Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine mediated regulation of c-Fos

CD4+ regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) and suppress the immune system. Whether and how metabolite availability in the TME regulates Treg cell differentiation is not understood. Here we measured 630 metabolites in the TME and found that substrates required for the synthesis of sphingolipids, serine and palmitic acid, were enriched. A serine-free diet or a deficiency in Sptlc2, the rate-limiting enzyme catalyzing sphingolipid synthesis, suppressed Treg cell accumulation and inhibited tumor growth. Sphinganine, an intermediate metabolite in sphingolipid synthesis, physically interacted with the transcription factor c-Fos. Sphinganine c-Fos interactions enhanced the genome-wide recruitment of c-Fos to regions near the transcription start sites of target genes including Pdcd1 (encoding PD-1), which promoted Pdcd1 transcription and increased inducible Treg cell differentiation in vitro in a PD-1-dependent manner. Thus, Sptlc2-mediated sphingolipid synthesis translates the extracellular information of metabolite availability into nuclear signals for Treg cell differentiation and limits anti-tumor immunity. To identify mechanisms via which sphinganine promoted the expression of FoxP3, we performed single cell RNA sequencing to analyze the transcriptional profiles of TCR-transgenic OT-II CD4+ T cells. To systematically study the influence of sphinganine on the chromatin accessibility and the binding of c-Fos, c-Jun, and NFAT2, we performed ATAC sequencing and CUT & RUN sequencing.

CD4+调节性T细胞（CD4+ regulatory T cells，简称Treg细胞）会在肿瘤微环境（tumor microenvironment，简称TME）中聚集并抑制机体免疫系统。目前学界尚未明确TME中的代谢物可获得性是否以及如何调控Treg细胞的分化过程。本研究对TME中的630种代谢物进行了检测，发现鞘脂（sphingolipids）合成所需的丝氨酸（serine）与棕榈酸（palmitic acid）在TME中显著富集。无丝氨酸饮食或鞘脂合成限速酶Sptlc2的缺失，均可抑制Treg细胞聚集并延缓肿瘤生长。鞘氨醇（sphinganine）作为鞘脂合成通路中的中间代谢物，可与转录因子c-Fos（transcription factor c-Fos）发生物理相互作用。这种鞘氨醇与c-Fos的相互作用能够增强c-Fos在全基因组范围内靶基因转录起始位点（transcription start sites）附近区域的募集水平，其中涵盖编码PD-1的Pdcd1基因；该过程可在体外以PD-1依赖的方式促进Pdcd1转录，并推动诱导性Treg细胞（inducible Treg cell）的分化。综上，Sptlc2介导的鞘脂合成可将胞外的代谢物可获得性信息转化为调控Treg细胞分化的核内信号，进而限制机体的抗肿瘤免疫（anti-tumor immunity）。为明确鞘氨醇促进FoxP3表达的具体机制，我们通过单细胞RNA测序（single cell RNA sequencing）分析了TCR转基因OT-II CD4+ T细胞（TCR-transgenic OT-II CD4+ T cells）的转录组特征。为系统研究鞘氨醇对染色质可及性（chromatin accessibility）以及c-Fos、c-Jun与NFAT2结合的影响，我们分别开展了ATAC测序（ATAC sequencing）与CUT&RUN测序（CUT & RUN sequencing）实验。