Immune Profile in Patients With COVID-19: Lymphocytes Exhaustion Markers in Relationship to Clinical Outcome

Abstract

The velocity of the COVID-19 pandemic spread and the variable severity of the disease course has forced scientists to search for potential predictors of the disease outcome. We examined various immune parameters including the markers of immune cells exhaustion and activation in 21 patients with COVID-19 disease hospitalised in our hospital during the first wave of the COVID-19 pandemic in Slovakia. The results showed significant progressive lymphopenia and depletion of lymphocyte subsets (CD3⁺, CD4⁺, CD8⁺ and CD19⁺) in correlation to the disease severity. Clinical recovery was associated with significant increase in CD3⁺ and CD3⁺CD4⁺ T-cells. Most of our patients had eosinopenia on admission, although no significant differences were seen among groups with different disease severity. Non-survivors, when compared to survivors, had significantly increased expression of PD-1 on CD4⁺ and CD8⁺ cells, but no significant difference in Tim-3 expression was observed, what suggests possible reversibility of immune paralysis in the most severe group of patients. During recovery, the expression of Tim-3 on both CD3⁺CD4⁺ and CD3⁺CD8⁺ cells significantly decreased. Moreover, patients with fatal outcome had significantly higher proportion of CD38⁺CD8⁺ cells and lower proportion of CD38⁺HLA-DR⁺CD8⁺ cells on admission. Clinical recovery was associated with significant decrease of proportion of CD38⁺CD8⁺ cells. The highest AUC values within univariate and multivariate logistic regression were achieved for expression of CD38 on CD8⁺ cells and expression of PD1 on CD4⁺ cells alone or combined, what suggests, that these parameters could be used as potential biomarkers of poor outcome. The assessment of immune markers could help in predicting outcome and disease severity in COVID-19 patients. Our observations suggest, that apart from the degree of depletion of total lymphocytes and lymphocytes subsets, increased expression of CD38 on CD3⁺CD8⁺ cells alone or combined with increased expression of PD-1 on CD3⁺CD4⁺ cells, should be regarded as a risk factor of an unfavourable outcome in COVID-19 patients. Increased expression of PD-1 in the absence of an increased expression of Tim-3 on CD3⁺CD4⁺ and CD3⁺CD8⁺ cells suggests potential reversibility of ongoing immune paralysis in patients with the most severe course of COVID-19.

Keywords: COVID-19; SARS-CoV-2; clinical outcome; immune cells exhaustion; immunologic predictors.

Figure 1

Representative flow cytometry plots of expression of lymphocyte exhaustion markers. Expression of PD-1 on CD4+ cells (A), CD8+ cells (B) and expression of Tim-3 on CD4+ cells (C), CD8+ cells (D).

Figure 2

Differences in the serum concentration of IgG (A), IgA (B) and IgM (C) among the groups of COVID-19 patients (differences not significant – Kruskal-Wallis test). Group A: n = 3 patients, Group B: n = 10 patients, Group C: n = 3 patients, Group D: n = 4 patients.

Figure 3

Absolute lymphocyte counts on admission to the hospital (group A versus group B: p = 0.043, group A versus group C: p = 0.038, group A versus group D: p = 0.007, group B versus group D: p = 0.043 – Kruskal-Wallis test, post-hoc analysis Conover test). Group A: n = 3 patients, Group B: n = 10 patients, Group C: n = 3 patients, Group D: n = 4 patients. *p < 0.05, **p < 0.01.

Figure 4

The lowest absolute lymphocytes counts found during hospitalization (group A versus group C: p = 0.004, group A versus group D: p = 0.0009, group B versus group C: p = 0.0103, group B versus group D: p = 0.0009 – Kruskal-Wallis test, post-hoc analysis Conover test). Group A: n = 3 patients, Group B: n = 10 patients, Group C: n = 3 patients, Group D: n = 4 patients. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

The differences in absolute neutrophil counts (A) and neutrophil-to-lymphocyte ratio (NLR) (B) among the patients’ groups on admission (4A – differences not significant − Kruskal-Wallis test; 4B – group A versus group B: p = 0.029, group A versus group C: p = 0.0014, group A versus group D: p = 0.0014; group B versus group C: p = 0.014, group B versus group D: p = 0.012 − Kruskal-Wallis test, post-hoc analysis Conover test). Group A: n = 3 patients, Group B: n = 10 patients, Group C: n = 3 patients, Group D: n = 4 patients. *p < 0.05, **p < 0.01.

Figure 6

The differences in absolute counts of CD3⁺ (A) – group A versus group B: p = 0.035, group A versus group C: p = 0.0012, group A versus group D: p = 0.0004, group B versus group C: p = 0.009, group B versus group D: p = 0.0012; CD3⁺CD4⁺ (B) – group A versus group D: p = 0.036; CD3⁺CD8⁺ (C) – group A versus group B: p = 0.043, group A versus group C: p = 0.002, group A versus group D: p = 0.004, group B versus group C: p = 0.007, group B versus group D: p = 0.033; and CD19⁺ (D) – group A versus group C: p = 0.017, group A versus group D: p = 0.017, group B versus group C: p = 0.011, group B versus group D: p = 0.011, among the patients’ groups on admission. Kruskal – Wallis test, post-hoc analysis Conover test). Group A: n = 3 patients, Group B: n = 10 patients, Group C: n = 3 patients, Group D: n = 4 patients. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

The dynamics of absolute counts of CD3⁺ (A) and CD3⁺CD4⁺ (B), in symptomatic surviving patients during the hospitalization (dynamics of CD3⁺: p = 0.022, dynamics of CD3⁺CD4⁺: p = 0.037 − Wilcoxon test for paired samples). Group B + C: n = 13 patients. *p < 0.05.

Figure 8

The differences in expression of PD-1 on CD3⁺CD4⁺ (A; p = 0.005) and CD3⁺CD8⁺ (B; p = 0.033) and Tim-3 on CD3⁺CD4⁺ (C; p = n.s.) and CD3⁺CD8⁺ (D; p = n.s.) between the survivors and non-survivors at admission. Mann-Whitney test for independent samples. Group A + B + C (i.e. survivors): n = 16 patients, Group D (i.e. non-survivors): n = 4 patients. *p < 0.05, **p < 0.01.

Figure 9

The changes in expression of Tim-3 on CD3⁺CD4⁺ (A) and CD3⁺CD8⁺ (B) during the hospitalization in symptomatic recovered patients (Tim-3 on CD3⁺CD4⁺: p = 0.008; Tim-3 on CD3⁺CD8⁺: p = 0.005 − Wilcoxon test for paired samples). Group B + C: n = 13 patients. **p < 0.01.

Figure 10

The differences in expression of CD38 on CD3⁺CD8⁺ (A; p = 0.003), co-expression of CD38 and HLA-DR on CD3⁺CD8⁺ (B; p = 0.037) − Mann-Whitney test for independent samples, Group A + B + C (i.e. survivors): n = 16 patients, Group D (i.e. non-survivors): n = 4 patients; and dynamics of CD38 expression on CD3⁺CD8⁺ during hospitalization among the symptomatic survivors (C; p = 0.008) − Wilcoxon test for paired samples, Group B + C: n = 13 patients.

Figure 11

ROC curves for expression of CD38 on CD8⁺ (AUC 0.984) cells, expression of PD1 on CD4⁺ cells (AUC 0.969) and expression of PD1 on CD8⁺ cells (AUC 0.852).