Background
IRG1——encodes the metabolic enzyme aconitate decarboxylase (ACOD1) that catalyzes the production of ITA.
The alterations of TME cause a metabolic reprogramming of tumor-associated immune cells, such as T cells and macrophages. One marked metabolic change is the rapid accumulation of a previously understudied metabolite, itaconic acid (ITA), from a barely detectable low level in resting cells to 3 to 4 mM in classically activated macrophages.
Results
1. Irg1 deficiency inhibits tumor growth in immune-competent mice
首先明确IRG1在cancer/tumor中的高表达，可以预示着IRG1起到促癌作用:
Irg1 KO mice model 在小鼠体内验证IRG1可以抑制tumor发生（多种实体瘤模型）
已知IRG1主要促进Acod1在髓系细胞中的ITA积累，所以进一步针对性KO髓系细胞Irg1。
2. Irg1 deficiency reverses the immunosuppressive TME
为什么Irg1 KO 可以抑制肿瘤发展？可能跟ITA代谢产物有关，所以继续检测Irg1对肿瘤微环境的影响。
B16-F10 tumors grown in Irg1+/+ and Irg1−/−
Irg1 mRNA expression was readily detected in myeloid cells, particularly in macrophages and neutrophils. Next, we used UMAP clustering and divided the population of “MФ and monocytes” into six distinct clusters.
Irg1 deficiency in mice led to the increase of Vcan+ monocytes and proinflammatory macrophages and a decrease of Vegfa+ macrophages in the TME of B16-F10 tumors, indicating that Irg1 may regulate TAM polarization during tumor development.
进一步通过流式验证：
总之，Irg1 KO可以改变TME中的免疫细胞命运，缓解肿瘤造成的免疫抑制的状态；也验证了Irg1已经被报道的免疫抑制的生物学功能。
3. Irg1-deficient macrophages acquire more proinflammatory features and promote antigen presentation and T cell chemotaxis
进一步的问题是，Irg1 KO如何改变TAM的极化和功能？
对以上6个亚群的细胞进行进一步统计学分析，发现：
Vcan+ monocytes exhibited up-regulation of IFN-stimulated genes. Proinflammatory macrophages exhibited up-regulation of antigen presentation and T cell migration.
流式确认antigen presentation能力增强：
qRT-PCR确认相关基因表达水平：
进一步在transwell和in vivo验证了irg1对T cell migration的影响：
4. Irg1-deficient macrophages promote T cell trafficking in a Tet2-dependent manner
进一步研究Irg1如何影响T cell migration and trafficking？
已知TET2通过甲基化修饰影响CXCL9,10,11炎症因子的表达，如何证明Irg1是通过TET2影响CXCL从而影响 T cell migration and trafficking？
首先通过免疫组化确认Irg1−/−小鼠tumor位置的5hmC production (TET的催化产物)
然后利用hMeDIP-qPCR确认5hmC enrichment at the promoter regions of cxcl9,10 genes
接下来，通过TET2 mutant mice确认mutant 来源的BMDM无法被ITA抑制CXCL表达
5. Deletion of Irg1 in mice enhances the efficacy of anti–PD-(L)1 immunotherapy
B16-F10 melanoma cells were inoculated subcutaneously into Irg1+/+ and Irg1−/− mice that were then subjected to PD-L1 blockade.
The combination of anti–PD-1 and Irg1 deficiency showed the most effective tumor inhibition and prolonged survival.
进一步研究真实病例，发现：PD-1 resistance 病人中Irg1 高表达，并和CD8 负相关。（与上述小鼠模型中一致）
6. Irg1 deficiency in macrophages, but not neutrophils, contributes to the enhanced antitumor immunity
ScRNA-seq results revealed that Irg1 was also expressed in tumor-associated neutrophils (TANs) in the TME of B16-F10 tumors.
如何确认到底是M还是N中的Irg1发挥作用？
首先体外antibody delete Macrophage 或者 Neutrophil：
很奇怪：在cre-组中，delete TAM和TAN反倒能够抑制肿瘤？？？
进一步的，adoptive transfer assay 分别在肿瘤中移植Macrophage 或者 Neutrophil：
最后，为了显示irg1的对肿瘤免疫治疗的应用前景，
combined the macrophage adoptive transfer assay together with anti–PD-L1 treatment in wild-type mice bearing B16-F10 melanoma.
除此之外，在胰腺癌模型上再次验证(known to be resistant to T cell–based immunotherapy)
(TUNEL:是一种用来检测细胞凋亡(apoptosis)的分析方法)
