Patient profile
The characteristics of 41 CCA, 10 HV, and 20 HCV SVR12 patients are shown in Table. 1 and Supplementary Table. 1. Of 41 CCA, the median age was 75 (48-90) years old, and 29 were male and 12 female. There were 23 patients with iCCA, 10 with GBCA, and 8 with dCCA. CCA were treated by surgery (n=18), chemotherapy (n=12), or BSC (n=11). As risk factors of cholangiocarcinoma, 2 had cirrhosis, 3 had HBV, 1 had HCV, 9 had obesity, 11 had DM, 3 had chronic alcohol use, and 22 had tobacco. Of 10 HV, the median age was 27 (23-32) years old, and 3 were male and 7 female. Of 20 HCV SVR12 patients, the median age was 75 (60-86) years old, and 10 were male and 10 female.
There were significant differences in sex (p=0.028) and age (p<0.001) between CCA and HV. There were no significant differences in sex (p=0.157) or age (p=0.847) between CCA and HCV SVR12 patients. There were significant differences in the values of aspartate transaminase (AST) (p<0.001), alanine transaminase (ALT) (p<0.001), and γ-glutamyltranspeptidase (γGTP) (p<0.001) between CCA and HV. There were also significant differences in the values of white blood cell (p<0.001), AST (p<0.001), ALT (p<0.001), alkaline phosphatase (p<0.001), γGTP (p<0.001), and total bilirubin (p=0.001) between CCA and HCV SVR12 patients.
The characteristics of each treatment group are shown in Table. 2. The surgery group consisted of 14 of 18 patients in whom ICP was able to be assessed before and after surgery. On comparison of laboratory test results before and after treatment in each treatment group, in the chemotherapy group, the values of carbohydrate antigen 19-9 (CA19-9) after chemotherapy was significantly higher than that before chemotherapy (p=0.015). In the surgery group, the values of ALT (p=0.039) and γGTP (p=0.022) after surgery were significantly lower than those before surgery. Recurrence was observed in 9 patients after surgery.
ICP in peripheral blood between CCA and HV including HCV SVR12 patients
CD4+ T cells and CD8+ T cells were isolated, and CD4+ T cells were further separated into helper T cells (defined as CD3+CD4+CD45RA+/-FoxP3-cells), naïve Tregs (nTregs) (defined as CD3+CD4+CD45RA+FoxP3+lowcells), and effector Tregs (eTregs) (defined as CD3+CD4+CD45RA-FoxP3+highcells), as previously reported5 (Figure. 1a). In addition, PD-1, CD25, CTLA-4, CXCR3, CCR4, CCR6, CD80, OX40, and 4-1BB expression levels were measured in each T cell fraction (helper T cells, nTregs, eTregs (Supplementary Figures. S1a-S1c), and CD8+ T cells (Figure. 1b)). Peripheral blood MDSCs were separated into CD14+HLA-DR-/lowMDSCs and CD14-CD15+MDSCs based on CD14, CD15, and HLA-DR expression levels. In addition, the PD-L1 expression level was measured in each fraction (Supplementary Figure. S1d).
There were significant differences between CCA and HV in the frequency of the following immune cells: OX40+ helper T cells, PD-1+ eTregs, CD80+ eTregs, OX40+ eTregs, nTregs, CD25+ nTregs, CCR6+ nTregs, CXCR3+ nTregs, CD25+ CD8+ T cells, (Figures. 1c, and 1d), lymphocytes, CD4+ T cells, CCR4+ helper T cells, CCR6+ helper T cells, CD80+ helper T cells, CTLA-4+ eTregs, PD-1+ nTregs, CCR4+ nTregs, CD80+ nTregs, OX40+ nTregs, CD80+ CD8+ T cells, CCR6+ CD8+ T cells, CCR4+ CD8+ T cells, PD-1+ CD8+ T cells, and monocytes. (Supplementary Figures. S2a-S2c).
There were significant differences between CCA and HCV SVR12 patients in the frequency of the following immune cells: lymphocytes, monocytes, nTregs, CD80+ nTregs, 4-1BB+ nTregs, eTregs, CTLA-4+ eTregs, PD-1+ eTregs, CCR4+ eTregs, CD80+ eTregs, OX40+ eTregs, 4-1BB+ eTregs, CTLA-4+ helper T cells, PD-1 helper T cells, CCR4+ helper T cells, CD80+ helper T cells, OX40+ helper T cells, CXCR3+ helper T cells, CD25+ CD8+ T cells, CTLA-4+ CD8+ T cells, PD-1+ CD8+ T cells, CXCR3+ CD8+ T cells, CD80+ CD8+ T cells, and OX40+ CD8+ T cells (Supplementary Figures. S3a-S3d).
There were significant differences between HV and HCV SVR12 patients in the frequency of the following immune cells: CCR4+ eTregs, CXCR3+ eTregs, 4-1BB+ eTregs, nTregs, PD-1+ nTregs, CCR4+ nTregs, CCR6+ nTregs, CXCR3+ nTregs, 4-1BB+ nTregs, OX40+ nTregs, CTLA-4+ CD8+ T cells, CCR6+ CD8+ T cells, CXCR3+ CD8+ T cells, and OX40+ CD8+ T cells (Supplementary Figures. S4a-S4c).
