Fig. 4. Lung Zbtb7a induction in response to MHC ligations and in post-LTx BOS patients is localized to AMs.

(A) Representative reverse transcription PCR analysis on total RNA isolated from BAL isolated AM (CD45+, CD11c+ and Siglec-F+) (lanes: 1, 4, 7 and 10), non-AM BAL cells (CD45+, CD11c- and Siglec-F-) (lanes: 2, 5, 8 and 11) and alveolar respiratory epithelial cells (REC, lanes: 3, 6, 9 and 12) on day seven from si-RNA lentivirus transduced and Ab-treated mice (n=6). (B) Freshly isolated human and mouse AMs were cultured at 1×10⁶ cells per well, as previously described (36). Ligation of surface MHC class I was done by addition of anti-H-2K^b (clone AF6-88.5.5.3, IgG2a) and anti-HLA (W6/32, IgG2a) to mouse and human AMs, respectively. For ligation of MHC class II, anti-I-A/I-E (clone M5/114, IgG2b) was used. Responses to MHC ligation were compared with those of isotype controls (C.1.18.4, IgG2a or LTF-2, IgG2b). Ligation of keratin was also evaluated by addition of anti-Keratin (AE3, IgG1). The final concentration of Abs were 100 µg/mL and cells were cultured at 37°C at 6% CO₂ for 4 h before mRNA analysis. Induction of Zbtb7a was normalized to that of Actb and presented as relative expression compared to untreated AMs. Data are presented as mean±SEM, two-tailed unpaired t-tests are applied, significance (p<0.05) is marked (*) and p values are indicated. (C) BAL cells from five BOS⁺ LTxRs (P-1 through P-5) and four BOS-free LTxRs (P-6 through P-9) with stable lung function were fractionated into AM and non-AM as described earlier. The lung biopsy specimens were obtained on same day of BAL fluid collection. Fold induction in ZBTB7A expression compared to the peripheral blood leukocyte for individual LTxRs is plotted as heat map.