Fig. 4. CALR-mutant-specific T-cell responses in different T-cell compartments.

a PBMCs from healthy individuals were enriched for CD4⁺ T_mem with MACS. Enriched cells, CD4⁺ T_mem-depleted cells, and unsorted cells were analyzed in a standard IFN-γ ELISPOT. (Middle) The number of spots per T-cell fraction and (top) a representative photograph of wells stimulated with either CALRLong4 or a negative scrambled peptide. (Bottom) Purity analyses of CD3⁺ gated cells in corresponding cell fractions. ELISPOT assays were performed in triplicate. b PBMCs from healthy individuals were enriched for T_naive cells with MACS. Enriched cells, T_naive-depleted cells, and unsorted cells were analyzed in a standard IFN-γ ELISPOT. (Middle) The number of spots per T-cell fraction; (top) a representative photograph of wells stimulated with either CALRLong4 or a negative scrambled peptide. (Bottom) Purity analyses of CD3⁺ gated cells in corresponding cell fractions. ELISPOT assays were performed in triplicates. c PBMCs from healthy individuals were enriched for T effector memory cells (T_EM) (CD3⁺, CD45RA^-, CD62L⁻) with FACS, and analyzed in an ex vivo IFN-γ ELISPOT. (Left) The number of spot-forming cells and a representative photograph of wells stimulated with either CALRLong4 or a negative scrambled peptide. (Right) FACS plots of unsorted PBMCs and T_EM-enriched fractions show the purity of T_EM-enriched fractions in CD3⁺ gated cells. Error bars display standard error of the mean