Change and predictive ability of circulating immunoregulatory lymphocytes in long-term outcomes of acute ischemic stroke

Abstract

Lymphocytes play an important role in the immune response after stroke. However, our knowledge of the circulating lymphocytes in ischemic stroke is limited. Herein, we collected the blood samples of clinical ischemic stroke patients to detect the change of lymphocytes from admission to 3 months after ischemic stroke by flow cytometry. A total of 87 healthy controls and 210 patients were enrolled, and the percentages of circulating T cells, CD4⁺ T cells, CD8⁺ T cells, double negative T cells (DNTs), CD4⁺ regulatory T cells (Tregs), CD8⁺ Tregs, B cells and regulatory B cells (Bregs) were measured. Among patients, B cells, Bregs and CD8⁺ Tregs increased significantly, while CD4⁺ Tregs dropped and soon reversed after ischemic stroke. CD4⁺ Tregs, CD8⁺ Tregs, and DNTs also showed high correlations with the infarct volume and neurological scores of patients. Moreover, these lymphocytes enhanced the predictive ability of long-term prognosis of neurological scores when added to basic clinical information. The percentage of CD4⁺ Tregs within lymphocytes showed high correlations with both acute and long-term neurological outcomes, which exhibited a great independent predictive ability. These findings suggest that CD4⁺ Tregs can be a biomarker to predict stroke outcomes and improve existing therapeutic strategies of immunoregulatory lymphocytes.

Keywords: Circulating lymphocytes; flow cytometry; ischemic stroke; regulatory B cells; regulatory T cells.

Figure 1.

Different clusters of regulating-cells in lymphocytes between control and different time points after acute ischemic stroke. (a) Representative flow cytometry analyses of total lymphocytes, B cells, T cells, CD4⁺ T cells, CD8⁺ T cells, Bregs, CD4⁺ Tregs, CD8⁺ Tregs, and DNTs. (b) Venn diagram of data at different time points. (c) Violin plots with median, 25 and 75% quartiles of the percentage of Bregs, DNTs, CD4+ Tregs, and CD8+ Tregs within lymphocytes of Controls (grey, n = 87) and different time points after acute ischemic stroke (Admission: blue, n = 210; Discharge: orange, n = 100; 1 month: red, n = 49; 3 months: purple, n = 66). Mann-Whitney test was employed to examine the difference between Controls and each time point. Decimals represent p-value. *: p < 0.05, ***: p < 0.001, ns: no significance.

Figure 2.

Correlation matrix between the percentage of lymphocyte subsets and neurological outcomes and Receiver operating characteristic (ROC) curves for predicting stroke severity and prognosis. (a) Heat map used to visualize correlations between the percentage of different clusters of lymphocytes at different time points and infarct volume, NIHSS and mRS score at admission, discharge, and 3 months. The color bar represents Spearman r value and star symbols (*) represents for statistical significance. Heatmap was drawn by R package pheatmap, clustered by columns. *: p < 0.05, **: p < 0.01, ***: p < 0.001, no symbol: no statistical significance. (b) ROC curves for prediction of poor admission NIHSS (NIHSS ≥5) using Random Forest (purple line), Logistic regression (red line), Support Vector Machine (SVM) (green line) and Decision Tree (blue line) models (Top left); for prediction of poor admission (Top right), discharge NIHSS (NIHSS ≥5) (Middle left), 3-month NIHSS (NIHSS ≥2) (Middle right), discharge mRS (mRS ≥3) (Bottom left) and poor 3-month mRS (mRS ≥2) (Bottom right) using Logistic regression model. “Basic” (dotted black line): factors including age, sex, past history of hypertension, diabetes and disorder of lipid metabolism, and habits of drinking and smoking, tPA treatment and infarct volume. “CD4+ Treg”(green line), “CD8+ Treg” (purple line), “DNT” (blue line), “Breg” (yellow line): “Basic” adding CD4⁺ Tregs/lymphocytes, CD8⁺ Tregs/lymphocytes, DNTs/lymphocytes, or Bregs/lymphocytes respectively; “All Regs” (red line): “Basic” adding Bregs/lymphocytes, CD4⁺ Tregs/lymphocytes, CD8⁺ Tregs/lymphocytes, and DNTs/lymphocytes. Decimals in brackets: area under the curve (AUC). NIHSS: National Institutes of Health Stroke Scale; mRS: modified Rankin Scale.

Figure 3.

Changes of neurological score from admission to 3 months in different levels of regulatory lymphocytes. Variation of NIHSS (left) or mRS score (right) from admission to 3 months grouped by the median of the percentage of regulatory lymphocytes within total lymphocytes at admission ((a): Breg: 2.466%; (b): DNT: 2.490%; (c); CD8+Treg: 1.363%; (d): CD4+Treg: 2.209%). Green circle symbols: patients with the percentage of regulatory lymphocytes lower than the median; red square symbols: patients with the percentage of regulatory lymphocytes higher than the median. Data are presented as mean ± SD. Two-way ANOVA and Bonferroni's multiple comparisons tests were employed to test significance. *: p < 0.05, **: p < 0.01, ns: no significance.

Figure 4.

Continuous changes in the percentage of CD4⁺ Tregs within lymphocytes in 28 patients. (a) Left: Violin plots with median, 25 and 75% quartiles of the percentage of CD4⁺ Tregs within lymphocytes of different time points after acute ischemic stroke (Admission: blue, Discharge: orange, 1 month: red, 3 months: purple) in the 28 patients with complete data (RM one-way ANOVA, p = 0.0005). Bonferroni's multiple comparisons tests were employed to examine the differences between time-points. Right: Spearman correlation between the percentage of CD4⁺ Tregs within lymphocytes and time after onset. The dotted line represents 95% confidence bands of the best-fit line. (b) Variation of the percentage of CD4⁺ Tregs within lymphocytes from admission to 3 months in patients with tissue plasminogen activator (tPA) treatment (n = 6), habits of drinking (n = 5), smoking (n = 4), history of hypertension (n = 19), diabetes (n = 11), blood low-density lipoprotein (LDL) higher than 3.37 mmol/L (n = 13) or not. All data are presented as mean ± SD. Two-way ANOVA and Bonferroni's multiple comparisons tests were employed to test significance. **: p < 0.01, ***: p < 0.001, “ns” or no symbol: no significance.