+/- and MyD88-/- littermate control (A-C) and Il23r-/- and Il23r+/- littermate control (D-F) mice was associated with M. pachydermatis. Vehicle treated groups were included for Il23r-/- and Il23r+/- mice. After 7 days, skin and dLN γδ T cells were quantified (A, D) and analyzed for IL-17 production (B, E), and the skin fungal load (CFU) was assessed (C, F). G.-K. Mice were treated daily with anti-Gr-1 depletion antibody (αGr-1) or control (PBS) starting one day prior to association with M. pachydermatis. Skin CD11b+ cell numbers (G), fungal load (H), transcript levels of Il23a (I) and Il1b (K) were assessed at 3 dpi. Data are pooled from three (A-F) or two (G-K) independent experiments with 2–5 mice per group. Each symbol represents one mouse. The median of each group is indicated. DL, detection limit. Statistics were calculated using unpaired Student’s t test (A-C, F, H-L), one-way ANOVA (G) or two-way ANOVA (D-E). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also S5 Fig.
Fig 8.
γδ T cells are responding directly and specifically to Malassezia-derived structures.
A.-F. The ear skin of WT mice was associated with M. pachydermatis for 7 days and γδ T cells were FACS-purified from dLN (A-C and E-F) or used as whole LN single cell suspension (D) and re-stimulated with the indicated compounds for 5 hours before quantification of IL-17A production by CD44+ Vγ4+ γδ T cells by flow cytometry. Stimulation with cell culture medium (medium) and PMA and ionomycin (PMA/iono) was included in all experiments as a reference for the response. Representative flow cytometry plots (A) and summary graphs (B-F) showing the proportion of IL-17A+ cells among CD44+ Vγ4+ γδ T cells after the indicated stimulation conditions. hk, heat killed fungal cells; sup, supernatant from fungal cultures; S. cer., S. cerevisiae; C. neo., C. neoformans; C. alb., C. albicans; A. fum., A. fumigatus; Bl-Eng, Blastomyces Endoglucanase-2 (a prototypical Dectin-2 agonist); TDB, Trehalose-6,6-dibehenate (a prototypical Mincle agonist); FICZ, 6-Formylindolo[3,2-b]carbazole (a prototypical AhR agonist). Data are from one representative of two independent experiments (A-B) or are pooled from two (C, D, F) or three (E) independent experiments with 2–3 mice per group. Each symbol represents one mouse, the mean+SD is indicated. Statistical significance between each stimulation condition and the respective medium control was determined using paired one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also S6 Fig.