Increased IL-17-producing CD8⁺ T cell frequency predicts short-term mortality in patients with hepatitis B virus-related acute-on-chronic liver failure

Figures & data

Table 1 Characteristics of participants enrolled in the study

Group	NC (n=17)	CHB (n=30)	HBV-ACLF (n=66)
Male, n (%)	16 (94)	29 (97)	60 (91)
Age (years)	38.76± 8.79	37.90±7.99	41 (18–75)
ALT (U/L)	21.47± 7.23	154.5 (27–1,658)	159.5 (15–1,986)
AST (U/L)	23.23± 7.55	136 (39–751)	172.5 (45–3,023)
Tbil (µmol/L)	N.D.	96.99 (15.51–602.08)	511.6 (183.8–1,301.7)
PTA (%)	N.D.	81.23±22.15	30 (17–40)
ALB (g/L)	N.D.	39.36±3.95	35.73±5.28
Cr (µmol/L)	N.D.	62.8 (41.7–142)	64 (34.5–161)
HBsAg positive	0	30	66
HBeAg positive	0	22	28
HBV-DNA (log10 IU/mL)	N.D.	4.98±1.07	4.85 (2.70–8.39)

Note: Data are shown as mean and standard deviations or medians and ranges.

Abbreviations: ACLF, acute-on-chronic liver failure; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CHB, chronic hepatitis B; Cr, creatinine; HBV, hepatitis B virus; NC, normal control; N.D., not determined; PTA, prothrombin time activity; Tbil, total bilirubin.

Figure 1 Tc17 cell frequency was significantly higher in patients with HBV-ACLF.

Notes: (A) Tc17 cells were analyzed by flow cytometry. In this study, Tc17 cells were defined as CD3⁺ CD8⁺ IL-17A⁺ cells. Gating strategy for the analysis of Tc17 cells was shown. (B) Representative dot plots of Tc17 cells from NC, patients with CHB, and patients with HBV-ACLF. The value in the upper right quadrant indicated the frequency of Tc17 cells. (C) Tc17 cells were significantly higher in patients with HBV-ACLF than in either patients with CHB (P=0.002) or NC subjects (P<0.001). Moreover, the frequency of Tc17 cells was significantly higher in cirrhotic patients with HBV-ACLF than in non-cirrhotic patients with HBV-ACLF (P=0.034). No differences were observed in Tc17 cells between HBeAg-positive and HBeAg-negative patients in the CHB group and in patients with HBV-ACLF. *P<0.05; **P<0.01; ***P<0.001.
Abbreviations: ACLF, acute-on-chronic liver failure; CHB, chronic hepatitis B; eAg-P, HBeAg-positive; FSC, forward scatter; eAg-N, HBeAg-negative; HBV, hepatitis B virus; ns, not significant; NC, normal control; SSC, side scatter; Tc17, IL-17-producing CD8⁺ T.

Figure 2 Tc17 cells may play a proinflammatory role in the pathogenesis of HBV-ACLF.

Notes: Tc17 cells were found to be associated with Tbil levels, PTA levels, AST levels, and ALB levels in patients with CHB (A) and patients with HBV-ACLF (B). The frequencies of Tc17 cells were significantly higher in non-surviving patients than in surviving patients (P<0.001). Moreover, the frequencies of Tc17 cells were positively correlated with MELD, MELD-Na, and CLIF-C ACLF scores (C). Lg (AST), AST levels were transformed into log₁₀. Solid line, linear growth trend; r, correlation coefficient. ***P<0.001.
Abbreviations: ACLF, acute-on-chronic liver failure; AST, aspartate aminotransferase; ALB, albumin; CHB, chronic hepatitis B; CLIF-C, chronic liver failure consortium; HBV, hepatitis B virus; MELD, model for end-stage liver disease; PTA, prothrombin time activity; TB, total bilirubin; Tc17, IL-17-producing CD8⁺ T.

Figure 3 Increased Tc17 cell frequency at admission indicated poor prognosis in HBV-ACLF.

Notes: (A) The prognostic value of Tc17 cell frequency was assessed by the ROC curve analysis. The AUROC for Tc17 cell frequency was 0.826 (95% CI: 0.712–0.908, P<0.001). The Youden index was used to identify the optimal cutoff value. With a cutoff value of 1.6%, the sensitivity was 86.84% (95% CI: 71.9%–95.6%) and the specificity was 75% (95% CI: 55.1%–89.3%). Moreover, no significant differences were found between AUROC values obtained with Tc17 cell frequency and those obtained with the MELD (AUROC=0.769, P=0.437), MELD-Na (AUROC=0.782, P=0.552), or CLIF-C ACLF (AUROC=0.773, P=0.474) scores, indicating that Tc17 cell frequency at admission may have a prognostic value equivalent to that of these accepted prognostic scores. (B) Survival was evaluated using Kaplan–Meier curves, and the statistics were compared by log-rank tests. Significant differences were found between the higher group (Tc17 cell frequency >1.6%) and the lower group (Tc17 cell frequency ≤1.6%) in both 30-day (chi-square=8.545, P=0.0035) and 90-day (chi-square=23.98, P<0.0001) survival.
Abbreviations: AUC, area under curve; ACLF, acute-on-chronic liver failure; AUROC, area under the ROC curve; CLIF-C, chronic liver failure consortium; Cum, cumulative; HBV, hepatitis B virus; MELD, model for end-stage liver disease; ROC curve, receiver operating characteristic curve; Tc17, IL-17-producing CD8⁺ T.

