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. 2023 Aug 8;7(15):4039-4045.
doi: 10.1182/bloodadvances.2022008224.

System-level immune monitoring reveals new pathophysiological features in hepatitis-associated aplastic anemia

Cecile Braudeau  1   2 , Laurence Delbos  2 , Marie-Laure Couec  3 , Gwenvael Danic  2 , Justine Chevreuil  1 , Camille Lecuroux  1   2 , Audrey Grain  3 , Marion Eveillard  4 , Fanny Rialland  3 , Flore Sicre de Fontbrune  5 , Gaelle Beriou  2 , Nicolas Degauque  2 , David Michonneau  5   6 , Regis Josien  1   2 , Régis Peffault de Latour  5   6   7 , Caroline Thomas  3 , Jerome C Martin  1   2
Affiliations

System-level immune monitoring reveals new pathophysiological features in hepatitis-associated aplastic anemia

Cecile Braudeau et al. Blood Adv. .
No abstract available

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
A Vδ1+ γδ T-cell acute response with long-term alterations in HAA. (A) Pie charts representing the percentage of indicated T-cell subsets within total blood T cells at 4 time points after disease onset for HAA case 1. (B) Opt-SNE projections of concatenated cytometry data for T cells from patient HAA-1 samples at early time points day 9, day 16, and day 40 (left), and HAA-1 day 78, HAA-2 and HAA-4 (right). Cells are grouped and annotated into color-coded metaclusters after FlowSOM clustering (top) or by sample origin (bottom). (C) Flow cytometry plots quantifying manually gated HLA-DR+ CD38+ activated cells within total CD8 T cells, γδ T cells, and CD4 T cells from HAA-1 and pediatric (ped.) HC. Numbers correspond to the percentage of activated cells within the corresponding T-cell subset. (D) Dot plots comparing the ratio of γδ1:γδ2 T cells between patients with HAA (n = 3 patients, >70 days after initial symptom onset) and pediatric HCs from Australia and Mali. Error bars indicate mean ± standard deviation; data analyzed by Kruskal-Wallis analysis of variance with Dunn posttest comparisons. ∗∗P = .0039 and ∗∗∗∗P < .0001. (E) Heatmap showing the relative expression of relevant membrane markers used to annotate FlowSOM metaclusters (color scale: row-adjusted z-score expression for each individual marker). (F) Pie charts representing the percentage of γδ T-cell metaclusters within total γδ T cells at 3 time points after disease onset for HAA-1. (G) Opt-SNE projections of concatenated cytometry data for γδ T cells from HAA-1 samples at early time points day 9, day 16, and day 40 (left), and HAA-2 and HAA-4 (right). Cells are grouped and annotated into color-coded metaclusters after FlowSOM clustering (top) or by sample origin (bottom). (H) Left: representative flow cytometry plots of manually gated CD45RA+ CD38+ γδ T cells within total CD57− γδ T cells for HAA-2 and control pediatric AA. Numbers represent the percentage of indicated CD45RA+ CD38+ γδ T cells. Right: representative flow cytometry plots of manually gated γδ1+, γδ2+, and γδ1−γδ2− cells within CD45RA+ CD38+ γδ T cells in HAA-2. Numbers represent the percentage of indicated subset in total CD45RA+ CD38+ γδ T cells. (I) Dot plot comparing the percentage of CD57− CCR7− CD45RA+ CD38+ γδ T cells between patients with HAA at late disease stage (n = 3) and controls (nonhepatitis pediatric AA: black dot; pediatric HC: black square; adult HC: white dot).
Figure 1.
Figure 1.
A Vδ1+ γδ T-cell acute response with long-term alterations in HAA. (A) Pie charts representing the percentage of indicated T-cell subsets within total blood T cells at 4 time points after disease onset for HAA case 1. (B) Opt-SNE projections of concatenated cytometry data for T cells from patient HAA-1 samples at early time points day 9, day 16, and day 40 (left), and HAA-1 day 78, HAA-2 and HAA-4 (right). Cells are grouped and annotated into color-coded metaclusters after FlowSOM clustering (top) or by sample origin (bottom). (C) Flow cytometry plots quantifying manually gated HLA-DR+ CD38+ activated cells within total CD8 T cells, γδ T cells, and CD4 T cells from HAA-1 and pediatric (ped.) HC. Numbers correspond to the percentage of activated cells within the corresponding T-cell subset. (D) Dot plots comparing the ratio of γδ1:γδ2 T cells between patients with HAA (n = 3 patients, >70 days after initial symptom onset) and pediatric HCs from Australia and Mali. Error bars indicate mean ± standard deviation; data analyzed by Kruskal-Wallis analysis of variance with Dunn posttest comparisons. ∗∗P = .0039 and ∗∗∗∗P < .0001. (E) Heatmap showing the relative expression of relevant membrane markers used to annotate FlowSOM metaclusters (color scale: row-adjusted z-score expression for each individual marker). (F) Pie charts representing the percentage of γδ T-cell metaclusters within total γδ T