Regulatory T cells induce a suppressive immune milieu and promote lymph node metastasis in intrahepatic cholangiocarcinoma

Abstract

Background: Emerging evidence indicates that immunogenicity plays an important role in intrahepatic cholangiocarcinoma (ICC). Herein, we systematically evaluated the clinical relevance of immunogenicity in ICC.

Methods: Highly immunogenic ICCs identified in the public dataset and the Cancer Immunome Atlas (TCIA) were assessed to determine the prognostic impact of immunogenicity in ICC and key components after curative resection. We also investigated the clinical relevance of the immune milieu in ICC.

Results: Using the Gene Expression Omnibus dataset 89749 and TCIA, we identified CD8⁺/forkhead box P3 (FoxP3)⁺ tumour-infiltrating lymphocytes (TILs), T-cell immunoglobulin and mucin domain 3 (TIM-3) and human leukocyte antigen-A (HLA-A) in highly immunogenic ICCs. Immunohistochemical analysis of the in-house cohort showed that intratumoral FoxP3⁺ TILs correlated with CD8⁺ TILs (P = 0.045, Fisher's exact test) and that high FoxP3⁺/CD8⁺ ratio (FCR) was an important marker for poor survival (P < 0.001, log-rank test). Furthermore, the FCR was higher in tumour-free lymph nodes in ICCs with lymph node metastases than in those without lymph node metastases (P = 0.003, Mann-Whitney U test).

Conclusions: FCR should be considered an important biomarker that represents the immune environment of ICC based on its potentially important role in tumour progression, especially lymph node metastasis.

Fig. 1. Immunophenogram analysis reveals the characteristics of highly immunogenic ICCs.

a Representative immunophenograms of highly or weakly immunogenic ICCs based on metagene analysis using the TCIA. Immunophenoscore ranges from 0 to 10 scale based on the sum of the weighted average z-score of the four categories. Act CD4/CD8 activated CD4 ⁺ /CD8 ⁺T cells, B2M beta-2 microglobulin, CP checkpoints/immunomodulators, CTLA4 cytotoxic T-lymphocyte-associated protein 4, EC effector cells, HLA human leukocyte antigen, IOCS induced T-cell co-stimulator, LAG3 lymphocyte activation gene 3, MDSC myeloid-derived suppressor cells, MHC major histocompatibility complex, PDCD1 programmed cell death 1, PDCD1LG2 programmed cell death 1 ligand 2, SC suppressor cells, TAP1/2 transporter associated with antigen processing 1/2, Tem CD4/CD8 cells effector memory CD4⁺/CD8⁺ T cells, TIM-3 T-cell immunoglobulin and mucin domain-containing protein 3, Treg regulatory T cells. b Heatmap of 48 fluke-negative ICCs. Highly immunogenic ICCs were characterised based on effector cells, suppressor cells and MHC-related molecules. ICC intrahepatic cholangiocarcinoma.

Fig. 2. Immunohistochemical analysis reveals the characteristics of local immune milieu of highly immunogenic ICCs.

a Upper images show representative CD8⁺ TIL counts in ICCs. Left side shows low counts of CD8⁺ TILs and right side high counts of infiltrating TILs. Lower bar graphs show the distribution of CD8⁺ TIL counts in intra-tumour and stroma of ICC. b Boxplot showing that CD8⁺ TIL counts are higher in the stroma than in the tumour. The upper and lower bars represent 5% and 95%, respectively. ***P < 0.001. c Upper images show representative FoxP3⁺ TIL counts in ICCs. Left side shows low counts of FoxP3⁺ TILs and right side high counts of infiltrating TILs. Lower bar graphs show the distribution of FoxP3⁺ TIL counts in intra-tumour and stroma of ICC. d Boxplot showing that FoxP3⁺ TIL counts are higher in the stroma than in the tumour. The upper and lower bars represent 5% and 95%, respectively. ***P < 0.001.

Fig. 3. Prognostic analysis with tumour-infiltrating lymphocytes (TILs) in intra-tumour of ICCs.

a CD8⁺ TILs, b FoxP3⁺ TILs, c combination of CD8⁺ and FoxP3⁺ TILs and d FoxP3⁺/CD8⁺ TIL ratio.

Fig. 4. FoxP3⁺/CD8⁺ TIL ratio of primary lesions and lymph nodes.

a Dot plot showing that the FoxP3⁺/CD8⁺ TIL ratio (FCR) is higher in metastatic lymph nodes than in primary lesions. ***P < 0.001. b Dot plot showing that the FCR in TFLN is higher in patients with ICC and lymph node metastasis than in those without lymph node metastasis. One FCR value in patients with ICC and lymph node metastasis was not shown in this figure because of outlier. TFLN tumour-free lymph node, **P < 0.01. c Dot plot showing the relatively strong correlation between the FCR in TFLN and that in the primary lesions (P = 0.01, rho = 0.478, Spearman’s rank correlation coefficient). d Dot plot showing the relatively strong correlation between the FCR in TFLN and the number of metastatic lymph nodes (P = 0.001, rho = 0.571, Spearman’s rank correlation coefficient).