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. 2022 Oct 6;108(6):1530–1543. doi: 10.3324/haematol.2022.280685

Figure 6.

Figure 6.

Unsupervised analysis of CD8+Dextramer+ T lymphocytes reveals earlier maturation in donor-restricted T cells. (A) Manual gating analysis for the distribution of differentiation T-cell subsets in CD8+Dextramer+ T lymphocytes restricted for shared or donor-specific HLA. Differentiation T-cell subsets were defined according to the expression profile of CD45RA, CD62L and CD95 markers (left panel) or to the combination of CD28 and CD27 (right panel). TN (T-naïve): CD45RA+CD62L+CD95–; TSCM (T stem cell memory): CD45RA+CD62L+CD95+; TCM (T central memory): CD45RA–CD62L+; TEM (T effector memory): CD45RA–CD62L–; TEMRA (T effector memory RA): CD45RA+CD62L–. (B) tSNE map (left panel) and Flow-SOM metacluster overlay (middle panel) of CD8+Dextramer+ T cells from all the concatenated samples. The right panel shows the different distribution of Dextramer+ T lymphocytes restricted for donor-specific (blue) or shared (red) HLA molecules. (C) Frequency of metaclusters expressed as the percentage of cells in each metacluster for each type of HLA restriction among total CD8+Dextramer+ T lymphocytes. Shared subpopulations (n=29), donor-specific subpopulations (n=26). (D) Percentage of events positive for the indicated markers in the metaclusters enriched in CD8+Dextramer+ T cells restricted for donor-specific (blue, metaclusters n. 9, 15, 20 and 21) or shared (red, metaclusters n. 1, 3, 8, and 11) HLA alleles. (E) Heatmap for the 25 Flow-SOM metaclusters; the ratio of fluorescence intensity of each marker with respect to the maximum is indicated in the color-legend. Blue and red rectangles highlight the clusters significantly enriched in CD8+Dextramer+ T cells restricted for donor-specific or shared alleles, respectively. (F) The frequency of each metacluster over time expressed as the percentage of events in each metacluster among total CD8+Dextramer+ T lymphocytes. Among the cytomegalovirus (CMV)-specific T-cell populations evaluated, three were obtained at 30 days after hematopoietic stem cell transplantation, 11 at day +60, 16 at day +90, 12 at day +180 and 13 at day +365. Red, blue and black contours highlight metaclusters enriched in shared or donor-restricted CMV-specific T cells or common metaclusters, respectively. The analyses in (A), (C) and (D) were conducted using two-way analysis of variance. *P<0.05; **P<0.01; ***P<0.001. DEX: Dextramer; tSNE: t-distributed stochastic neighbor embedding; HSCT: hematopoietic stem cell transplantation.