TISEs were purified from plasma using anti-donor HLA class I–specific antibodies in patients A–E (n = 5). Representative results in patient E are shown. (A) C-peptide production in the peritransplant period is shown. The patient had acute rejection of the donor islets, with rise in alloantibody titer and drop in C-peptide by day 5. (B) As confirmation that the isolated microvesicles contained exosomes, samples tested on Western blot showed exosome markers CD63 and flotillin 1, but absence of cellular/apoptotic body marker cytochrome c. (C) NanoSight fluorescence for donor-specific HLA-A2 signal demonstrated TISEs on post-transplant day 2, but not in pretransplant sample. Donor islet culture exosomes are used as positive control (P = 0.008; 1 of 5 shown). (D) FXYD2 coexpression in HLA-bound exosomes showed high signal in donor islet culture and recipient post-transplant day 2 samples, but not the pretransplant sample. (E) Western blot of HLA-A2–bound exosomes showed islet endocrine hormone protein and FXYD2 expression in post-transplant day 2 sample but not pretransplant sample (P = 0.008). Islet graft obtained from xenoislet experiments served as positive tissue control. TSG101 is an exosome marker. (F) RT-PCR showed islet endocrine hormones and FXYD2-γa in post-transplant 20-minute and 2-day samples but not in pretransplant sample (P = 0.008). (G) Postrejection analysis (6 weeks) on NanoSight for HLA-A2 in recipient plasma (top) and for FXYD2 in HLA-A2–bound fraction (bottom) were both negative. (H) Western blot analysis of HLA-A2–bound fractions from recipient plasma after rejection showed absence of insulin and FXYD2. Normoglycemic HLA-A2–positive blood donor (HLA-A2–positive human plasma) is shown to validate that the bead-bound exosomes were not nonspecifically binding islet exosomes (P = 0.008; 1 of 5 shown).