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

Single lung cell suspensions were adhered to sterile tissue culture dishes for 1 hr at 37uC. Non-adherent cells were washed and removed from the plates. CD4 + and CD8 + T cells were obtained from the non-adherent cell fraction by magnetic cell separation (BD IMAG anti-CD4 + particles GK1.5, anti-CD8 + particles 53-6.7) and either placed directly into TRIzol reagent (Invitrogen, Grand Island, NY), homogenized, and frozen at 280uC or used for culture as described below. CD8 + Vb8 + T cells were obtained from the CD4 neg fraction after treatment with anti-CD4 magnetic beads (BD), then stained with PE anti-Vb8 (eBioscience) and purified using anti-PE magnetic particles (BD). Purity of all CD4 + and CD8 + T cell populations was determined to be greater than 90% for all experiments by flow cytometry using an LSRII flow cytometer (BD Biosciences, San Jose, CA).