Fig. 6. CALR-mutant-specific responses in FACS-sorted T_mem, but not in T_naive.

a PBMCs from a healthy individual were enriched for T_mem (CD3⁺, CD62L⁺, CD45RA⁻ or CD3⁺, CD62L⁻, CD45RA⁺ or CD3⁺, CD62L⁻, CD45RA⁻) and T_naive (CD3⁺, CD62L⁺, CD45RA⁺). Cells were analyzed in an ex vivo IFN-γ ELISPOT to determine the numbers of spot-forming cells (left) in each cell fraction that responded to CALRLong4. (Middle) photographs of wells. (Right) Analysis of purity of the different T-cell fractions in CD3⁺ gated cells. b PBMCs isolated from freshly drawn blood from an older healthy individual with a clear CALRLong4-specific response were rested overnight in an incubator. Cells were sorted into T_mem and T_naive, and analyzed for peptide-specific T-cell responses in an ex vivo IFN-γ ELISPOT assay. The number of spot-forming cells was determined in each cell fraction for cells stimulated with (left) CALRLong4 or (middle) CALRLong36. (Right) FACS purity analysis results. Due to difficulties in separating T_naive (CD3⁺, CD62L⁺, CD45RA⁺) and T_EMRA (CD3⁺, CD62L^-, CD45RA⁺) cells, the sorting gates were set differently from those set in a, to avoid contamination of T_naive and T_EM enrichments. c T_mem isolated in b were intracellularly stained after one week of in vitro stimulation with the CALRLong4 peptide. Panel shows the cytokines produced by (left) CD4⁺ T-cells and (right) CD8⁺ T-cells upon stimulation with CALRLong4 or a negative control scrambled peptide. Error bars display standard error of the mean