γδ tcr

For cellular phenotyping, cells were washed, incubated with anti-CD16 to block Fc receptors and stained with VIVID (Invitrogen, Carlsbad, CA) to exclude dead cells. Cells were surface stained in cocktails with different combinations of fluorescent dye-conjugated monoclonal antibodies (mAbs) depending on the panel that included antibodies raised against CD3ε, CD4, CD8, CD19, CD11b, Ly6C, Ly6G, F4/80, NKp46, CD127 (IL-7Rα), CCR6 and γδTCR (BD Biosciences, San Jose, CA). For intracellular cytokine staining (ICS) experiments, single cell suspensions of spleen, MLN, small intestinal IELs (sIEL) and sLPLs, colonic IELs (cIEL) and cLPLs were re-stimulated for 4 hours with 40ng/mL phorbol-12-myristate-13-acetate (PMA) and 2μg/mL ionomycin (Millipore, Billerica, MA, USA) in the presence of brefeldin A (BD Biosciences, San Jose, CA). Cells were surface stained as above then were fixed and permeabilized using the eBioscience Foxp3 buffer system and stained with antibodies against mouse IL-10, IFN-γ, IL-17, IL-22, RORγt and/or Foxp3 (BD Biosciences, San Jose, CA). Cells were acquired on a BD LSRII. Data were analyzed with FlowJo software (Tree Star, Ashland, OR).

Lymphocyte phenotypes in BM and PB were serially investigated by flow cytometry during therapy using antibodies against CD3, CD4, CD8, CD16, CD25, CD45RO, CD45RA, CD56, CD127, CD197, CD279, CD335 (NKp46), CD337 (NKp30), and γδTCR (BD Pharmingen), as previously described.^{21 (link)} Gating strategies are depicted in supplemental Figure 5. A complete blood count on the same day as flow analysis provided percentage of lymphocytes and an absolute lymphocyte count to calculate the absolute number of cells.

Cells, isolated from whole lung or MPs and EXs as described above, were stained with the following FITC- or PE- or PerCP-Cy5.5 labeling antibodies to CD3, CD4, CD8, NK1.1, γδTCR, CD69 and Annexin V (BD Pharmigen) and analyzed by Attune® Acoustic Focusing Cytometer (Thermo Scientific-Applied Biosystems, Foster City, CA, USA). Isotype control was used for all the samples. MPs and EXs were defined as Annexin V positive cells. To measure the size of MPs and EXs, we used calibration beads from 0.1 to 3 µm in diameter, as previously described. ^{48 (link), 49 (link)}