After overnight incubation with polyclonal antibodies against FOXP3 (sc-21072; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-human IL-17 monoclonal antibodies (R&D Systems Inc., Minneapolis, MN), the samples were incubated with the secondary antibodies, biotinylated with anti-IgG for 20 min and then incubated with a streptavidin–peroxidase complex (Vector, Peterborough, UK) for 1 hr. This was followed by incubation with 3,3’-diaminobenzidine (Dako, Glostrup, Denmark). The sections were counterstained with haematoxylin, and samples were photographed with an Olympus photomicroscope (Tokyo, Japan). The LDE225 research buy positivity for each immunohistochemistry stain was examined in a blind fashion relative
to the clinical information. Analysis was performed by counting the total number of infiltrating cells that express FOXP3 or IL-17 in the cortex. The area of cortex was measured with a loupe and the data were expressed as
the number of cells/mm2. The counting of the FOXP3+ and IL-17+ cells was performed by HistoQuest Experiment (TissueQuest Software, TissueGenostics, Vienna, Austria). A pathologist blinded to the results of the HistoQuest Experiment, manually counted the cell number. The FOXP3+ cell and IL-17+ cell numbers counted by pathologist and HistoQuest Experiment were highly correlated (r = 0·901, P = 0·00) AT9283 cell line and the result did not change the classification of the patient. Indirect immunofluorescence staining was Protein kinase N1 performed using monoclonal antibodies against complement protein C4d (Biogenesis, Poole, UK) in 48 (68%) biopsies. In 23 (32%) biopsies where no C4d staining was performed on frozen sections, sections were obtained from paraffin blocks and stained for immunohistochemistry with C4d using a rabbit polyclonal antibody (Biogenesis, Poole, UK). C4d positivity was defined as diffuse (> 50%) and linear staining of peritubular capillaries. Figure 1(a,b) shows representative stains of FOXP3 and IL-17. The cell numbers of the FOXP3+ cell and IL-17+ cell infiltrations were 11·6 ± 12·2 cells/mm2 and 5·6 ±
8·0 cells/mm2, respectively. The average value of the ratio between FOXP3+ cell and IL-17+ cell (FOXP3/IL-17) was 5·6 ± 8·2. We used log transformation to correct data skewness for the FOXP3/IL-17 ratio. When log transformation of the FOXP3/IL-17 ratio (Log FOXP3/IL-17) is 0·45, it conferred the highest sensitivity (0·713) and specificity (0·724) in the prediction of allograft failure by receiver operating characteristic analysis. Therefore, when Log FOXP3/IL-17 was > 0·45, the biopsy was considered as the FOXP3 high group (n = 30) and when it was < 0·45, the biopsy was considered as the IL-17 high group (n = 26). Only the first biopsy tissues were considered in the evaluation of the clinical outcome after ATCMR and the long-term allograft survival. Clinical information was collected by retrospective chart review.