Figure 6.

Allogeneic AM reconstitution induces lung-associated antigen specific autoimmunity. (A) Elicitation of serum Ab to Kα1T and Col V at 16 wk post AM transfer was measured by ELISA (33). (B) Accumulation of Kα1T and Col V specific T cells was evaluated by IL-17A and IFNγ ELISPOT assays (BD Biosciences). Single cell suspension was prepared from lungs by digestion with Liberase TL (300μg/mL, Roche Life Science) and DNaseI (5U/mL, Sigma-Aldrich) at 37°C for 25 min. Lung leukocytes were isolated from the single cell suspension by Ficoll-Paque (Sigma-Aldrich) gradient centrifugation. One million lung leukocytes were added per well and were supplemented with 5×10⁴ C57BL/6 splenocytes (irradiated at 30 Gy) as antigen presenting cells. The cells were stimulated with Col V or Kα1T protein at 1μg/mL and cytokine producing spots were enumerated. Individual serum titer from five mice is plotted (in A) and stimulation of T cells (in B) is expressed as mean±SEM (n=5). Multiple t-tests were applied and statistical significance was determined using Holm-Sidak method (p value indicated).