T-cell responses differentiate nonaggregated from heat-aggregated rhEPO. (A) T cells from HLA-typed HX575-naïve healthy donors (n = 24) were primed with either nonstressed or heat-stressed HX575, and restimulated with either nonstressed or heat-stressed HX575, respectively (for individual SI data and HLA-haplotypes, see supplemental Table 1). Donors whose T cells responded to heat-stressed HX575 were categorized as “responders” (closed red circles), whereas those donors whose T cells did not respond to heat-stressed HX575 were categorized as “nonresponders” (open red circles). Two individuals showed responses to nonstressed rhEPO, possibly due to epitope spreading. (B) Representative experiment showing T-cell responses to heat-stressed HX575 (closed red circles), nonstressed HX575 (open blue circles), and TT (green squares). (C) Specificity of anti-HX575 T-cell responses was demonstrated by priming PBMCs from HX575-naïve healthy donors with either TT or heat-stressed HX575, and restimulation with either nonstressed, heat-stressed HX575, or TT.