GPR56 increases after non-self recognition by allo-reactive T cells after allo-HCT. (A) Percentage of CD8⁺ T_EM in BM (left) and percentage of GPR56⁺ on CD8⁺ T_EM (right). Numbers below plots show the median percentage. Numbers between groups of patients without (no-Allo), before (pre-Allo), and after (post-Allo) allo-HCT indicate the P values (unpaired Wilcoxon test). Note that the total fraction of CD8⁺ T_EM in BM does not significantly differ between the 3 groups. Box plots showing medians and quartiles, each dot represents an individual sample. (B) Representative FACS plots showing CD27 and GPR56 expression on CD3⁺CD8⁺ cells in healthy BM (left), in a patient with low GPR56 upregulation (patient 1 [middle]), and a patient with a dominant GPR56⁺CD27^– fraction (patient 2 [right]). (C) Time course of percentage of GPR56^–CD27⁺ (blue), GPR56⁺CD27⁺ (purple), and GPR56⁺CD27^– (pink) in the CD8⁺ compartment in patients with CR after allo-HCT. Numbers above the box plots indicate the median percentages. Box plots represent medians, quartiles, and outliers. (D) Proposed model of CD8⁺ T-cell phenotype switch after allo-HCT. (E) Percentage of GPR56⁺ on CD8⁺ T_EMRA in recipients with CMV IgG negativity (left) and positivity (right). Numbers above box plots indicate the median percentages. Box plots represent medians, quartiles, and outliers. (F) Time course of the percentage of the indicated cell types in patient GXW165. As indicated, CMV status was positive for the recipient and negative for the donor (CMV R/D pos/neg); donor sex was male. The text below the x-axis provides clinical information on the course of the disease. Note the drop in chimerism, neutrophils, and GPR56 positivity accompanied by FACS MRD positivity around day +100. IS was reduced, and the patient developed skin GVHD requiring steroids, which was accompanied by a steady increase in GPR56⁺, re-establishment of full donor chimerism, and CR. (G) Schematic illustrating the experimental strategy for measuring IFN-γ production using ELISpot. GPR56⁺ and GPR56^– CD8⁺ T cells were sorted using FACS from PBMCs of 6 patients with AML in CR after allo-HCT. Sorted populations were then cocultured for 24 hours with primary AML blasts from initial diagnosis of the same patients. ELISpot assay was performed to detect and quantify IFN-γ production by the T cells in response to AML blasts. (H) Representative wells from the ELISpot assay after coculturing primary AML blasts with GPR56^– (orange) and GPR56⁺ (purple) CD8⁺ T cells for 24 hours. (I) ELISpot results showing the numbers of spots generated in the GPR56⁺ and GPR56^– T-cell fractions of 6 patients with AML in remission within 24 hours of contact with matched AML blasts. P value was calculated using paired Wilcoxon test. Individual points indicate biological replicates (mean across technical replicates) and connected points indicate fractions originating from the same sample. Chim, donor chimerism; IgG, immunoglobulin G; IS, under immunosuppression; MRD, minimal residual disease.