cells at 3 time points after disease onset for HAA-1. (G) Opt-SNE projections of concatenated cytometry data for γδ T cells from HAA-1 samples at early time points day 9, day 16, and day 40 (left), and HAA-2 and HAA-4 (right). Cells are grouped and annotated into color-coded metaclusters after FlowSOM clustering (top) or by sample origin (bottom). (H) Left: representative flow cytometry plots of manually gated CD45RA+ CD38+ γδ T cells within total CD57− γδ T cells for HAA-2 and control pediatric AA. Numbers represent the percentage of indicated CD45RA+ CD38+ γδ T cells. Right: representative flow cytometry plots of manually gated γδ1+, γδ2+, and γδ1−γδ2− cells within CD45RA+ CD38+ γδ T cells in HAA-2. Numbers represent the percentage of indicated subset in total CD45RA+ CD38+ γδ T cells. (I) Dot plot comparing the percentage of CD57− CCR7− CD45RA+ CD38+ γδ T cells between patients with HAA at late disease stage (n = 3) and controls (nonhepatitis pediatric AA: black dot; pediatric HC: black square; adult HC: white dot).
Figure 2.
Figure 2.
A type-1 immune environment contributed by γδ T and CD8 T cells in HAA. (A) Spearman correlation and hierarchical clustering of indicated cytokines measured in the serum of HAA cases (n = 4 patients; 4 time points were collected for HAA-1; 7 samples in total) and HC (pediatric, n = 1; adult, n = 6). (B) Dot plots representing the level of indicated cytokine at 4 time points after disease onset for HAA-1. (C) Comparison of cytokine levels between HAA (n = 4 patients, >70 days after initial symptom onset) and HC. Error bars indicate mean ± standard deviation. (D) Peripheral blood mononuclear cells were stimulated for 3 hours with PMA and ionomycin and analyzed for intracellular cytokine production. Flow cytometry plots show the expression of indicated cytokines in CD4+ and CD4− T cells obtained from HAA-1 at day 9 after initial symptom onset. (E) Comparison of cytokine levels between HAA (n = 4 patients, >70 days after symptom onset) and HC. Error bars indicate mean ± standard deviation; data analyzed by 2-sided Mann-Whitney U test. ∗P < .05 and ∗∗P < .01. (F) HAA-1 BM smear was obtained at day 9 and colored with May-Grünwald-Giemsa (MGG) stain. Representative pictures of activated macrophages. Arrows indicate hemophagocytosis. (G) Left: ratio of classical-to-nonclassical monocytes defined after FlowSOM metaclustering in patients with HAA at late-stage disease (n = 3) and HCs. Right: representative manual gating of blood monocyte subsets in patients with HAA and HCs. (H) Comparison of CCL5 levels between patients with HAA (n = 4 patients at >70 days after symptom onset) and HCs. Error bars indicate mean ± standard deviation; data analyzed by 2-sided Mann-Whitney U test. ∗∗P < .01. (I) Dot plots representing the level of indicated cytokine at 4 time points after disease onset for HAA-1.
Figure 2.
Figure 2.
A type-1 immune environment contributed by γδ T and CD8 T cells in HAA. (A) Spearman correlation and hierarchical clustering of indicated cytokines measured in the serum of HAA cases (n = 4 patients; 4 time points were collected for HAA-1; 7 samples in total) and HC (pediatric, n = 1; adult, n = 6). (B) Dot plots representing the level of indicated cytokine at 4 time points after disease onset for HAA-1. (C) Comparison of cytokine levels between HAA (n = 4 patients, >70 days after initial symptom onset) and HC. Error bars indicate mean ± standard deviation. (D) Peripheral blood mononuclear cells were stimulated for 3 hours with PMA and ionomycin and analyzed for intracellular cytokine production. Flow cytometry plots show the expression of indicated cytokines in CD4+ and CD4− T cells obtained from HAA-1 at day 9 after initial symptom onset. (E) Comparison of cytokine levels between HAA (n = 4 patients, >70 days after symptom onset) and HC. Error bars indicate mean ± standard deviation; data analyzed by 2-sided Mann-Whitney U test. ∗P < .05 and ∗∗P < .01. (F) HAA-1 BM smear was obtained at day 9 and colored with May-Grünwald-Giemsa (MGG) stain. Representative pictures of activated macrophages. Arrows indicate hemophagocytosis. (G) Left: ratio of classical-to-nonclassical monocytes defined after FlowSOM metaclustering in patients with HAA at late-stage disease (n = 3) and HCs. Right: representative manual gating of blood monocyte subsets in patients with HAA and HCs. (H) Comparison of CCL5 levels between patients with HAA (n = 4 patients at >70 days after symptom onset) and HCs. Error bars indicate mean ± standard deviation; data analyzed by 2-sided Mann-Whitney U test. ∗∗P < .01. (I) Dot plots representing the level of indicated cytokine at 4 time points after disease onset for HAA-1.
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