实验方法

构建了黑色素瘤患者细胞系，scRNA-seq+scTCR+CITE-seq(真有钱啊，用了好多抗体）

supplemetary table2 里面有所用CITE抗体，可供参考

一些要点

• 可以参考其CD4+ cell annotation

  
  
  
• Highly expanded clones were distributed among exhausted CD4+ TILs (TTE, TFH/TPE and TProl), but also among TReg TILs, as confirmed by the skewing of TCR diversity in these clusters.
• normalized shannon index: 可用于评价异质性或者clonoality，感觉已经看到好几次了
• 四个黑色素瘤样本中，有两个是高表达MHC-II分子的，提示免疫原性较高？
• 他们假说：肿瘤能够通过MHC-II提呈抗原肽直接激活CD4+ T细胞，从而激活效应T和Treg。验证方法：构建了19+56+38+56个TCR-T（healthy donor CD4+ T）,与他们构建的黑色素瘤患者细胞系进行共培养。看到这里我就觉得，之前老板也提到让我做TCR-T，但我总有一种畏难情绪。看到别人做的工作，觉得自己的态度确实不太端正……加油加油！需要注意的是，他们这里的共培养配对都是autologous的，比如自体的TCR序列与自体肿瘤细胞和PBMC， B， EBV-LCL共培养。
  
  共培养结果：高表达MHC-II分子的肿瘤细胞可诱导肿瘤反应性Treg
  他们看反应性没有看杀伤作用，而是看CD137,CD107a,IFN-g,TNF-a,IL-2等
• IFN-γ可以诱导肿瘤细胞上调MHC-II
• TCR reactivities based on CD137 upregulation of TCR tzransduced T cell

• 是否可以通过APC间接诱导？

  
  
• TCRs were screened against autologous EBV-LCLs pulsed with hundreds of peptides corresponding to: (1) personal neoantigens defined by HLA class I or II prediction pipelines (Methods; Supplementary Table 6) or detected within the HLA class II immunopeptidomes of pdMel-CLs; (2) previously identified melanoma-associated antigens (MAAs), tested as pools of overlapping peptides; or (3) collections of common viral antigens
• HLA class II upregulation as the preferred mechanism in the setting of extreme TMB, since this expression state would endow melanoma cells with the ability to present mutated peptides to CD4+ TILs, thus increasing the chances of engaging immunosuppressive TReg cells.黑色素瘤MHC-II表达高，突变负荷高，可提呈识别的新抗原更多，TIL浸润更多，诱导的Treg越多（可能的思路）

• 总结

  
  Proposed mechanism of HLA class II-driven immune evasion in melanoma: as well as being able to the indirectly elicit CD4+ responses through APCs, HLA class IIpos melanoma with extreme TMB can directly trigger and expand immunosuppressive TReg cell clones (delineated by different TCR colours) through exposure of HLA class II–neoantigen complexes.

• 黑色素瘤免疫逃逸：对于低表达MHC-II分子的，可以通过APC提呈诱导Treg。对于高表达MHC-II分子的，可以直接诱导肿瘤特异性Treg
  -TCR-T构建方法

  
  

1. Furin linker
2. 用小鼠的骨架区，避免错配

第一遍看完了，感觉这篇工作量大，思路也很清晰，很多方法也值得学习

值得二刷