Supplementary Data
CTLA-4-mediated regulatory phenotype of T cells intolerant lung recipients
Karine Botturi, Yannick Lacoeuille, Pascal Thomas, Stéphanie Boniface, Martine Reynaud-Gaubert, Antoine Magnan.
Material and methods
Study design
Blood samples were collected in consecutive LTR included from July 2005 to April 2007. To avoid bias related to perioperative complications, LTR transplanted for less than 6 month were not included. This project was approved by the Sud-Méditerannée II Ethic Committee and written informed consent was obtained from all the patients.
LTR were regularly followed for clinical, functional and radiological evaluation. Spirometry was performed with an 830-L whole-body plethysmograph (Masterlab Jaeger, Wurzburg, Germany). Lung function results were expressed as percentages of predicted values and of baseline values. LTR were considered as healthy when they displayed normal and stable respiratory function (FEV1 ³ 80% of baseline) and no clinical and radiological abnormality. Bronchiolitis obliterans syndrome was diagnosed on a progressive bronchial obstruction according to guidelines (E1).
Patients received cyclosporine (whole blood levels adjusted between 250 and 300 ng/ml), and azathioprine (1 mg/kg per day, adjusted to white blood cell count above 4000/mm3). Patients were switched from cyclosporine to tacrolimus (adjusted to maintain whole blood trough levels between 10 and 15 ng/ml) and from azathioprine to mycophenolate mofetil (500 mg to 1g, bid, according to AUC values) after the first AR or in case of BOS (E1). Azithromycin (250 mg/d) was added after the second AR episode occurring within 3 months after the first episode of treated AR, and as soon as the diagnosis of BOS was made.
Bacterial stimulation
To assess the characteristics of DC/T cell interactions in presence of microbial compounds, lysed Pseudomonas aeruginosa was added to co-cultures. Pseudomonas aeruginosa was chosen as a source of recall antigens for the majority of patients displaying CF or bronchectasis. We used a Pseudomonas aeruginosa KK1 mucoïd strain (a kind gift from S. De Bentzamm, CNRS, Marseille) isolated from a CF patient, witch was cultured in Luria-Bertani broth overnight at 37°C. Culture supernatants were then spin down, filtered over a 0.45µm Durapore membrane filter (Millipore, Marlborough, MA), and conserved at 4°C. DC were incubated with 10 µl of Pseudomonas aeruginosa culture supernatant on day 2. After 12h of incubation, DC were co-cultured with autologous lymphocytes.
References
E1. Estenne M, Maurer JR, Boehler A, Egan JJ, Frost A, Hertz M, Mallory GB, Snell GI, Yousem S. Bronchiolitis obliterans syndrome 2001: An update of the diagnostic criteria. J Heart Lung Transplant 2002;21:297-310.