Identification and isolation of CD4⁺ T cells that proliferated specifically in response to the L33 peptide. PBMCs drawn from an HPA-1a–alloimmunized woman were stimulated in vitro with L33 peptide for weeks in tissue culture, then labeled with CFSE dye, and stimulated again with L33 peptide or Lol P1 control peptide in parallel cultures. After 13 days, the cultures were assayed for proliferation by flow cytometry. A sort gate was set to define an area for optimal enrichment of HPA-1a–specific proliferating CD4⁺ T cells by FACS. CD3⁺CD4⁺ lymphocytes that underwent enhanced proliferation in response to L33 peptide (1.4%; left) represented a considerably enriched population compared with the control culture (0.3%; right). The displays are biexponential (see “Cell culture and generation of HPA-1a–specific T-cell lines”).