Altered compositions of monocyte, T lymphocyte and NK cell subsets in heart failure of adult congenital heart disease

Fig. 1. Increased circulating levels of CD16+monocytes in heart failure of ACHD (A–E) Gating strategy for monocyte subsets: Mon1 (classical, HLA-DR+CD14++CD16−), Mon2 (intermediate, HLA-DR+CD14++CD16+), Mon3 (non-classical, HLA-DR+CD14+CD16++). Gating was verified by back-gating to CCR2 (D) and CX3CR1 (E) expression. (F–H) Representative flow cytometry density plots. (J–L) Frequency of monocyte subsets in controls vs. ACHD according to New York Heart Association (NYHA) class. (M) Circulating Mon2 in subgroups of ACHD malformations, coarctation (CoA), pulmonary artery lesions (PA), Eisenmenger syndrome and pulmonary hypertension (PH), n = 21/26/79/76/15/13. (J–M) n = 21/134/45/30. Values expressed as mean ± SEM. Statistical analysis performed using ANOVA with post hoc Tukey's test: §p < 0.05 vs. control, *p < 0.001 vs. control, †p < 0.001 vs. NYHA I. (N) Spearman-correlation matrix of Mon1–3 (cells/μL), CRP (mg/L) and NT-proBNP (ng/L), correlation coefficient is expressed by colour intensity and numeric R values are shown in the top right half, p value indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. Expansion of a CD14+HLA-DRneg/lowmonocyte subset in ACHD patients. (A–F) Gating strategy to quantify CD14+HLA-DRneg/low monocytes. (E–F) Gating was verified in a subgroup by CD33 expression of CD14+ monocytes. (B–C) Representative flow cytometry density plots of a control and adult congenital heart disease (ACHD) patient classified to New York Heart Association (NYHA) class III. (J + M) Linear regression analysis between CD14+HLA-DRneg/low counts and (J) S100A8/A9 plasma or (M) serum CRP levels in ACHD patients. (G-H, K-L) CD14+HLA-DRneg/low in control vs. ACHD and according to patient's NYHA class. Percentages indicate HLA-DRneg/low cells within CD14+ monocytes. Absolute counts were calculated by multiplying percentages from gated monocytes with monocyte frequencies from blood cell counts. n = 21/209 (G, K) or n = n = 21/134/45/30 (H,L). Values expressed as median (IQR). Statistical analysis performed using Mann-Whitney U or Kruskal-Wallis test: §p < 0.05 vs. control, *p < 0.001 vs. control.

Fig. 3. ACHD patients exhibited decreased T cell subset counts next to increased frequencies of CD56brightNK cells. (A–D) Frequency of T and NK subpopulation cell counts in controls vs. ACHD, n = 20/160. (E–H) Absolute CD8+ and CD4+ T cell and NK subset counts in controls vs. ACHD according to New York Heart Association (NYHA) heart failure class, n = 20/101/40/19. Values are expressed as median [interquartile range]. Statistical analysis performed using Mann-Whitney U or Kruskal-Wallis test: §p < 0.05 vs. control. (J) Linear regression analysis between CD8+ T cell counts and Mon3 frequency in ACHD patients.

Fig. 4. Kaplan-Meier survival plots of the primary and secondary outcome in ACHD. (A–B) The top tertile of the Mon2 (intermediate, HLA-DR+CD14++CD16+) and CD14+HLA-DRneg/low monocyte subset war compared to the bottom two tertiles in all ACHD patients presented according to the primary outcome (all-cause mortality and hospitalisation for cardiac decompensation). (C–E) The top tertile of the Mon2 (C) and the bottom tertile of total circulating lymphocytes (D) and T helper cells (E) was compared to the respective two remaining tertiles. The cut-off ranges of the tertile groups are shown in the figure. Maximum follow-up period was 8.25 years. Log-rank test was performed, n = 209.