Cd3 percp cy5

Peripheral blood mononuclear cell (PBMC) was resuspended in PBS containing 0.5% BSA and stained with commercially available antibodies (CD8-FITC, CD38-PE, CD3-PerCPCy5, HLADR-APC, all from BD Biosciences) and analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences). After gating the lymphocyte population in the FSC/SSC-plot, the following cell populations were defined: T-cells (CD3 +), CD8 + T-cells (CD3 + /CD8 +) and activated CD8 + T cells (CD3 + /CD8 + /CD38 + /HLADR +). PBMC were collected at baseline (week 0) and week 24 and stained with anti-CD3/CD8 to define CD8 + T cells and counterstained with anti-CD38/HLADR to detect the frequency of activated CD8 + T cells.

Cells in BAL fluid were stained with CD3 FITC, CD11c PercP, Siglec F Alexa 647, CD11b PE-Cy7, viability dye APC Cy7 (all BD Biosciences, San Jose, CA, USA), Ly6G Alexa700 (Biolegend, San Diego, CA, USA), MHCII PE, and CD45 PE-eFluor610 (eBiosciences, San Diego, CA, USA) in the presence of Fc blocker (CD16/CD32, eBiosciences). Single cell suspensions from lungs were stained with CD4 FITC, CD45 PerCP-Cy5.5 (eBiosciences), GATA-3 Alexa 647, and viability dye APC-Cy7 (BD Biosciences). The following markers were used for the analysis of ILCs in lung tissue: Lineage (Lin) markers including CD3e, CD19, GR1, B220, Ter119, FcaR1 (all FITC, Biolegend), CD45 Alexa700, CD90 PE, ST2 Brilliant Violet 421 (Biolegend), CD49b PE-Cy7 (eBiosciences) and CD3 Percp-Cy5.5 (BD biosciences). Mediastinal lymph nodes (mLN) cells were stained with CD45 PerCP-Cy5.5, CD4 FITC, GATA-3 Alexa 647 (BD Biosciences) and IL-4 APC (Biolegend). For intracellular/intranuclear staining, cells were permeabilized and fixed using a FOXp3 Staining Buffer set (eBioscience) and subsequently stained with the appropriated markers. All appropriate Fluorescence Minus One (FMO) controls were used. Data were collected on a BD Biosciences Canto II flow cytometer or BD FACSAria™ III and analyzed using FlowJo software (Treestar, Palo Alto, CA, USA).

Anti-CD123 CAR T cells were harvested prior to and post lentiviral transduction and phenotypically analyzed. Cells were washed twice with 1× PBS supplemented with 2 mM EDTA, 2% FCS, and 5% sodium azide, resuspended in 100 µL, and stained with live dead aqua viability dye-V500 (1:500), CD3-PerCPCy5.5, CD8-APC, CD27-APC-ef780, CD45RO-PeCy7 (BD Biosciences) (1:100) for 30 min on ice, in the dark. Cells were washed twice and resuspended in a final volume of 200 µL prior to flow cytometric analysis. Unstained and fluorescence minus one (FMO) controls were used to identify gating boundaries.

Mixed leukocyte culture (MLC) was used to test the capacity of DCs while stimulating the allogeneic T cell proliferation (26 (link)). BTLA⁺ DCs and BTLA⁻ DCs were sorted by flow cytometry. PBMCs were isolated from eight active TB patients with a positive sputum smear and from eight volunteers, and stained with mouse anti-human antibody-fluorochrome cocktails, including Lin1 (BU15; eBioscience), mouse anti-human HLA-DR, CD11c, and CD123, BTLA-APC (MIH26; Biolegend), and BTLA-PE (J168-540, BD Pharmingen) monoclonal antibodies, followed by sorting on a FACS Aria II. CD3⁺ T cells were isolated from an allogenic donor using a human T cell isolation kit, according to the manufacturer's instructions (StemCell Biotech, Vancouver, Canada). Isolated cells, with >97% purity, were used for subsequent experiments. To evaluate the T cell proliferation, some of the purified CD3⁺ T cells were labeled with 20 μM CFSE (Beyotime), and 5 × 10⁴ cells/well were cultured with sorted BTLA⁺DCs or BTLA⁻DCs (5 × 10³ cells/well) in 96-well round-bottom plates for 7 days. T cells alone served as the negative control. The cells were harvested, stained with CD3-PerCp-Cy5.5 (BD Pharmingen) and CD4-APC (BD Biosciences) for 30 min at 4°C, acquired using a BD FACS-Canto II flow cytometer, and analyzed using the FlowJo software. The CFSE-low cells were quantified as a percentage of proliferating cells.

Production of IFNγ and TNFα by T, NKT-like and NK cells was determined on blood and BAL samples from all subjects as previously reported [11 (link)–13 (link)].
Briefly, one mL blood diluted 1:2 in RPMI and prepared BAL cells were stimulated with phorbol myristate (25 ng/mL) (Sigma, Sydney, Australia) and ionomycin (1 μg/mL) (Sigma). Brefeldin A (10 μg/mL) was added as a “Golgi block” (Sigma) and the tubes re-incubated in a humidified 5% CO₂/95% air atmosphere at 37°C for 16 h. Aliquots of blood and BAL were added to FACS tubes (BD) and treated with FACSLyse and FACSPerm as above and five μL of appropriately diluted anti- IFNγ FITC (BD), CD3 perCP.CY5.5 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), TNFα V450 (BD) and CD45 V500 (BD) monoclonal antibodies were added for 15 min in the dark at room temperature. Two mL of 0.5% bovine serum albumin (Sigma) / Isoflow (Beckman Coulter) was then added and the tubes centrifuged at 300 ×g for 5 min. After decanting, cells were analyzed as above.

To systematically investigate the in vivo antitumour immune responses against mimic distant tumours, the tumours were harvested and treated with the tissue dissociation kit (Miltenyi Biotec, Germany) to produce a single-cell suspension according to the specified procedures. The harvested cells were further stained with several fluorochrome-conjugated antibodies: CD45-FITC (BD, Catalog: 561088), CD3-PerCP-Cy5.5 (BD, Catalog: 551163), CD4-BV510 (BD, Catalog: 563106), CD8-BV421 (BD, Catalog: 563898), NKp46-APC (Biolegend, Catalog: 137608), B220-PE-Cy7 (BD, Catalog: 552772) and Foxp3-PE (eBioscience, Catalog: 12–4771) and then analysed by FCM. For the analysis of the memory T cells, spleen cells of mice were harvested and stained with anti-CD3-FITC (Biolegend, Catalog: 100306), anti-CD4-PE-Cy7 (eBioscience, Catalog: 25–0041), anti-CD8-PerCP-Cy5.5 (eBioscience, Catalog: 45–0081), anti-CD44-PE (eBioscience, Catalog: 12–0441) and anti-CD62L-APC (eBioscience, Catalog: 17–0621) antibodies and then analysed by FCM. All antibodies were diluted ~100 times.