Cd44 apc

Cell suspensions from iliac lymph nodes, blood and tumors were first incubated with 1µg/10⁶ cells of Fc block treatment (BD Pharmigen) and then stained for surface markers in FACS buffer (PBS + 1% FBS). For Treg staining on cells isolated from iliac lymph nodes: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD3 FITC, clone 145-2C11 (Biolegend); CD4 Pacific Blue, clone RM4-5 (Biolegend); Foxp3 APC, clone FJK-16S (eBioscience). For Treg staining on cells isolated from tumors: Live/Dead (Fixable Near-IR Dead Cell stain kit, Life Technologies); CD45 PerCP-Cyanine 5.5 (eBioscience); CD4 Pacific Blue, (Biolegend); CD8 Brilliant Violet 510, clone 53-6.7 (Biolegend); Foxp3 APC, (eBioscience). For effector memory staining on cells isolated from blood: Live/Dead (Fixable Near-IR); CD8 PerCP, (Biolegend); CD44 APC (Biolegend); CD62L FITC, clone MEL-14 (BD Pharmigen).

For splenic T cell sorting, magnetic bead-mediated positively selected CD4⁺ cells were stained with combinations of antibodies (0.5μg/mL per) to CD62L-PE (BioLegend, San Diego, CA) or CD62L-BB515 (BD Bioscience), CD44-APC (BioLegend, San Diego, CA), CD45Rb-APC/Cy7 (BioLegend, San Diego, CA), CD124 (1μg/mL, BD Biosciences, San Jose, CA), and CD25-BV421 (BD Biosciences, San Jose, CA). For Tregs, FoxP3-RFP reporter signal was used for FACS. Cells were washed twice in MACS buffer (Miltenyi Biotec, San Diego, CA) before sterile cell sorting using a FACSAria (BD Biosciences, San Jose, CA) with the support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Analysis of all FACS data was performed using FlowJo v10 (Tree Star, Inc., Ashland, OR).

To analyze cell-surface expression of typical MSC surface marker proteins, cells were detached, counted and labeled with the following anti-human antibody conjugates: CD44 - APC; CD73 - APC; CD90 - PE; CD14 - PerCp/Cy5.5; CD45 - PerCp/Cy5.5; CD31 - FITC; CD34 - FITC; CD19 - Pacific Blue; HLA-DR - Pacific Blue (all from Biolegend) and also CD105 PE (eBioscience, Inc., San Diego, CA, USA). The mouse isotype antibodies used as the respective controls were: Pacific Blue IgG1; Pacific Blue IgG2a; IgG1k PErCp/Cy5.5; IgG2a PerCp/Cy5.5; IgG1k PE; IgG1k APC and IgG1k FITC (all from Biolegend®, San Diego, CA, USA). A total of 10,000 labeled cells were acquired using a Gallios Flow cytometer (Beckman Coulter) and results analyzed with Kaluza software (Beckman Coulter, Inc., Carlsbad, CA, USA).

Surface markers on MSC-EVs were analyzed using Apogee A50-Micro flow cytometer (Apogee Flow Systems, UK). The following fluorochrome-conjugated antibodies against murine antigens were used according to the manufacturer’s protocols: CD29-APC (clone: HMβ1-1, Biolegend), CD44-APC (clone: IM7, Biolegend), CD81-APC (clone: Eat2, BD Bioscence), CD90-APC (clone: 30-H12, Biolegend), CD309-APC (clone: Avas12, Biolegend) and Sca-1-APC (clone: E13-161.7, Biolegend) as well as the following isotype controls: Armenian hamster IgG-APC (clone: HTK888, Biolegend), Rat IgG2a, κ-APC (clone: RTK2758, Biolegend) and rat IgG2b, κ-APC (clone: RTK4530, Biolegend). MSC-EVs were co-stained with SYTO RNA Select dye (ThermoFisher Scientific), which binds RNA molecules. Staining was conducted for 30 min in the dark at 4 °C. The obtained results were analyzed using Apogee Histogram software (Apogee Flow Systems). In order to confirm the presence of the indicated antigens on MSC-EVs, an ImageStream X Mark II imaging cytometer (Merck) was additionally used.

Harvested cells were stained for flow cytometry in PBS for 20 min at RT. The following antibodies were purchased from BioLegend (San Diego, United States) and used for flow cytometric analysis: CD8a-BV510 (cat: 100752, clone: 53-6.7, dilution 1:200), CD45.1-PacificBlue (cat: 110722, clone: A20, dilution 1:200), CD44-APC (cat: 559250, clone: IM7, dilution 1:100), CD11c-PerCP (cat: 117326, clone: N418, dilution 1:200), CD69-PeCy7 (cat: 104512, clone: H1.2F3, dilution 1:100), CD25-FITC (cat: 553072, clone: 3C7, dilution 1:100). The viability of cells was determined by using fixable near-IR dead cell staining (LifeTechnologies, Carlsbad, United States). Biotinylation was assessed by streptavidin-PE (BioLegend) staining for 10 min at RT. T-cell activation was determined by the expression of GFP expression under the control of the Nur77 promoter. Data was acquired on a Canto^TM flow cytometer (BD Bioscience, Allschwil, Switzerland) and analyzed using the FlowJo software (Treestar, Ashland, OR, USA) (see Supplementary Fig. 12 for gating strategy).

The T cell transfer colitis model has been described previously.¹⁸ (link) Briefly, for transfer of CD4⁺ naïve T cells, splenic CD4⁺ cells from CD4-Cre^+/TgMettl14^FL/FL conditional knockout mice and littermate control mice were isolated with CD4⁺ T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol. The isolated CD4⁺ T cells were then stained with CD4-FITC, CD25-PE, CD62L-BV421 and CD44-APC (BioLegend, San Diego, CA). Naïve T cells were fluorescence-activated cell sorter (FACS) sorted as CD4⁺, CD25^–, CD62L⁺, and CD44^– cells. Each recipient Rag1^–/– mice was injected with 5 × 10⁵ naïve T cells. For co-transfer of CD4⁺ naïve T cells and T_reg cells, naïve T cells were isolated as described previously from C57BL6-CD45.1 mice in which the CD45.1 marker can be used as a donor marker for naïve T cells. For isolation of T_reg cells, splenic CD4⁺ cells from Foxp3^YFP–CreMettl14^FL/FL conditional knockout mice and littermate control mice were isolated with CD4⁺ T cell isolation kit II (Miltenyi Biotec). The T cells were then stained with CD4-APC-Fire750 (BioLegend). T_reg cells were sorted as YFP⁺ CD4⁺ cells. Recipient Rag1^–/– mice were injected with naïve T cells and T_reg cells at a 2:1 ratio. The mice were then weighed every 3–4 days. Mice were scarified 7–8 weeks after T cell transfer.