Supplemental Information. Human CD1c + Dendritic Cells Drive. the Differentiation of CD103 + CD8 + Mucosal Effector T Cells via the Cytokine TGF-

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1 Immunity, Volume 38 Supplemental Information Human CD1c + Dendritic Cells Drive the Differentiation of CD103 + CD8 + Mucosal Effector T Cells via the Cytokine TGF- Chun I. Yu Christian Becker Yuanyuan Wang, Florentina Marches, Julie Helft, Marylene Leboeuf, Esperanza Anguiano, Stephane Pourpe, Kristina Goller, Virginia Pascual, Jacques Banchereau, Miriam Merad, and Karolina Palucka Supplemental Inventory 1. Supplemental Figures and Tables Figure S1, Related to Figure 1 Figure S2, Related to Figure 2 Figure S3, Related to Figure 3 Figure S4, Related to Figure 4 Figure S5, Related to Figure 5 Figure S6, Related to Figure 6 2. Supplemental Experimental Procedures

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3 Figure S1. Human DC Subsets in Tissues of Humans and Humanized Mice, Related to Figure 1 This figure provides additional data on the characterization of DC subsets. (A-E) Single-cell suspensions were stained with specific antibodies and analyzed by flow cytometry. DCs were gated as human CD45 +, CD3 -, CD19 -, CD4 +, HLA-DR +, CD14 - and CD11c + cells with differential expression of CD1c and CD141. Flow cytometry characterization of sorted CD1c + and CD141 + DCs in the lungs (A), draining LNs (B), and spleen of humanized mice (C), human lungs (D), and human blood (E). (F) Immunofluorescence staining of DCs in human lung frozen tissue sections (scale bar = 100 m). Nucleated cells were defined by DAPI staining (blue); epithelial cells were identified by cytokeratin staining (green); DCs were identified by CD1c staining (red, upper plots) and CLEC9A staining (red, lower plots). CLEC9A is expressed by CD141 + DCs. Representative data from 5 different donors.

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5 Figure S2. Autologous CD8 + T Cell Responses Mediated by Subsets of Human Lung DCs in Humanized Mice, Related to Figure 2 (A-D) provides additional data on the characterization of DC subsets in humanized mice 3 days after PBS or LAIV inoculation. Panel E-F provides additional data illustrating the capacity of both DC subsets to trigger T cell proliferation and expansion. (A-D) Humanized mice were inoculated intranasally with PBS or LAIV. Three days later, lungs (A-B) and draining LNs (C-D) were harvested. Single cell suspensions were stained with specific antibodies. DCs were gated as human CD45 +, CD3 -, CD19 -, CD4 +, HLA-DR +, CD14 - and CD11c + cells with differential expression of CD1c and CD141. Representative dot plots illustrating the gating strategy for CD1c + and CD141 + DCs in the lungs and draining LNs of humanized mice at 3 days after PBS (A, C) or LAIV (B, D) inoculation. (E) Humanized mice were treated with PBS or LAIV intranasally for 3 days. Different subsets of DCs were sorted from the lungs of humanized mice and cocultured with autologous T cells. Dot plots illustrating T cell expansion by 3000 DCs sorted from the lungs of humanized mice treated with PBS or LAIV for 3 days. (F) Sorted DCs from the lungs of naïve humanized mice were treated with or without LAIV in vitro and then cocultured with autologous T cells for 8 days. Dot plots illustrating CD8 + T cells specific to FluM1 epitope expanded by 3000 DCs.

