Cutaneous diffuse large B-cell lymphoma induce a macrophage immunosuppressive phenotype through IL-10 secretion Free

Background : Tumor associated macrophages (TAM) may endorse a large range of phenotype and functions owing to their plasticity and the specificities of each tumor's microenvironment (TME). Enrichment in TAM has been associated with worse prognosis in diffuse large B-cell lymphoma (DLBCL), but disease diversity in term of genetics and TME prevents to draw disease-scale conclusions, therefore TAM functions should be specifically studied in narrowed, homogeneous models. We previously demonstrated that primary cutaneous DLBCL (PCDLBCL-LT), a MYD88-mutated disease genetically falling into the group of “MCD” DLBCL, were particularly enriched in PD-L1+ CD163+ TAM – these markers being linked to immunosuppressive and pro-tumoral macrophage behaviors. This led us to hypothesize that dialog between tumor B cells and TAM may be a critical feature of PCDLBCL-LT, whose study would helpt to improve the biological understanding of this rare disease and unravel specific therapeutical vulnerabilities.

Aims : To describe the changes induced by PCDLBCL-LT tumor B-cells on macrophages (MF) and the effects of MF on tumor B-cells, as well as to identify the main factors involved in this interplay.

Methods : We used ARSI cell line, a unique PCDLBCL-LT cell line previously established in our institute, along with cells from 4 PCDLBCL-LT patients-derived xenograft (PDX). For MF, we used primary monocytes from healthy donors and THP-1 monoblastic cell line, differentiated following well established cytokines pipelines. MF were co-cultured for 1 to 4 days with tumor cells, according to the analysis, or with tumor cell supernatant. MF changes were analyzed by flow cytometry (FCM), using a panel of canonical MF activation markers (CD163, PD-L1, CD80, HLA-DR), and by RNAseq in order to identify gene signature changes. LegendPlex assay was used to identify cytokines levels in cells media. Proliferation assays were used to study the effects of activated MF on T-cells from healthy donors. Finaly, supportive effects of MF on tumor B-cell were exploring by assessing their proliferation in co-culture (by FCM) and the cytotoxicity induced by doxorubicin.

Results : We first showed that ARSI cell line promotes phenotypic changes in MF consisting in an increase in CD163 (a major M2-like marker) and PD-L1 surface expression, along with a slight increase in CD80 and a decrease in HLA-DR expression (both being M1-like markers) when compared with MF cultured alone. RNAseq of MF co-cultured with ARSI showed a differential expression of many genes translating, in GSEA, in an enrichment in the “C1Q signature” according to MoMac-VERSE (this gene expression profile being associated with immunosuppressive functions). LegendPlex analysis in medium of MF co-cultured with ARSI identified an increase in IL-10, a pivotal immunosuppressive cytokine, at the protein level in comparison with MF cultured alone. Finally, MF cultured with ARSI showed a stronger ability to inhibit T-cell division in culture, confirming their immunosuppressive phenotype. We also showed that the MF phenotypic changes induced by ARSI (notably the pivotal CD163 and PD-L1 increased expression) were also found when co-cultured with PCDLBCL-LT cells from PDX. On the contrary, MF co-cultured with Ri-1, SUHDL4 and U2932 did not exhibit any significant phenotypic changes, while co-culture with the MYD88-mutated OCI-Ly3 cell line reproduce FCM changes similar to those induced by ARSI, suggesting that these changes are disease-specific and may be linked to genetics. We also showed that soluble factors produced by tumor B cells were implicated in MF changes by demonstrating that culture with tumor supernatant are sufficient to promote an increase in CD163 expression and drop in HLA-DR expression. We hypothesized and confirmed that IL-10 was involved in this effect by showing that IL-10RA blockade in co-culture could fully reverse the phenotypic changes induced by ARSI cells. Finally, we demonstrated that MF exhibit strong supportive effect on ARSI cells by increasing their proliferation and reducing cell death under exposure of doxorubicin. Survival of PCDLBCL-LT cells from PDX were also improved when co-cultured with MF.

Conclusions : PCDLBCL-LT exhibit close interplay with MF, tumor cells favoring acquisition of immunosuppressive phenotype while macrophages enhance tumor cell survival and drug resistance. We also identified IL-10 as a potent key-driver of MF behavior within the LME of DLBCL.