Prediction of tumor recurrence by distinct immunoprofiles in liver transplant patients based on mass cytometry

Abstract

Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is a marker of poor prognosis. However, the reliable biomarkers of post-LT HCC recurrence remain to be identified. In this study, serial peripheral blood samples from the LT recipients with and without HCC recurrence were collected at five time points. Single-cell mass cytomertry (CyTOF) was utilized for the in-depth analysis of peripheral blood monocellular cells (PBMCs). CyTOF analysis showed that at 3 weeks post-LT, the activated immune cell population was increased, while the fraction of immune cells with suppressive functions (myeloid-derived suppressive cells) was reduced. The post-LT immune composition in patients with LT for HCC was enormously different from that in patients with LT for causes other than HCC. Furthermore, at 3 weeks after LT, compared with patients without recurrence, the patients with HCC recurrences were high in two subsets of T cells: CD57⁺ HLA-DR⁺ CD8⁺ and CD28⁺γδ. The CD57⁺ HLA-DR⁺ CD8⁺ T cells presented high levels of perforin, granzyme B, and Ki-67 and displayed a highly cytotoxic and proliferative phenotype, while the CD28⁺γδ T cells had reduced levels of IFN-γ and, hence, were less activated compared to CD28^- cells. Based on these findings, we concluded that analyzing the PBMCs of LT recipients by CyTOF can predict the post-LT HCC recurrence. The distinct immune features can stratify patients with the risk of HCC recurrence at 3 weeks after LT, which will help clinician in further management plan and improve the prognosis of patients.

Keywords: Hepatocellular carcinoma; liver transplantation; mass cytometry.

Figure 1

Immune profiles of peripheral blood monocellular cells (PBMCs) from liver transplantation recipients. A. Pseudocolor contour plot showing the gating strategy used to obtaining CD45⁺ CD15^- immune populations. B. Heatmap showing normalized mean expression of the markers of 56 X-shift-defined clusters. Clusters are grouped by expression profiles and cell types are indicated by the color (pDC: plasmacytoid dendritic cells). C. All CD45⁺ CD15^- cells are plotted on a t-SNE map colored by 56 X-shift-defined clusters.

Figure 2

Immunophenotyping of peripheral blood monocellular cells (PBMCs) from liver transplantation recipients. A. Sample collection and analysis pipeline, PBMCs were collected before surgery (BS) and at 3 days (3D), 1 week (1W), 2 weeks (2W), 3 weeks (3W) after surgery from patients who received liver transplantation for hepatocellular carcinoma (HCC-LT, n=9), or other non-HCC reasons (NHCC-LT, n=3). HCC-LT recipients are classified into tumor recurrence (TR, n=4) group and non-recurrence (NR, n=5) group. Time of flight masscytometry (CyTOF) was used to analyze collected PBMCs. CyTOF results and clinical data were co-analyzed using multiple bioinformatics methods to search for immune features unique to TR HCC-LT recipients. B. Visualization of all CD45⁺ CD15^- CyTOF events from 12 enrolled liver transplantation recipients, colored by normalized expression of indicated markers. C. t-SNE map colored by main cell populations based on manual annotation of X-shift clustering. D. Box plots showing the percentage of main immune cell populations at 5 peri-transplant time points (BS, n=12; 3D, n=10; 1W, n=9; 2W, n=6; and 3W, n=12). P-values were calculated using unpaired two-sided Student’s T-test and indicated by numbers.

Figure 3

Immune profiles of peripheral blood monocellular cells (PBMCs) from liver transplantation recipients. A. Cells colored by normalized expression of indicated markers on the t-SNE map. B. Box plots showing the percentage of main immune cell populations at 4 peri-transplant time points (BS, 3D, 1W, and 3W), 9 patients with their PBMCs collected at all 4 time points were included. P-values were calculated by two-sided paired Student’s T-test (*: P<0.05, **: P<0.01 and ***: P<0.001). The horizontal line of the box plot indicates the median, the boxes represent the interquartile range (IQR), and the whiskers reach the farthest data within 1.5×IQR from the median. C. Plotting PBMCs collected at 5 different time points (BS, 3D, 1W, 2W, and 3W) from liver transplantation recipients on a tSNE map reveals the peripheral immune composition changes after liver transplantation. D. Pie plot showing the composition of plasmacytoid dendritic cells (pDCs) and myeloid cells.

