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

For ELISpot, bone marrow-derived dendritic cells (BMDCs) were obtained from the tibiae and femora of age and sex matched non-infected wild-type or IL-10 2/2 C57BL/6 or CBA/J mice. Cells were differentiated into dendritic cells using complete DMEM supplemented with 10% conditioned media derived from GM-EL4 cells, a GM-CSF-producing clone kindly provided by Arthur A. Hurwitz (NCI). 2610 6 bone marrow cells were plated at 37uC in 1 ml of GM-EL4 conditioned media in sterile 24-well tissue culture plates. GM-EL4 conditioned media was replaced on days 2, 4 and 6. 3610 4 BMDCs were infected overnight with Mtb Erdman at an MOI of 1:1 then fixed in 2% paraformaldehyde (CBA/J IL-10 2/2 BMDCs were used unfixed). Infected BMDCs were cultured with 2610 5 CD8 + T cells or CD8 neg T cells for 72 hr at 37uC in media containing either tissue culture media alone or 10 mg/mL anti-CD3 (145-2C11) and 1 mg/mL anti-CD28 (37.51). ELISpot reagents were obtained from eBioscience (e, f) 24 hr prior to necropsy mice were injected with BrdU, and lung cells were analyzed for expression of BrdU + CD4 + or CD8 + T cells. (g, h) Absolute numbers of CD4 + or CD8 + T cells in wild-type or IL-10 2/2 CBA/J mice as determined by flow cytometry. Results representative of at least three independent experiments with 5 mice per group, per timepoint. * p,0.05, ** p,0.01, *** p,0.001 as obtained by Student's t test. (b, h) + p,0.05, ++ p,0.01, +++ p,0.001 as obtained by twoway analysis of variance comparing day 90 to day 150 post-infection. doi:10.1371/journal.pone.0058612.g001