Measurement of DSA

As described previously, DSA levels were determined by flow cytometric crossmatching using donor splenocytes or PBMCs, incubated with recipient serum from serial blood draws (26). DSA titer measurement was made on serum samples which were stored at –80°C and batched for analysis. For IgG measurement, samples were serially diluted (1:50) in each run, while for IgM measurements, neat sera were used. No serum, ‘naïve’ (pre-sensitization) as well as ‘peak’ (2 weeks following second skin transplantation) sensitization samples were run as negative and positive controls. Donor PBMCs or splenocytes were incubated with recipient serum, washed, and then stained with FITC-labeled anti-monkey IgG (Sera Care, 5210-0216), or FITC-labelled anti-monkey IgM (Sera Care, 5230-0423), anti-CD20 mAb (2H7), anti-CD3 mAb (SP34–2) (both BD Bioscience), and Live/Dead Fixable Blue staining (Life Technologies, Carlsbad, CA). The mean fluorescence intensity (MFI) of anti-monkey IgG or IgM on T or B cells was measured on BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software version 9 or 10 (Tree Star). Results were expressed as either MFI, or MFI fold-change from the pre-sensitized (naïve) time point. A MFI value lower than the neat naive sample was considered negative.