Table 2 Characteristics of patients with HBV-ACLF according to clinical outcome

Group	Surviving (n=28)	Non-surviving (n=38)	P-value
Female, n (%)	0 (0)	6 (16)	0.035
Age (years)	38.5 (18–67)	43.5 (29–75)	0.086
Cirrhosis	3	13	0.041
Antiviral therapy	19	24	0.796
MELD	24.67±2.80	28.53±4.28	<0.001
MELD-Na	25.74±2.79	29.62±4.18	<0.001
CLIF-c ACLF	38.44±5.13	44.59±6.19	<0.001
Tc17 cells (%)	1.38 (0.36–3.35)	2.11 (0.40–7.48)	<0.001
PLT (10^9/L)	119.5 (29–249)	86 (26–250)	0.065
Tbil (µmol/L)	379.7 (193–957.9)	565.5 (183.8–1,301.7)	0.009
PTA (%)	36 (23–40)	27 (17–40)	<0.001
AST (U/L)	161.5 (45–1,154)	195.5 (46–3,023)	0.371
ALT (U/L)	161 (15–1,688)	151 (23–1,986)	0.804
ALB (g/L)	36.60±5.84	35.08±4.80	0.250
Na (mmol/L)	138.4 (127.6–143.3)	137.3 (124.6–143.1)	0.195
Cr (µmol/L)	63 (44–102)	66.3 (34.5–161)	0.476
AFP (ng/mL)	85.85 (4–1,000)	23.18 (5.6–1,332.6)	0.039
HBV-DNA (log10 IU/mL)	4.39 (2.70–7.44)	5.21 (2.70–8.39)	0.128

Note: Data are shown as mean and standard deviations or medians and ranges.

Abbreviations: ACLF, acute-on-chronic liver failure; AFP, alpha-fetoprotein; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CLIF-c, chronic liver failure consortium; Cr, creatinine; HBV, hepatitis B virus; MELD, model for end-stage liver disease; PLT, platelet counts; PTA, prothrombin time activity; Tbil, total bilirubin; Tc17, IL-17-producing CD8⁺ T.

Table 3 Factors associated with mortality in patients with HBV-ACLF by using the Cox regression analysis

Parameters	Wald	P-value	HR	95% CI
Univariate analysis
Age (years)	2.939	0.086	1.022	0.997–1.048
Gender (female)	10.931	0.001	4.771	1.889–12.047
Cirrhosis	4.733	0.03	2.128	1.078–4.203
Tc17 cells (%)	12.581	<0.001	1.340	1.140–1.575
MELD	29.483	<0.001	1.290	1.177–1.414
MELD-Na	29.766	<0.001	1.324	1.197–1.464
CLIF-c ACLF	17.655	<0.001	1.101	1.053–1.152
Tbil (µmol/L)	8.502	0.004	1.002	1.001–1.003
PTA (%)	20.597	<0.001	0.882	0.835–0.931
PLT (10^9/L)	2.846	0.092	0.994	0.988–1.001
AFP (ng/mL)	0.565	0.452	1.000	0.998–1.001
Multivariate analysis
Gender (female)	12.181	<0.001	5.450	2.103–14.122
Tc17 cells (%)	12.106	0.001	1.401	1.159–1.694
MELD-Na	26.546	<0.001	1.344	1.201–1.503

Abbreviations: ACLF, acute-on-chronic liver failure; AFP, alpha-fetoprotein; CLIF-c, chronic liver failure consortium; HBV, hepatitis B virus; MELD, model for end-stage liver disease; PLT, platelet counts; PTA, prothrombin time activity; Tbil, total bilirubin; Tc17, IL-17-producing CD8⁺ T.

Figure 4 Tc17 cell frequency was positively correlated with serum IL-17A and IL-22 levels.

Notes: (A) Serum levels of cytokines (IL-17A, IL-22, and IL-23) were determined by a FlowCytomix assay. IL-17A levels and IL-22 levels were higher in patients with HBV-ACLF than in patients with CHB and NC subjects. However, the IL-23 level did not differ between patients with HBV-ACLF and those patients with CHB. (B) Positive correlations were found between the frequency of Tc17 cells and the levels of both IL-17A (r=0.341, P=0.007) and IL-22 (r=0.260, P=0.032). However, no correlation was found between Tc17 cell frequency and IL-23 level (r=0.030, P=0.867). These data indicated that Tc17 cells may be an additional source of IL-17 in HBV-infected patients. **P<0.01; ***P<0.001. Solid line, linear growth trend; r, correlation coefficient.
Abbreviations: ACLF, acute-on-chronic liver failure; CHB, chronic hepatitis B; HBV, hepatitis B virus; NC, normal control; ns, not significant; Tc17, IL-17-producing CD8⁺ T.