Tumor-Associated Macrophages Promoting PD-L1 Expression in Regulatory B Cells Through the CXCL12/CXCR4 Axis in Human Hepatocellular Carcinoma

Background: The inflammation-related tumor microenvironment (TME) is a major driving force of hepatocarcinogenesis. Many studies have focused on different immune cells; however, cell-to-cell communication within the TME of hepatocellular carcinoma (HCC) has not been extensively investigated yet.Methods: We aimed to investigate cell-to-cell communication among HCC-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and infiltrated regulatory B cells (Bregs) through re-analyzing HCC single-cell RNA-seq data, and to confirm this communication using multiple-color immunohistochemistry (mIHC) staining and software-based single-cell data analysis.Findings: We found a subset of IgA-secreting Bregs with PD-L1 expression (PD-L1+ Bregs), mainly located in adjacent HCC tissues. In co-localization with PD-L1+ Bregs, a subset of TAMs with high expression of CXCL12 (CXCL12+ TAMs) was also mainly located in adjacent HCC tissues. Moreover, CXCL12+ TAMs can be stimulated in vitro using an HCC conditional medium. Using CellChat analysis and mIHC staining, CXCL12+ TAMs were found to be first recruited by CAFs through a CD74/macrophage migration inhibitory factor (MIF) pattern, and further differentiated into TGF-β-enriched tissues. Furthermore, CXCL12+ TAMs recruited PD-L1+ Bregs via the CXCL12/CXCR4 axis, and CXCR4 expression was significantly positively correlated to PD-L1 expression in PD-L1+ Bregs.Interpretation: Immunosuppressive HCC TME involving cell-to-cell communications comprised MIF-secreting CAF, CXCL12-secreting TAMs, and PD-L1-producing Bregs, and their regulation could be promising therapeutic targets in future immunotherapy for human HCC.Funding Information: This work was supported by grants from the National Natural Science Foundation (Grant Number: 81772569 and 81972266 to R.J.).Declaration of Interests: The authors declared no competing interests.Ethics Approval Statement: All experiments were approved by the Ethics Committee of The Affiliated Drum Tower Hospital of Nanjing University Medical School (approval number: 2021-304). All patients gave informed consent to participation.