Abstract
Glioblastoma multiforme (GBM) is a heterogeneous malignant brain tumor, the pathological incidence of which induces the accumulation of tumor-infiltrating lymphocytes (TILs). As a tumor suppressor gene, LRRC4 is absent in GBM cells. Here, we report that the recovery of LRRC4 in GBM cells inhibited the infiltration of tumor-infiltrating regulatory T cells (Ti-Treg), promoted the expansion of tumor-infiltrating effector T (Ti-Teff) cells and CD4+CCR4+ T cells, and enhanced the chemotaxis of CD4+CCR4+ T cells in the GBM immune microenvironment. LRRC4 was not transferred into TILs from GBM cells through exosomes but mainly exerted its inhibiting function on Ti-Treg cell expansion by directly promoting cytokine secretion. GBM cell-derived exosomes (cytokine-free and programmed cell death 1 containing) also contributed to the modulation of LRRC4 on Ti-Treg, Ti-Teff, and CD4+CCR4+ T cells. In GBM cells, LRRC4 directly bound to phosphoinositide-dependent protein kinase 1 (PDPK1), phosphorylated IKKβser181, facilitated NF-κB activation, and promoted the secretion of interleukin-6 (IL-6), CCL2, and interferon gamma. In addition, HSP90 was required to maintain the interaction between LRRC4 and PDPK1. However, the inhibition of Ti-Treg cell expansion and promotion of CD4+CCR4+ T cell chemotaxis by LRRC4 could be blocked by anti-IL-6 antibody or anti-CCL2 antibody, respectively. miR-101 is a suppressor gene in GBM. Our previous studies have shown that EZH2, EED, and DNMT3A are direct targets of miR-101. Here, we showed that miR-101 reversed the hypermethylation of the LRRC4 promoter and induced the re-expression of LRRC4 in GBM cells by directly targeting EZH2, EED, and DNMT3A. Our results reveal a novel mechanism underlying GBM microenvironment and provide a new therapeutic strategy using re-expression of LRRC4 in GBM cells to create a permissive intratumoral environment.
Highlights
Glioblastoma multiforme (GBM) is the most common malignant brain tumor and has a median survival of only 14.6 months even after treatment
The conditioned medium from primary cultured GBM and astrocytoma cells transfected with pcDNA3.1 LRRC4 induced much greater CD4+CCR4+ T cell chemotaxis and expansion than that from untransfected LRRC4 control cells (Figures 1C,D); the expansion of tumor-infiltrating CD4+CD25+Foxp3+ Treg cells (Ti-Treg) was inhibited (Figure 1E), especially the proportion of CD4+CD25+CD127−neuropilin− Ti-iTreg cells (Figure 1F, CD127 expression inversely correlates with Foxp3 expression, and the combined use of CD4+, CD25+, and CD127− can define the Treg cell population with suppressive functions), but drove CD4+ tumor-infliltrating effector T cells (Ti-Teff) expansion (Figure 1G)
We detected the effect of the conditional medium derived from IDH1wt U251 Tet-on-LRRC4 cells on CD4+CCR4+ T cells, tumor-infiltrating regulatory T cells (Ti-Treg) cells and tumor-infiltrating effector T (Ti-Teff) cells and obtained results that were consistent with those obtained for primary cultured GBM cells (Figures S1C–F in Supplementary Material)
Summary
Glioblastoma multiforme (GBM) is the most common malignant brain tumor and has a median survival of only 14.6 months even after treatment. The pathological incidence of a brain tumor induces the accumulation of tumorspecific immune cells, which constitute a part of the brain tumor mass. TILs include CD4+CD25+FoxP3+ regulatory T (Treg) cells, effector T (Teff) cells, and cytotoxic T cells, among others [2,3,4,5]. Each of these TIL subsets exerts different functions and secretes specific cytokines that can affect tumor growth. Tumor-infiltrating Treg cells expressing FoxP3 produce IL-10 and exert antitumor effects by inhibiting tumor-infiltrating effector T cells in the tumor microenvironment [6,7,8,9,10]. A detailed understanding of communication between GBM cells and tumor-specific TILs in the GBM microenvironment will help to unveil the key regulatory hub of immunosuppressive mechanisms and contribute to GBM immunotherapy
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