The changes in ICP after biliary drainage in CCA compared with HV
The changes of patient characteristics of 3 CCA after biliary drainage are shown in Supplementary Table. 2. Cholangitis was improved by biliary drainage in these CCA. These patients were not included in the 41 CCA because they have not been followed for a long time. On comparison of ICP of 3 CCA before biliary drainage with those of HV, the frequencies of CD4+ T cells (p=0.028), PD-1+ eTregs (p=0.014), OX40+ eTregs (p=0.014), PD-1+ nTregs (p=0.007) and OX40+ helper T cells (p=0.007) in CCA were significantly higher than those in HV (Supplementary Figure. S5). After biliary drainage, the frequency of CD4+ T cells of CCA was comparable to that of HV (p=0.287), but the frequencies of PD-1+ eTregs (p=0.007), OX40+ eTregs (p=0.007), PD-1+ nTregs (p=0.014), and OX40+ helper T cells (p=0.007) in CCA remained higher than those in HV (Supplementary Figure. S5).
The differences in ICP based on the anatomic locations and risk factors of cholangiocarcinoma
ICP based on obesity, DM, and tobacco are shown in Supplementary Figures. S6a-S6d. In particular, CCA with DM exhibited significant differences in the frequencies of immune cells compared with CCA without DM. ICP for each location are shown in Supplementary Figure. S6e. The frequencies of CCR6+ eTregs, CD80+ nTregs, CXCR3+ CD8+ T cells, and OX40+ CD8+ T cells were significantly different among iCCA, GBCA, and dCCA.
The changes in ICP after surgery in CCA compared with HV
The immune cells that demonstrated a significant difference before surgery compared with HV but reached a level comparable to that in HV after surgery were lymphocytes, PD-1+ CD8+ T cells, CXCR3+ nTregs, CD80+ nTregs, eTregs, CTLA-4+ eTregs, PD-1+ eTregs, and CXCR3+ eTregs (Figures. 2a-2c).
In contrast, the frequencies of CD4+ T cells, helper T cells, CD25+ CD8+ T cells, CD80+ CD8+ T cells, PD-1+ nTregs, CCR4+ nTregs, CCR6+ nTregs, OX40+ nTregs, and OX40+ eTregs were significantly higher in CCA even after surgery (Figures. 2a-2c). These items were compared among three groups: non-recurrent CCA (n=5), recurrent CCA (n=9), and HV (n=10). The frequencies of CD4+ T cells, PD-1+ nTregs, CCR6+ nTregs, and OX40+ nTregs before surgery were higher in recurrent CCA than in HV, and comparable between non-recurrent CCA and HV (Figure. 2d).
The changes in ICP after surgery in CCA
The changes in ICP after surgery in CCA are shown in Supplementary Figure. S7. The frequencies of CTLA-4+ eTregs (p=0.030), CXCR3+ eTregs (p=0.004), CD80+ eTregs (p=0.035), CD80+ nTregs (p=0.004), CTLA-4+ helper T cells (p=0.048), CXCR3+ helper T cells (p=0.041), 4-1BB+ helper T cells (p=0.017), CD80+ helper T cells (p=0.019), CD25+ CD8+ T cells (p=0.004), PD-1+ CD8+ T cells (p=0.002), CXCR3+ CD8+ T cells (p=0.002), 4-1BB+ CD8+ T cells (p=0.048), and CD80+ CD8+ T cells (p=0.003) after surgery were significantly lower than those before surgery. The frequency of lymphocytes after surgery was significantly higher than that before surgery (p=0.013).
The relationship between ICP and overall survival (OS) in each treatment group
The relationships between OS and disease stage, tumor marker, or the frequency of immune cells before treatment were examined.
In the BSC group, there was no association between disease stage and OS (p=0.249). A higher carcinoembryonic antigen (CEA) level was weakly associated with a shorter OS (p=0.053). There was no association between CA19-9 levels and OS (p=0.312). Univariate analysis of ICP revealed that a lower frequency of helper T cells (p=0.001), higher frequency of eTregs (p=0.008), and lower frequency of CD80+ eTregs (p=0.024) was associated with a shorter OS (Figure. 3a).
In the chemotherapy group, no association between disease stage and OS was found (p=0.515). A higher CEA level was associated with a shorter OS (p=0.025). There was no association between CA19-9 levels and OS (p=0.143). Univariate analysis of ICP demonstrated that a lower frequency of CD25+ nTregs before chemotherapy was associated with a shorter OS (p=0.005) (Figure. 3b).
In the surgery group, no association between disease stage and OS was found (p=0.240). There was no association between CEA levels and OS (p=0.599), nor was there an association between CA19-9 levels and OS (p=0.541). Univariate analysis of ICP revealed that lower frequencies of OX40+ helper T cells (p=0.022), CD25+ CD8+ T cells (p=0.017), and OX40+ CD8+ T cells (p=0.032) before surgery were associated with a shorter OS (Figure. 3c).
The relationship between ICP and recurrence-free survival (RFS) in the surgery group
The relationships between RFS and disease stage, tumor marker, or the frequency of immune cells before surgery were examined.
A higher disease stage was weakly associated with a shorter RFS (p=0.053). There was no association between CEA levels and RFS (p=0.800), nor was there an association between CA19-9 levels and RFS (p=0.860). Univariate analysis of ICP revealed that higher frequencies of CD4+ T cells (p=0.009), CCR6+ nTregs (p=0.014), and CXCR3+ nTregs (p=0.012), and lower frequencies of PD-1+ helper T cells (p=0.006), OX40+ helper T cells (p=0.004), CD8+ T cells (p=0.001), and CTLA-4+ CD8+ T cells (p=0.036) before surgery were associated with a shorter RFS (Figure. 4).