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7 Figure S3. CD103 + CD8 + T Cell Responses in Cultures with CD1c + DCs, Related to Figure 3 (A-C) shows that, as expected, CD141 + DCs induce higher allogeneic T cell proliferation at different DC:T cell ratios. Panel D-F shows additional data corroborating the conclusion that CD1c + DCs are uniquely able to elicit the expansion of allogeneic CD103 + CD8 + T cells regardless of their tissue origin. Panel G-I shows additional data corroborating the conclusion that CD1c + DCs are uniquely able to elicit the expansion of autologous CD103 + CD8 + T cells. (A-C) Different subsets of DCs were sorted from the lungs of humanized mice treated with LAIV intranasally for 3 days (A-B), or from human lungs (C), and cocultured with 10 5 CFSE-labeled allogeneic naïve T cells. (A) Representative dot plots illustrating CFSE-dilution on T cells at day 6. (B-C) CD8 + T cell proliferation measured by the percentage of CFSE - CD8 + T cells (ordinate) in cocultures with CD1c + (red) or CD141 + (blue) DCs at titrated doses (abscissa). Representative data from more than three experiments. Two-way ANOVA with Bonferroni posttests. Data are shown as mean ± SEM. (D-E) CD1c + and CD141 + DCs were sorted from the spleen of humanized mice. Sorted DCs were cocultured with 10 5 CFSE-labeled allogeneic naïve T cells for 6 days. T cell proliferation measured by CFSE dilution and CD103 expression in CSFE-negative CD8 + T cells were assessed by flow cytometry analysis. (F) Sorted CD1c + or CD141 + DCs from human blood were activated with LAIV in vitro and cocultured with 10 5 allogeneic naïve T cells. T cell proliferation measured by CFSE dilution and CD103 expression in CD8 + T cells were assessed every two days by flow cytometry analysis. (G-I) Sorted CD1c + and CD141 + DCs from human blood were loaded with LAIV in vitro and then cocultured with autologous T cells. (G) Representative dot plots illustrating the phenotype of proliferation-gated CFSE-negative CD8 + T cells in cocultures with CD1c + or CD141 + DCs at day 8 in comparison to the phenotype of freshly isolated CD8 + T cells from human lungs. (H) T cell proliferation measured by CFSE dilution and CD103 expression in CD8 + T cells were assessed every two days by flow cytometry analsysis. (I) LAIV-loaded human blood DC subsets were cocultured with CD103-depleted autologous blood T cells. Representative dot plots illustrating CFSE-dilution and CD103 expression on CD8 + T cells at day 8 of the cocultures.

8 Figure S4. CD103 + CD8 + T Cells Expanded by CD1c + DCs Express Granzyme and Kill Peptide-Pulsed Target Cells, Related to Figure 4 Figure S4 shows additional data corroborating the conclusion that CD103 + CD8 + T cells expanded by CD1c + DCs express an effector phenotype. CD1c + and CD141 + DC subsets were sorted from the lungs of humanized mice after LAIV inoculation (A) and from human blood (B-C). (A-B) DCs were cocultured with CFSE-labeled naïve allogeneic T cells. After 6 days, CFSE - CD8 + T cells were stained to assess intracellular expression of granzyme. Dot plots illustrating the expression of effector molecules in gated CFSE - CD8 + T cells stimulated by CD1c + or CD141 + DC subsets as indicated in the figure. (C) DC subsets were sorted from HLA-A* human blood, loaded with LAIV and used in cocultures with autologous T cells. At day 8 of coculture, proliferating CFSE - CD8 + T cells were sorted and used as effector cells in a flow cytometry-based four-hour cytotoxicity assay following the manufacture s protocol (Invitrogen). Target cells were HLA-A*0201 transduced T2 cells that were used either unpulsed (control) or pulsed with HLA-A*0201-restricted FluM1 peptide. Specific lysis of T2 cells (ordinate) was assessed at different effector-to-target (E:T) ratios (abscissa). Data are shown as mean ± SEM.

9 Figure S5. CD1c + DCs Elicit the Differentiation of CD103 + CD8 + T Cells In Vivo Figure S5 shows additional data corroborating the conclusion that CD1c + DCs elicit the differentiation of CD103 + CD8 + T cells in vivo regardless of the survival of T cells. Sorted CD1c + or CD141 + DCs from human blood were co-injected with allogeneic naïve CFSElabeled T cells (3x10 5 DCs and 3x10 6 T cells) into human epithelial grafts created as described in Figure 5A. The frequencies of CD3 + CD8 + human T cells in the spleen were analyzed by flow cytometry at day 6. Figure S6: CD1c + DCs elicited the differentiation of CD103 + CD8 + T cells independent of GM-CSF. Figure S6 shows additional data corroborating the conclusion that CD1c + DCs elicit the differentiation of CD103 + CD8 + T cells via TGF-. Sorted blood DCs were first stimulated with LAIV for one hour and then cocultured with CFSElabeled allogeneic naïve T cells at a 1:10 ratio in the presence of GM-CSF-neutralizing Abs (30 g/ml) or TGF-β receptor I kinase inhibitor (1 M). At day 6, CFSE - CD8 + T cells were analyzed for CD103 expression by flow cytometry. The percentage of CFSE - CD8 + T cells (left, ordinate) and the percentage CD103-expressing CFSE - CD8 + T cells (right, ordinate) in cocultures with DCs with different conditions as indicated (abscissa). Data are shown as mean ± SEM.

