Immunity, Volume 38 Supplemental Information IRF4 Transcription Factor-Dependent CD11b+ Dendritic Cells in Human and Mouse Control Mucosal IL-17 Cytokine Responses Andreas Schlitzer, Naomi McGovern, Pearline Teo, Teresa Zelante, Koji Atarashi, Donovan Low, Adrian W.S. Ho, Peter See, Amanda Shin, Pavandip Singh Wasan, Guillaume Hoeffel, Benoit Malleret, Alexander Heiseke, Samantha Chew, Laura Jardine, Harriet A. Purvis, Catharien M.U. Hilkens, John Tam, Michael Poidinger, E. Richard Stanley, Anne B. K rug, Laurent Renia, Baalasubramanian Sivasankar, Lai Guan Ng, Matthew Collin, Paola Ricciardi-Castagnoli, Kenya Honda, Muzlifah Haniffa, and Florent Ginhoux Supplemental Inventory 1. Supplemental Figures and Tables Figure S1, Related to Figure 1 Figure S2, Related to Figure 2 and 3 Figure S3, Related to Figure 4 Figure S4, Related to Figure 5 Figure S5, Related to Figure 5 Figure S6, Related to Figure 7 Table S1, Related to Figure 3 Table S2, Related to Figure 6 Table S3, Related to Figure 6 2. Supplemental Experimental Procedures 3. Supplemental References Supplementary figure 1 Sorting strategy for mouse lung and small intestinal DC A Sorting strategy, Lung Singlets Dapi-CD45+ 250K 250K 250K 105 200K 200K 200K 104 A I - 150K 150K 150K C P C C 3 10 S A S S 100K 100K S 100K D S S 102 50K 50K 50K 0 0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 103 104 105 0 103 104 105 FSC FSC-W CD45 GR1 Auto Fluor.- MHCII+ GR1- SSClow CD11c+ CD11b+ 250K 105 105 105 200K 4 104 10 104 150K I 3 I 3 4 3 0 10 3 C 2 H 10 10 100K 1 S D C D S C M 2 C 10 50K 0 0 0 0 0 102 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 Auto fluor. CD11c CD11b CD11b Purity, Lung sorting Sorting gate 1 Sorting gate 2 ! Sorting gate 3 CD103+ DC CD11b+ DC CD11b+ MAC 105 105 ! 105 4 10 104 104 3 103 4 3 4 3 0 10 10 2 2 1 ! D D D 2 10 C C C 0 0 0 90 ! 98 97 0 103 104 105 0 103 104 105 0 103 104 105 CD11b CD11b! CD11b B + Sorting strategy, Small intestine MHCII Singlets Dapi-CD45+ CD11c+ CD11b+ 250K 250K 105 105 105 105 200K 200K 4 4 4 4 10 10 10 10 I A 3 I I 150K - 150K 4 0 P H 2 C 1 C 3 3 3 3 A 10 10 10 10 C D S S 100K 100K D D S C M S C 50K 50K 0 0 0 0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 102 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 FSC FSC-W CD45 CD11c CD11b CD64 Purity, SI sorting Sorting gate 1 Sorting gate 2 Sorting gate 3 Sorting gate 4 CD103+ DC CD103+CD1!1b+ DC CD11b+ DC CD11b+ MAC 105 105 ! 105 105 104 104 104 104 3 3 3 3 3 3 10 10 4 10 4 10 0 0 ! 2 2 1 1 D D D D 0 0 C 0 C 0 C 98 C !95 87 93 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 CD11b CD11b ! CD64 CD64 Supplementary figure 1 Sorting strategy for mouse lung and small intestinal DC Sorting strategy of lung CD103+ and CD11b+ DC as well as CD11b+ Macs from steady state WT mice (A). Sorting strategy of small intestinal CD103+, CD103+CD11b+ and CD11b+ DC as well as CD11b+ Macs from steady state WT mice (B). Supplementary figure 2 Chimerism of blood granulocytes in mixed BM chimeras A Blood, Granulocytes 100 WT : FLT3-/- 2 -/- . WT : CSF-1R 5 -/- 4 WT : IRF4 BM D 50 C % 0 Supplementary figure 2 Chimerism of blood granulocytes in mixed BM chimeras Blood chimerism of granulocytes of FLT3-/-, CSF-1R-/- and IRF4-/- mixed BM chimeras 4 weeks after irradiation (mean ± SEM, n=4) (A). Supplementary figure 3 Flow cytometric analysis of mouse colonic and small intestinal lamina propria A Colon Dapi-CD45+ MHCII+ + + + SSClow CD11c+ CD11b CD24 CD64 5 5 10 105 8 79 10 12 105 105 4 4 10 4 10 4 4 10 10 10 I 3 3 3 I 0 4 2.8 0 0 C 3 1 1 2 1 3 10 H 10 3 3 3 10 10 D 10 D D D 0 0 0 M C C 0 C C 0 64 80 96 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 CD11c CD11b CD64 CD11b CD11b B Small intestine, Dendritic cell and Macrophage suBsets 5 6 6 8 6 6 4 6 4 4 3 4 + 4 4 CD103 DC 2 2 2 2 2 2 1 0 0 0 0 0 0 3 4 5 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 10 10 10 3 5 5 8 10 10 4 4 8 6 8 + + 2 CD103 CD11b 6 3 3 6 4 4 2 2 DC 4 1 2 2 2 1 1 0 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 6 8 5 2 0.8 6 4 6 1.5 0.6 4 3 + - 4 CD11b CD103 4 1 0.4 2 2 DC 2 0.5 2 1 0.2 0 0 0 0 0 0 0 103 104 105 0 103 104 105 3 4 5 0 103 104 105 0 10 10 10 3 4 5 0 103 104 105 6 0 10 10 10 4 3 5 8 6 4 3 6 4 2 4 3 + 4 2 CD11b MAC 2 2 2 1 2 1 1 0 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 CD11c CD24 CD26 BTLA GR1 MHCII 5 4 6 3 6 4 3 4 2 3 4 + 2 CD103 DC 2 2 1 2 1 1 0 0 0 0 0 3 4 5 3 4 5 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 0 10 10 10 5 3 12 6 10 4 9 8 + + 2 4 CD103 CD11b 3 6 6 2 DC 4 1 2 3 1 2 0 0 0 0 0 3 4 5 3 4 5 0 103 104 105 0 103 104 105 0 103 104 105 0 10 10 10 0 10 10 10 2.5 3 3 2 2 6 1.5 2 2 CD11b+CD103- 1.5 4 1 1 DC 1 1 2 0.5 0.5 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 5 2 20 6 8 4 1.5 15 6 4 3 + 1 10 CD11b MAC 4 2 2 0.5 5 1 2 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 F4/80 CD172 CD14 CX3CR1 LysozyMe Supplementary figure 3 Flow cytometric analysis of mouse colonic and small intestinal lamina propria Analysis of the colonic lamina propria DC compartment for the depicted markers by flow cytometry. Dot plots were gated as indicated (n=5) (A). DC and macrophage populations within the SI-LP were labeled with antibodies specific for the indicated markers, or an isotype-matched control antibody and analyzed by flow cytometry (n=3) (B). Supplementary figure 4 Lung CD24+CD11b+ DCs and gut LP CD24+CD103+CD11b+ DCs are absent in Itgax-cre Irf4fl/fl but not in any other organ A B C D Total MHCII+ CD11c+ Lung, DCs Small intestine, DCs Blood 100 100 100 100 P P P P F F F F G 50 G 50 G 50 G 50 % % % % 0 0 0 0 Spleen CD103+ CD103+ Granulocytes Lung CD11b+ CD24+ CD11b+ CD103+ B-cells Small intestine CD11b+ CD64+ CD11b+ CD24+ T-cells CD11b+ CD64+ GR1high Monocytes GR1low Monocytes E H Lung Small intestine CD45+ MHCII+ CD45+ MHCII+ CD45+ MHCII+ CD11c+ CD11c+ CD11b+ CD11c+ CD24+ 5 10 105 11 67 105 46 24 31 4 4 10 4 10 10 WT WT 3 3 10 3 10 10 0 0 0 29 19 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 103 104 105 5 105 10 23 44 105 11 37 7 4 4 3 3 10 10 104 0 0 4 1 1 ItgaX-cre Irf4fl/fl 2 ItgaX-cre Irf4fl/fl 3 3 D 10 3 D 10 D 10 0 0 0 C 46 C C 24 3 4 5 0 103 104 105 0 103 104 105 0 10 10 10 CD11b CD64 CD11b F I Lung Small intestine CD103+ CD11b+ CD11b+ CD103+ CD103+ CD11b+ CD11b+ DC DC MAC DC CD11b+ DC DC MAC *** + *** *** *** + B B + + 80 + 60 60 40 40 + 40 50 I 1 1 I I I I WT I I I 1 1 + + C C C C fl/fl c c D D ItgaX-cre Irf4 H 1 H 1 H H C C 1 1 40 + 30 30 M 20 + M M 20 M 20 25 D D c c f f f f C 1 1 C o o o o 1 1 D D % % 0 % 0 0 0 0 % 0 0 C C G J Lung dr.