Characteristics of circulating immune cells in HBV-related acute-on-chronic liver failure following artificial liver treatment

Abstract

Background and aim: Liver failure, which is predominantly caused by hepatitis B (HBV) can be improved by an artificial liver support system (ALSS). This study investigated the phenotypic heterogeneity of immunocytes in patients with HBV-related acute-on-chronic liver failure (HBV-ACLF) before and after ALSS therapy.

Methods: A total of 22 patients with HBV-ACLF who received ALSS therapy were included in the study. Patients with Grade I according to the ACLF Research Consortium score were considered to have improved. Demographic and laboratory data were collected and analyzed during hospitalization. Immunological features of peripheral blood in the patients before and after ALSS were detected by mass cytometry analyses.

Results: In total, 12 patients improved and 10 patients did not. According to the immunological features data after ALSS, the proportion of circulating monocytes was significantly higher in non-improved patients, but there were fewer γδT cells compared with those in improved patients. Characterization of 37 cell clusters revealed that the frequency of effector CD8⁺ T (P = 0.003), CD4⁺ T_CM (P = 0.033), CD4⁺ T_EM (P = 0.039), and inhibitory natural killer (NK) cells (P = 0.029) decreased in HBV-ACLF patients after ALSS therapy. Sub group analyses after treatment showed that the improved patients had higher proportions of CD4⁺ T_CM (P = 0.010), CD4⁺ T_EM (P = 0.021), and γδT cells (P = 0.003) and a lower proportion of monocytes (P = 0.012) compared with the non-improved patients.

Conclusions: Changes in effector CD8⁺ T cells, effector and memory CD4⁺ T cells, and inhibitory NK cells are associated with ALSS treatment of HBV-ACLF. Moreover, monocytes and γδT cells exhibited the main differences when patients obtained different prognoses. The phenotypic heterogeneity of lymphocytes and monocytes may contribute to the prognosis of ALSS and future immunotherapy strategies.

Keywords: Acute-on-chronic liver failure; Artificial liver support system; Hepatitis B; Immunophenotype; Mass cytometry.

Fig. 1

Peripheral blood immune cell cluster sorting according to marker expression levels. A Heatmap of normalized immune cell marker expressing in 37 immune cell clusters. B T-SNE map was colored by clusters, displaying 40 000 cells from PBMC analyze with immune cell clusters in all samples. 37 immune cell clusters were divided into 9 cell lineages. T-SNE maps displaying 40 000 cells from PBMC analyzed with immune cell clusters in two groups before and after ALSS treatment. t-SNE: t-distributed stochastic neighbor embedding; PBMC: peripheral blood mononuclear cells; ALSS: artificial liver support system

Fig. 2

The frequency of CD8⁺ T-cell and CD4⁺ T-cell subsets between improved and non-improved patients before and after ALSS therapy. A The frequency of effector CD8⁺ T in non-improved patients was significantly lower after ALSS therapy. B The frequency of CD8⁺ T_EM (C31, C36, C37) and CD8⁺ T_EMRA (C32) in non-improved patients was significantly lower after ALSS therapy. C Heatmap of PD1, PD1, CTLA4, CD69, CD27, CD127, and Granulozyme B expression in CD4T⁺ and CD8T⁺ cell clusters. D The frequency of CD4⁺ T_CM and CD4⁺ T_EM in non-improved patients decreased significantly after ALSS and was lower than in improved patients. E The frequency of CD127⁺ T_CM and CD4⁺ T_EM in improved patients was higher than that before ALSS therapy. p < 0.05 was considered statistically significant, *p < 0.05. **p < 0.01. ALSS: artificial liver support system; EM: effector memory; EMRA: terminally differentiated effector memory; CM: central memory

Fig. 3

Comparison of NK cells and clusters in non-improved patients before and after ALSS therapy. A Boxplots displaying the variation of immune cells in non-improved patients at the total immune cells level. B Pie charts displaying the frequency of immune cell subsets in the PBMC before and after ALSS therapy. C The frequency of C13 (CD56^high NK cell) significantly reduced in non-improved patients after ALSS. D The expression of CD56 in 37 clusters. C13–16 were marked with red dotted box. p < 0.05 was considered statistically significant, *p < 0.05. **p < 0.01. NK: natural kill; ALSS: artificial liver support system; PBMC: peripheral blood mononuclear cells

Fig. 4

Comparison of γδT cells, monocytes, granulocytes, and clusters between improved and non-improved patients after ALSS therapy. A Boxplots displaying the difference between immune cell subsets of two groups in total immune cell level after ALSS therapy. B The frequency of γδT cell clusters (C28 and C29) of improved patients was significantly higher compared to non-improved patients. C T-SNE maps displaying the expression of CD27 and CD45RA in 37 clusters. C28 and C29 were marked with red boxes. D The frequency of monocytes (C03) in non-improved patients with ALSS was significantly higher than that before treatment, and was significantly higher compared to improved patients. E Sequential gating strategy for identification of classical monocytes (CD14⁺⁺ CD16^–), intermediate monocytes (CD14⁺⁺ CD16⁺), and non-classical monocytes (CD14⁺ CD16⁺⁺) using immunophenotyping followed by CyTOF. F The expression of HLA-DR between improved and non-improved patients before and after ALSS therapy. G The frequency of HLA-DR expression in classical monocytes in the improved and non-improved groups before and after ALSS. H The frequency of eosinophils and basophils in all samples decreased but neutrophils increased after ALSS. p < 0.05 was considered statistically significant, *p < 0.05. **p < 0.01. ***p < 0.001 ALSS: artificial liver support system; t-SNE: t-distributed stochastic neighbor embedding; CyTOF: mass cytometry