Cd8α percp

T cell proliferation was evaluated by CellTrace CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester) cell proliferation kit (Invitrogen, Carlsbad, CA, United States). Briefly, single-cell suspensions of WT rat spleens were prepared and stained with CD3-APC (Clone: 1F4) for 40 min at 4°C. CD3 + T cells were isolated by flow cytometry sorting (BD LSRII; BD Biosciences) and were then labeled with 5 mM CFSE. The labeled T cells were cultured in replicate wells with or without previously plated BMSCs at a 20:1 ratio and were stimulated with α-CD3 (0.5 mg/mL) and α-CD28 (1 mg/mL) mAbs (EBioscience, San Diego, CA, United States) for 96 h. Cells were then stained for surface marker expression with CD4-PE (Clone: OX-38) and CD8α-PerCP (Clone: OX-8) antibodies (BD Biosciences) and incubated 40 min at 4°C. T cell proliferation was detected by flow cytometry.

Human blood was obtained from healthy volunteers recruited after obtaining informed written consent in accordance with QIMR Berghofer Medical Research Institute Human Research Ethics Guidelines and approved by the QIMR ethics committee, ethics approval number P158. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinised blood by Ficoll-Paque PLUS (GE Healthcare, Little Chalfont, UK) density centrifugation and labelled with CD8α-PerCP, CD4-PE Cy7, CD3-APC, CCR7-Alexa Fluor700, and CD45RA-FITC (BD Biosciences, San Jose, CA) antibodies and subsequently sorted for CD4⁺ naïve (CD45RA⁺ CCR7⁺ CD27⁺) and memory (CD45RA⁻) cells using the FACS Aria (BD Biosciences) cell sorter.

Spleen, lung, and MLN single cell suspensions from naïve and infected mice were stained with conjugated monoclonal antibodies targeting murine CD3, CD8, CD4, CD314, CD11c, CD11b, MHCII, F4/80, and B220 for 30 min at 4°C and analysed on a BD-LSRII (BD-USA). The following antibodies were purchased from BD Pharmingen: CD8α-PERCP and CD8α-FITC (53–6.7), CD4α-FITC (RM4-4), CD3-APC (145-2C11), F4.80-PE (T45-2342) CD314-PE (CX5), CD11c-APC (HL3), MHCII-PERCP (M5/114), CD11b-FITC (M1/70), and CD45r-PERCP (RAB3-6B2). Results were analysed using Flowjo software version 7 (Flowjo; Ashland, USA).