Table 1.
Patient characteristics.
Figure 1.
Reconstitution of CD161-expressing T cells in patients after allo-SCT.
(A) Percentage of circulating CD161+ within CD4+ and CD161hi within CD8+ T cells in patients at 1 (n = 11, 20), 3 (n = 50, 71), 6 (n = 19, 24), and 12 (n = 19, 22) months after allo-SCT. (B) Absolute levels of circulating CD161+CD4+ and CD161hiCD8+ T cells in patients at 1 (n = 8, 20), 3 (n = 50, 71), 6 (n = 19, 24), and 12 (n = 19, 22) months after allo-SCT. (C) Absolute number of circulating CD161−CD4+ and CD161neg/lowCD8+ T cells in patients at 1 (n = 8, 20), 3 (n = 54, 69), 6 (n = 19, 24), and 12 (n = 19, 22) months after allo-SCT. (D) Correlation between the percentage of circulating CD161+CD4+ and CD4+ T cells (n = 58), and CD161hiCD8+ T cells and CD8+ T cells (n = 70) at 3 months after allo-SCT. Lines represent median value, grey areas represent the reference range of healthy controls (mean ± SD). Statistical analysis was performed using a One-way ANOVA followed by a Bonferroni post-hoc test (CD4) or non-parametric One-way ANOVA followed by a Dunns post-hoc test (CD8). Correlations were determined by calculating the Spearman correlation coefficient (R). *P<0.05, **P<0.01, ***P<0.001.
Figure 2.
CD161hiCD8+ T cells escape from functional inhibition by CsA.
(A) Relative ABCB1 expression determined by qPCR on sort-purified CD161−CD4+, CD161neg/lowCD8+ (white bars) and CD161+CD4+, CD161hiCD8+ (black bars) T cells. Expression is normalized to that of CD161−CD4+ T cells. Data represent the mean ± SEM of 2 independent healthy donors. (B) PBMCs were labelled with Rh123, cultured for 30 minutes, and measured by flow cytometry for CD3, CD4, CD8, and CD161. A representative sample is shown gated on CD3+CD4+ and CD3+CD8+ T cells. (C-D) CFSE-labelled PBMCs were stimulated in an MLR with irradiated allo-PBMCs in the presence or absence of CsA. CFSE levels were determined after 7 days of culture. (C) Flow cytometry data of one representative experiment is shown gated on CD3+CD8+ T cells. Numbers represent percentages of proliferation per subset. (D) Relative proliferation is depicted as the percentage proliferating CD161neg/lowT cells (white bars) and CD161hiCD8+ T cells (black bars) compared to the condition without CsA, as mean+SD (n = 3). One representative experiment out of 3 independent healthy donors is shown. Statistical analysis was performed using One way ANOVA followed by a Bonferroni post-hoc test. *P<0.05, **P<0.01, ***P<0.001.
Figure 3.
Circulating CD161+CD4+ and CD161hiCD8+ T cells are decreased in allo-SCT patients who develop GVHD.
(A) Comparison of percentage and absolute levels of circulating CD161+CD4+ T cells in patients 3 months after allo-SCT and the occurrence of acute GVHD (Percentage, No n = 28, Yes n = 22; Absolute, No n = 28, Yes n = 22) and chronic GVHD (Percentage, No n = 35, Yes n = 15; Absolute, No n = 35, Yes n = 15). (B) Comparison of percentage and absolute numbers of circulating CD161hiCD8+ T cells in patients 3 months after allo-SCT and the occurrence of acute GVHD (Percentage, No n = 34, Yes n = 34; Absolute, No n = 34, Yes n = 32) and chronic GVHD (Percentage, No n = 46, Yes n = 22; Absolute, No n = 46, Yes n = 20). Lines represent mean (CD4) or median (CD8) values. Statistical analysis was performed using univariate logistic regression analysis. *P<0.05, **P<0.01.
Table 2.
Multi-variable analysis of GVHD.
Figure 4.
Memory phenotype of CD161-expressing T cells.
