Btla pe

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.

Cells were thawed and cultured in AIM-V culture medium with 5% heat-inactivated human serum in 24-well plates at a concentration of 3–5 × 10⁶ cells/ml. Plates were incubated in a humidified incubator at 37°C, with 5% CO₂. After 48 hours of resting, cells were harvested and washed in FACS buffer before surface staining. For exhaustion profile analysis, cells were stained at 4°C for 30 min., washed, and, for tubes only intended for surface staining, resuspended in FACS buffer. For tubes intended for intracellular staining, cells were permeabilized using BD Bioscience Cytofix/CytopermTM Kit according to manufacturer's instructions. The following antibodies were used: CD4-PerCP, CD57-FITC, CD27-PE, CD56-PE-Cy7, CD28-APC, CD8-AmCyan, ICOS-PE, BTLA-PE, CTLA-4-APC, PD-1-PE-Cy7 (all from BD Bioscience, San Jose, CA, USA), Near Infra Red dead cell marker (Dako), LAG-3-FITC (LifeSpan Biosciences), TIM-3 (eBioscience), CD107a; PECy7-conjugated IFN-c; PerCP-conjugated CD8; APC-conjugated CD4, TNF-a; APCCy7-conjugated CD3. Cells were resuspended in FACS buffer prior to acquisition, and the cells were acquired using a BD FACSCanto II flow cytometer. A minimum of 100 000 events were recorded per sample. Analysis was performed with the BD FACSDiva Software (BD Bioscience,)

The following mouse anti-human antibodies were used for flow cytometry and cell sorting: CD11c-APC-eFluor780 (BU15, eBioscience), CD11c-PE-Cy7 (B-ly6, BD Pharmingen), Lin1-FITC (SK7, 3G8, SJ25C1, L27, eMfP9, NCAM16.2, BD Biosciences), Lin1-APC (UCHT1, HCD14, 3G8, HIB19, 2H7, HCD56, BioLegend), CD123-PerCP-CyTM5.5 (7G3, BD Biosciences), HLA-DR-APC-eFluor780 (LN3, eBioscience), HLA-DR-APC (G46-6, BD), CD86-FITC (FUN-1, BD Biosciences), CD80-FITC (2D10.4, eBioscience), CD83-FITC (HB15e, BD Biosciences), CCR7-FITC (150503, BD Biosciences), BTLA-APC (MIH26, Biolegend), BTLA-PE (J168-540, BD Pharmingen), CD3-PerCp-Cy5.5 (SP34-2, BD Biosciences), CD4-PerCP-Cy5.5 (RPA-T4, eBioscience), CD4-APC (L200, BD Biosciences), CD27-PE-Cy7 (0323, eBioscience), CD45RA-FITC (HI100, Biolegend), CD27-PE-Cy7 (0323, eBioscience), CD45RA-FITC (HI100, Biolegend), Foxp3-PE (PCH101, eBioscience), IFN-α-FITC (MMHA-11, InterferonSource), IL-12-FITC (MHCIL1201, Invitrogen), IL-4-FITC (MP4-25D2, Biolegend), and IFN-γ-APC-eFluor780 (45.B3, eBioscience).