Figure 4

Peripheral immune composition change induced by liver transplantation. A. Box plots showing the percentage of indicated B cell and Myeloid cell clusters at 4 peri-transplant time points (BS, 3D, 1W, and 3W), 9 patientswith their PBMCs collected at all 4 time pointswere included. P-values were calculated by two-sided paired Student’s T-test andindicated by numbers. B. Heatmap showing normalized marker expression of X-shift-defined B cell and Myeloid cell clusters. C. Heatmap showing normalized marker expression of X-shift-defined T cell clusters. Clusters were grouped by expression profiles and cell types were indicated by the color. D. Boxplots showing the frequencychanges of naïve T cells (TN), effector T cells (TEFF), central memory T cells (TCM), and effector memory T cells (TEM) inCD4 and CD8 T cells. P-values were calculated by two-sided paired Student’s T-testand indicated by numbers. E. Box plots showing the percentage of indicated T cell clusters at 4 peri-transplant time points (BS, 3D, 1W, and 3W), 9 patients with their PBMCs collected at all 4 time points were included. P-values were calculated by two-sided paired Student’s T-test andindicated by numbers. F. Box plots showing the frequency comparisons of HLA-DR⁺/HLA-DR^- CD8⁺ T cellsat 5 peri-transplant time points (BS, 3D, 1W, 2W, and 3W). P-values were calculated using two-sided unpaired Student’s T-testandindicated by numbers.

Figure 5

Identification of clusters unique to tumor recurrence LT recipients. A. tSNE map of PBMCs isolated from tumor recurrenceHCC-LT recipients (TR, orange), non-recurrenceHCC-LT recipients (NR, blue), and patients received LT not for HCC (NHCC-LT, pink). B. Principal Component Analysis of all blood samples grouped by TR (blue), NR (green), and NHCC-LT (red) recipients. The shadedarea representedthe 95% confidential ellipseof each sample group. C. Heatmap depicting the immune composition difference between (1) NHCC-LT and HCC-LT recipients (up) and between (2) TR HCC-LT and NR HCC-LT (bottom) recipients. Heatmaps were colored by P-values calculated using two-sided unpaired Student’s T-test. Each unit of the heatmap represents the significance of the immune composition difference for the indicated cluster at the indicated time point and was painted to red if P-value <0.05. D. Box plots showing the frequency comparisonsof cluster T14 (up) and T21 (bottom) in NHCC recipients, TR recipients, and NR recipients at 3 weeks after transplantation. P-values are calculated using two-sided unpaired Student’s T-test andindicated by numbers. E. Biaxial plots showing manually gated T14 and T21 from representative samples. T14 was manually gated by high expression of CD28 and CD127 (up), T21 was manually gated by high expression of CD57 and HLA-DR (bottom). F. Box plots showing the positive percentages of PD-1/CD38/CD27/CD28 in 4 groups divided according to the expression of CD57 and HLA-DR. P-values were calculated using two-sided paired Student’s T-test andindicated by numbers.

Figure 6

Functional assessment of CD57⁺ HLA-DR⁺ CD8⁺ T cells and CD28⁺γδ T cells using CyTOF. A. Sample collection and analysis pipeline, PBMCs were collected 3 weeks (3W) after transplantation from patients who received liver transplantation for hepatocellular carcinoma (HCC-LT, n=3). Collected PBMCs were analyzed by Time of flight mass-cytometry (CyTOF) using a specially designed 30-maker functional assessment panel. B. Histograms showing the expressiondistributions of indicated cytokines and functional biomarkers in 4 divided groups: CD57^- HLA-DR^- CD8⁺ T cells, CD57^- HLA-DR⁺ CD8⁺ T cells, CD57⁺ HLA-DR^- CD8⁺ T cells, and CD57⁺ HLA-DR⁺ CD8⁺ T cells. C. Boxplots showing the positive percentages of the indicated cytokines and functional biomarkersin 4 CD8⁺ T cell subsets divided according to the expression of CD57 and HLA-DR. P-values were calculated using two-sided paired Student’s T-testandindicated by numbers. D. Boxplots showing the positive percentages of the indicated cytokines and functional biomarkersin 2 γδ T cell subsets divided according to the expression of CD28. P-values were calculated using two-sided paired Student’s T-test andindicated by numbers.

Figure 7

Correlation Analysis of X-shift-defined immune clusters in tumor recurrence (TR) and non-recurrence (NR) recipients. A. Heatmap showing pairwise Pearson correlation coefficients of immune cell phenotypes in TR and NR groups. Green rectangles indicatedstrong correlations unique to TR recipients. The red rectangle indicatedstrong correlations unique to NR recipients. B. Scatterplots showing strong correlations unique to TR or NR groups. Pearson correlations and P Values were indicated.