Following stimulation and processing, 5 μl of appropriately Cell Cycle inhibitor diluted IFN-γ Alexa488 (BD), CD3 PerCP·Cy5.5 (BD), CD28 PE-Cy7 (BD), TNF-α V450 (BD), IL-2 Alexa488 (BD), CD45 V500 (BD) and PE-conjugated monoclonal antibodies to CD40L, CD152,

CD137, CD134 or isotype control were added for 15 min in the dark at room temperature. Cells were washed and events acquired and analysed as described above. Aliquots of whole blood were incubated with 10−6 M methylprednisolone for 18 h then stimulated for cytokine production and analysed as reported previously [8]. Statistical analysis was performed using the Kruskal–Wallis test and post-hoc analysis using Mann–Whitney and Spearman’s rho correlation tests using spss software and differences between groups of P < 0·05 were considered significant. Corrections for multiple comparisons were not performed. There was no significant difference

in the absolute lymphocyte counts for controls and transplant patients [1·5 (1·4–1·9), 1·6 (1·3–2·1), 1·6 (1·3–2·2) × 109/l, check details median and range for controls, stable patients and patients with BOS, respectively, P > 0·05]. There was no change in the percentage of CD4 or CD8 T cells between controls or transplant groups (61 ± 11·7, 62 ± 12·8, 60 ± 11·9 CD4 and 39 ± 6·7, 38 ± 6·8, 39 ± 8·1 CD8 T cells for controls, stable transplant and BOS patients, respectively). The percentage of CD28null/CD4+ T cells in stable transplant patients was decreased significantly compared to control subjects (Fig. 1). In BOS, there were significant increases in the percentage GBA3 of both CD28null/CD4+

and CD28null/CD8+ T cells compared with both controls and stable transplant patients (Fig. 1). CD28null/CD8+ T cells were increased significantly when compared to CD28null/CD4+ in patients with BOS (Fig. 1). There was a significant increase in the percentage of both CD28null/CD4+ and CD28null/CD8+ T cells expressing perforin in stable transplant patients and in patients with BOS compared with controls (Fig. 2a). A similar increase was noted in the CD28+ subgroup (0·2%, 1·0% and 1·1%; and 0·3%, 2·3% and 2·5% CD28+/perforin+/CD4+ and CD28+/perforin+/CD8+ for controls, stable patients and patients with BOS, respectively) (all P < 0·05). There was an increase in the percentage of both CD28null/CD4+ and CD28null/CD8+ T cells expressing granzyme B (GB) in patients with BOS compared with controls (Fig. 2b). For CD4+ T cells expressing GB, the increase was significantly greater in BOS patients compared with stable transplant patients and controls, and in stable transplant patients compared with controls (Fig. 2b). The percentage of CD28null/GB+/CD8+ T cells was higher in all groups compared to the CD4+ subset (Fig. 2b).