M1 70

CD11b⁺Gr-1^high Ly6C^int MDSC were isolated to >90% purity and incubated with 1 µM CFSE (Invitrogen) at 37°C for 20 min in serum-free media. Excess dye was removed by washing labeled cells with complete media. A total of 1 × 10⁵ Gr-MDSC were incubated with basic media (DMEM/F12/10% FBS) or with CM isolated from the supernatants of primary PDA cells (PDA-CM) or metastatic PDA cells (Met-CM); the recombinant murine cytokines, GM-CSF, G-CSF, or M-CSF (R&D Systems, 50 ng/ml); or blocking antibodies to GM-CSF, G-CSF, or M-CSF (R&D Systems, 10 µg/ml). After 48h, cells were stained for Gr-1-e450 (BD Biosciences RB6-8C5, 1:200) and CD11b-PE (BD Biosciences M1/70, 1:200), and Annexin-V-APC and CFSE dilution were measured by gating on live Gr-1⁺CD11b⁺ cells to assess for apoptosis and proliferation, respectively.

Mice were infected with H37Rv Mtb-mCherry and sacrificed at D10 and D14. Lungs were excised and submerged in BD Cytofix fixative solution diluted 1:3 with PBS for 24hr at 4°C. Lungs were washed 2x in PBS and dehydrated in 30% sucrose for 24hr prior to OCT embedding and rapid freezing in a methylbutane-dry ice slurry. 20um sections were stained overnight at RT (with the following antibodies: CD11c BV480; HL3 (BD), CD11b BV510; M1/70 (BD), CD45.2 AF700; 104 (BioLegend), MHC-II APC-Fire750; M5/114.15.2 (BioLegend), Siglec-F BV421; E50-2440 (BD), pS6 AF488; 2F9 (Cell Signaling), CD3 CF633; 17A2, CD4 CF660C; RM4-5) and coverslipped with Fluoromount G mounting media (Southern Biotec). Images were acquired on a Leica SP8X confocal microscope, compensated for fluorophore spillover using LAS X (Leica), and analyzed with Imaris (Bitplane) and FlowJo (11 (link)). More details can be found in Supplemental Fig. 3.

Slides containing tissue sections (10–25 μm thickness) were blocked with 3% normal goat serum (NGS) in 1X PBS + 0.05% Tween-20 (PBS-T). For intracellular antigens 0.2% triton was added to the blocking buffer. Primary antibody dilutions were prepared as follows in 5% NGS in 1X PBST: rat anti mouse CD11b clone M1–70 (BD Pharmingen; #553308) – 1:100, F4/80 clone A3–1 (BioRAD; MCA497G) – 1:400, and IL-33 (abcam; ab187060) – 1:200. Sections were stained with primary antibodies overnight at 4°C, and subsequently washed with 3% NGS in 1X PBS-T. Secondary antibodies (Goat anti-rat Alexa-647, Invitrogen) were all prepared in 5% Normal Goat Serum (NGS) in 1X PBST at a dilution of 1:500. Three 5-minute washes with PBST were performed after each antibody incubation. Sections were incubated in secondary antibodies for 1 hour at room temperature, and subsequently washed with 1X PBST. For multicolor immunofluorescence staining for primary and secondary of each antigen were performed in sequence. Sections were either mounted with antifade mounting medium with DAPI (Fisher Scientific; H1200) or counterstained with 2 μg/ml DAPI in 1X PBST for 30 mins at room temperature and then mounted in Antifade Gold mounting medium.