Clonal Expansions of CD8(+) T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

Cyktor, Joshua C.; Carruthers, Bridget; Beamer, Gillian L.; Turner, Joanne

Isolated lung cells or MLN were suspended in deficient RPMI (Mediatech, Manassas, VA) supplemented with 0.1% sodium azide (Sigma-Aldrich). Surface targets were detected as previously described. Specific antibodies and isotype controls were purchased from BD Biosciences: PerCP-Cy5.5 anti-CD3e (145-2C11), allophycocyanin-Cy7 anti-CD4 + (GK1.5), PE-Cy7 anti-CD8 + (53-6.7), PerCP-Cy5.5 anti-CD8 + (53-6.7), PE-Cy7 anti-IFN-c (XMG1.2), PE anti-PD-1 (J43), FITC anti-CD122 (TM-Beta 1), and FITC Vb screening kit. PE anti-Tim-3 (HAVCR2) antibody was purchased from eBiosciences. Cytokine levels were determined according to the manufacturer's instructions for intracellular cytokine staining (Cytofix/Cytoperm fixation/permeabilization solution kit with BD GolgiStop, BD Biosciences), following a 4 hr incubation with 1 mg/mL anti-CD3 (145-2C11) and 0.1 mg/ mL anti-CD28 (37.51). Samples were read using an LSRII flow