10 Supplemental Experimental Procedures Antibodies and Reagents Antibodies (Abs) to human CD1a (HI149), CD3 (UCHT1), CD4 (SK3), CD8 (SK1), CD11c (B-ly6), CD19 (HIB19), CD36 (CB38), CD80 (L307.4), CD83 (HB15e), CD86 (IT2.2), CD87 (VIM5), and granzyme B (GB11) were from BD (Franklin Lakes, NJ); CCR7 (112509), HLA-DR (LN3) were from ebioscience (San Diego, CA); CD1c (L161), CD103 (Ber-ACT8), CD105 (43A3), GM-CSF (BVD2-23B6), granzyme A (GB9), and perforin (dg9) were from Biolegend (San Diego, CA); CD303 (AC144), CD141 (AD5-14H12), and cytokeratin (AE1/AE3) Abs were from Mitenyi Biotec (Auburn, CA). Human CD14 (Tuk4), CD45 (HI30), and HLA-ABC (W6/32) Abs were from Invitrogen (Carlsbad, CA). Anti-human CLEC9A (4C6) Ab was a gift of M. Lahoud and K. Shortman (Walter and Eliza Hall Institute, Melbourne, Victoria, Australia). Anti-human CLEC9A (683409), NKG2A (131411), NKG2D (149810), TGF-, TGF- receptor I, and TGF- receptor II Abs, and recombinant human IL-2, IL-7, CD40L, and TGF- 1 were from R&D Systems (Minneapolis, MN). CD40 (MAB89) Ab and HLA-A*0201 tetramers loaded with influenza A virus M (GILGFVFTL), NS (AIMDKNIIL), and HIV-1 gag (SLYNTVATL) were purchased from Beckman Coulter (Brea, CA). TGF-β RI Kinase Inhibitor II was purchased from EMD Chemicals (Gibbston, NJ). Trivalent live-attenuated influenza virus (LAIV) vaccine FluMist ( season; MedImmune, Gaithersburg, MD) was obtained from the hospital pharmacy. Cytotoxicity Assay Killing activity was measured using a flow cytometry-based Live/Dead Cytotoxicity assay following the manufacturer s protocol (Invitrogen). Briefly, target cells including both unloaded or peptide-loaded T2 (ATCC, Manassas, VA) were first labeled with 5 l of DiOC for 20 mins at 37 C and then cultured with effector cells at different effector:target (E:T) ratios in a 96-well U-bottom plate in the presence of propidium iodide (PI). The plate was centrifuged at 1200 rpm for 30 sec before incubating at 37 C for 4 hours. Total cytotoxicity was measured as the percentage of PI + in DiOC + target cells. The spontaneous cytotoxicity was determined as the percentage of PI + in DiOC + target cells without the presence of effector cells. The specific cytotoxicity was calculated as [(total cytotoxicity)-(spontaneous cytotoxicity)] x 100 percentages. Immunohistofluorescence Staining Tissues were embedded in OCT (Sakura Finetek U.S.A., Torrance, CA) and snap frozen in liquid nitrogen. Frozen sections were cut at 6 μm and air dried on Superfrost slides (CardinalHealth, Dublin, Oh). Frozen sections were fixed with cold acetone for five minutes and air-dried. Tissue sections were first treated with Background Buster and Fc Receptor Block (Innovex Bioscience, Richmond, CA). The sections were then stained with primary mouse monoclonal Abs for one hour at room temperature, followed by staining with fluorochrome-labeled secondary Abs. Respective isotype Abs were used as the control. Finally, sections were mounted with ProLong Gold with DAPI (Invitrogen), visualized using a fluorescence microscope (Olympus, Japan) with MetaMorph software (Molecular Devices, Sunnyvale, CA) or a Leica SP 5 confocal microscope with Leica LAS AF 2.0 software (Buffalo Grove, IL). Flow Cytometry Analysis Cells were first treated with Fc blocker (Mitenyi Biotec) and then stained for 30 min on ice with fluorochrome-conjugated specific Abs. After washing twice with PBS, the samples were acquired on an LSRII (BD), and analyzed with FlowJo software (Tree Star, Ashland, OR). For tetramer analysis, samples were stained at room temperature for 30 min with fluorochrome-conjugated Abs for surface markers and APC-conjugated FluM1-HLA-A*0201 tetramer for FluM1-specific CD8 + T cells. To assess the expression of intracellular proteins, samples were first stained with fluorochrome-conjugated Abs against surface markers at room temperature for 15 min, followed by fixation and permeabilization. They were then stained for intracellular proteins at room temperature for 30 min.

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