(A) Memory phenotype of total CD4+ T cells (left panel) and CD161+CD4+ T cells (right panel) in healthy controls (○, n = 18) and patients 3 months after allo-SCT (•, n = 55). (B) Memory phenotype of total CD8+ T cells (left panel) and CD161hiCD8+ T cells (right panel) in healthy controls (○, n = 18) and patients 3 months after allo-SCT (•, n = 55 and n = 52 respectively). Tn, naïve (CCR7+CD45RA+); Tcm, central memory (CCR7+CD45RA-); Tem, effector memory (CCR7−CD45RA-); TemRA, CD45RA+ effector memory (CCR7−CD45RA+). Statistical analysis was performed using One-way ANOVA followed by a Bonferroni post-hoc test. *P<0.05, ***P<0.001.
Figure 5.
IL17 and IFNγ production of CD161-expressing T cells after allo-SCT.
(A) Representative IL17 and IFNγ production by intracellular cytokine staining from a healthy control and allo-SCT patient. Numbers plot indicate the percentage of cells in each quadrant. (B) Comparison of IL17 (left panels) and IFNγ (right panels) production, measured by intracellular cytokine staining, between CD161− and CD161+CD4+ T cells, and CD161neg/low and CD161hiCD8+ T cells in healthy controls (○) and patients 1–3 months after allo-SCT (•). Data is shown as the percentage of positive cells, lines represent mean value. Statistical analysis was performed using One-way ANOVA followed by a Bonferroni post-hoc test, or a one-tailed paired student t-test. *P<0.05, ***P<0.001.
Figure 6.
Preferential migration of CD161-expressing T cells to CCL20.
(A) Comparison of CCR6 expression between CD161− and CD161+CD4+ memory T cells, and CD161neg/low and CD161hiCD8+ Tem cells of healthy donors (о) and patients 3 months after allo-SCT (•). The frequency of CD161+CD4+ T cells in the measured samples was 34.6–55.7% in allo-SCT patients (n = 10) and 36.6–46.4% in healthy controls (n = 3), and the frequency of CD161hiCD8+ T cells was 0.05–23.8% in allo-SCT patients (n = 8) and 11.5–42.4% in healthy controls (n = 3). Data is shown as mean fluorescent intensity (MFI). Lines represent mean value. (B) Migration of CD4+ and CD8+ T cell subsets to CCL20. CD4+ or CD8+ T cells were applied to the upper chamber and migrated to the lower chamber containing medium with or without CCL20. Migrated cells were analysed by flow cytometry. Migration is shown relative to input (mean ± SD) for CD161−CD4+ and CD161neg/lowCD8+ T cells (dashed line), and CD161+CD4+ and CD161hiCD8+ T cells (solid line) (n = 3). One representative experiment out of 2 (CD4) or 3 (CD8) independent healthy donors is shown. (C) Migration of CD4+ T cell subsets to CCL20. CD3+ T cells were applied to the upper chamber and migrated to the lower chamber containing medium with or without CCL20. Migrated cells were analysed by flow cytometry. Migration is shown relative to input (mean ± SD) for CD161−CD4+ (dashed line), and CD161+CD4+ (solid line) (n = 3). Two independent patients 3 months after allo-ST are shown. Statistical analysis was performed using Two-way ANOVA followed by a Bonferroni post-hoc test. *P<0.05, **P<0.01, ***P<0.001.
Figure 7.
CCL20 is expressed in GVHD tissues and selectively attracts CCR6+ T cells.
(A) CCL20 staining in skin biopsies of patients diagnosed with acute GVHD at respectively 49 (UPN 833) and 30 days (UPN 877), or chronic GVHD 127 days (UPN 722) and 321 days (UPN 741) days after allo-SCT. Squares indicate examples of single cells in the dermis in close proximity to the epidermal-dermal junction (acute GVHD) or in the epidermis which are situated in close proximity to or at the epidermal-dermal junction (chronic GVHD). Images were captured at 400X. (B) CD3 (red), CD4 (blue), and CCR6 (green) triple staining in skin biopsies of patients diagnosed with acute GVHD at respectively 49 (UPN 833) and 30 days (UPN 877), or chronic GVHD 127 days (UPN 722) and 321 days (UPN 741) days after allo-SCT. White arrows and squares indicate examples of CD3+CD4+CCR6+ cells, yellow arrows and squares indicate examples of CD3+CD4−CCR6+ cells. Single stainings of the cells in squares are depicted under the image. Images were captured at 400X.