Supplementary Figures IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia Yaming Wang, Kristy J. Szretter, William Vermi, Susan Gilfillan, Cristina Rossini, Marina Cella, Alexander D. Barrow, Michael S. Diamond & Marco Colonna Supplementary Figure 1. Schematic diagram of the generation of Il34 LacZ/LacZ and Il34 f/f mice. Il34 mutant mice were generated from a EUCOMM JM8.N4 ES clone carrying the targeted Il34 tm1a(eucomm)wtsi allele designed to allow generation of either a null, LacZ reporter allele or a conditional floxed allele for subsequent Cre-mediated deletion. Mice carrying the targeted Il34 allele were bred either to: 1) CMV-Cre transgenic mice to remove the neo r gene and generate Il34 LacZ/+ reporter mice in which exons 3-5 are deleted and replaced by an IRES-LacZ gene, or 2) CAG-FLPe transgenic mice to remove the IRES-LacZ/neo r cassette and generate mice carrying a conditional Il34 allele in which exons 3-5 are flanked by loxp sites (Il34 f/+ ). Offspring were intercrossed to generate homozygote Il34 LacZ/LacZ or Il34 f/f mice. Exons are represented by grey boxes. 1
Supplementary Figure 2. IL-34 expression in different brain areas of Il34 LacZ/+ mice and lack of IL-34 expression in skin and brain of Il34 LacZ/LacZ (KO) mice. Staining for X-gal (blue) in Il34 LacZ/+ mice was performed in different brain regions. Punctate signals are detected in the cerebral cortex (a), choroid plexus (b) and hippocampus (c); no signal was observed in the cerebellum (d) and meninges (M, in a). Original magnification 200x (a; scale bar 100µm) and 400x (b-d; scale bar 100µm). (e, f) Quantitative-RT-PCR (qpcr) analysis of Il34 mrna in the (e) skin and (f) brain of wild-type (WT) and KO mice (n=2/group). *P = 0.0008, **P = 0.002 (unpaired Student s t-test). Bars represent mean ± SEM. Data were obtained from two independent experiments. 2
Supplementary Figure 3. IL-34 deficiency leads to the absence of LCs. (a, b) Immunofluorescence staining of MHCII of epidermal sheets isolated from WT and KO mice. Magnification 40x (scale bar 40µm). Quantification of the frequencies of MHCII + cell per microscopic field is shown in (b). *P < 0.0001 (unpaired Student s t-test). (c,d) Percentages and quantification of CD11c + MHCII + cells in epidermal sheets of WT and KO mice (n=9/group) was determined by flow cytometry. **P = 0.0006 (unpaired Student s t-test). (e,f) Percentages and quantification of CD11c + MHCII + cells in dermal sheets of WT and KO mice (n=3/group). (g,h) Frequencies and quantification of CD11c + CD11b + cells within MHCII + gate in the dermis of WT and KO mice (n=2/group). (I,j) Frequencies and quantification of CD11c + CD103 + cells within MHCII + gate in the dermis of WT and KO mice (n=3/group). (k) Immunohistochemical analysis of Langerin expressing cells in the spleen and thymus of WT and KO mice. Original magnification 400x. Data represent two independent experiments. 3
Supplementary Figure 4. Impact of IL-34 deficiency on T cells in steady-state, after CHS to DNFB and after delayed-type hypersensitivity (DTH) to C. albicans. (a) Total numbers of inguinal lymph node cells of WT and KO mice (n=3-5/group) were quantified day-7 post DNFB treatment. *P = 0.0032 (unpaired Student s t- test). (b) IL-34 deficiency does not affect T cell development in the thymus and T cell numbers in skin-draining lymph nodes (SLN). (c,d) The skin of WT and KO mice was infected with 2 10 8 blastoconidia of the SC5314 strain of C. albicans. (c) DTH on day-7 was measured by specific footpad swelling 24 h after injection of 10 7 heat killed C. albicans (n=6-7/group). **P = 0.0237, ***P = 0.0476 (unpaired Student s t-test). (d) Total numbers of inguinal lymph node cells of WT and KO mice were quantified day-7 post infection (n=4/group). Data represent two independent experiments. 4
Supplementary Figure 5. (a) Cytokine production by microglia after TLR ligand stimulation. 2X10 5 purified microglia from WT and KO mice were cultured for 16 h in the presence of LPS (1 µg/ml), CpGB (1 µg/ml) or Imiquimod (6 µm). Cytokine production in the supernatants was determined by bead arrays and flow cytometry. (b) Viral burden in different regions of the CNS of WT and KO mice infected intracranially with 10 5 PFU of WNV-E218A. Viral titers in various regions were determined on day 3 and day 6 post-infection (n=3-5/group). (c-e) Leukocyte accumulation in the CNS at day-6 post WNV-E218A infection. Brains from WT and KO mice (n=5/group) were harvested on day 6 after infection, and leukocytes were isolated by percoll gradient centrifugation. The total number of (c) microglia (CD11b hi /CD45 int ) and macrophages (CD11b hi /CD45 hi ), or (d) CD3 + CD4 + or CD3 + CD8 + T cells was determined. (e) Brain leukocytes were also stained with an H2-D b -NS4b tetramer and then stained for CD3, CD8, granzyme B, and analyzed by flow cytometry. *P = 0.0079 (unpaired Student s t-test). Bars represent mean ± SEM. Data represent two independent experiments. 5
Supplementary Figure 6. (a) Frequencies of myeloid cells in peripheral blood, and bone marrow of WT and KO mice. Myeloid cells were identified by expression of CD11b, F4/80, Ly6C and Ly6G. In blood Ly6C hi Ly6G - cells correspond to monocytes whereas Ly6C int Ly6G hi correspond to neutrophils. Ly6G - Ly6c hi CD11b hi cells represent inflammatory monocytes, whereas Ly6G - Ly6C int CD11b + cells represent resident monocytes. In bone marrow, Ly6C + Ly6G + cells correspond to granulocytes and the Ly6C hi CD31 - population mostly contains monocytic cells 1. (b) Subsets of splenic DCs in WT and KO mice as analyzed by flow cytometry. CD11c hi SiglecH - cells correspond to classical DCs, whereas SiglecH + CD11c int cells represent plasmacytoid DCs (pdcs). By gating on the CD11c hi population and evaluating the expression of CD8α and CD11b/CD4, we determined the relative frequency of the two major splenic classical DC populations. Data represent one experiment with 2-3 mice/group. 6
Supplementary Figure 7. Disappearance of LCs day-7 post UV treatment in WT mice. WT mice were treated with UV light for 30 min. Frequency of LCs (CD11c + MHCII + within CD45 + gate) in epidermis was determined by flow cytometry at day-7 post treatment. Data represent one experiment with 3 mice/group. References 1. Leenen, P.J., de Bruijn, M.F., Voerman, J.S., Campbell, P.A. & van Ewijk, W. Markers of mouse macrophage development detected by monoclonal antibodies. J Immunol Methods 174, 5-19 (1994). 7