Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure

Immunity, 2023

Background:

Tex cells are epigenetically programmed by the transcription factor Tox. This Tox-dependent program allows exhausted CD8+ T (Tex) cells to persist during chronic viral infection and cancer but precludes further developmental plasticity toward functional effector (Teff) or memory (Tmem) CD 8+ T cells.

Tcf1 (Tcf7)-expressing progenitor-intermediate ‘‘effector-like’’ subset (Tex int)- terminally exhausted CD8+ T cells (Tex term) upon entering peripheral tissues.

Results:

1. Tox restrains Stat5a activity in virus-specific CD8+T cells during chronic infection

基于已知Tox-Tex关系，发现新的Tox相关信号通路

model：wild-type (WT) and Tox-deficient CD8+ T cells

转录因子TF相互作用预测分析工具：

  A. Ingenuity pathways analysis (IPA)

跟着Cell学作图| 11.Ingenuity Pathway Analysis(IPA) - 知乎 (zhihu.com)

  B. Taiji rank analysis

https://www.science.org/doi/10.1126/sciadv.aav3262

2. Stat5a reduces Tox expression and fosters effector-like CD8+ T cell differentiation during chronic viral infection

（长期感染模型，耗竭建立阶段）

过表达 in vivo model

流式分析 FlowSOM clusters

基因敲除 in vivo model

Stat5 fosters differentiation of the effector-like Ly108-/Tim-3+ subset

3. Constitutive Stat5a activation promotes an epigenetic state with features of effector and exhausted CD8+ T cells during chronic infection

Stat5如何调控Tox？是否通过染色质表观调控？

ATAC-seq 染色质开放性测序技术

一文详解ATAC-seq原理+读图：表观遗传的秀儿 - 知乎 (zhihu.com)

进一步通过PCA、K-means 聚类分析，其中关键是找到阴性（P14 Empty）和阳性（Eeff，通过急性感染T cell获得）对照，

发现：the altered epigenetic program in P14 STAT5CA cells was consistent with a shift toward effector biology at a stage when epigenetic imprinting of exhaustion typically occurs.

ATAC-seq

4. Constitutive Stat5a activation promotes a durable effector-NK-like transcriptional state with improved therapeutic potential

Stat5对耗竭维持的作用？

day 27 p.i.

详细的细胞亚型分析：

scRNA-seq

肿瘤治疗/耗竭逆转的潜力？

荷瘤小鼠治疗模型（day 10）

5. Stat5 is essential for Tex int cell formation and response to PD-L1 blockade

Stat5 KO model for fully established exhaustion

PD-L1 blockade did not rescue Tex int cellformation

详细转录因子表观遗传分析：

CITE-seq 

一文讲明白ChIP-seq（上）：高分文章里为什么做ChIP-seq? (qq.com)

一文讲明白ChIP-seq（下）：这些文献里ChIP-seq结果图是啥意思？ (qq.com)

CITE-seq

6. Temporal reactivation of Stat5 in Tex cells drives Tex int cell accumulation and synergizes with PD-L1 blockade

Temporal reactivation of Stat5 in Tex cells：通过IL2实现？why？

IL2-Stat5 通路

temporary activation of IL2

orthogonal IL-2:IL2Rb system：对T细胞进行基因改造使之表达受体ortho-hIL-2Rβ，这种受体只能被ortho-hIL-2激活

PD-L1 combined

7. Temporal reactivation of Stat5 in Tex progenitors enables functional recovery and partial epigenetic rewiring toward the Teff-Tmem lineage upon rechallenge

Stat5 could rewire the epigenetic program of established Tex cells？

Teff-Tmem

(慢性感染模型：LCMV clone 13; 急性感染模型：LCMV Armstrong) 

IL2/αPD-L1 could rescue the exhausted T cells

ATAC-seq

Methods:

mice：

A. Six-week old C57BL/6 female mice (CD45.2) 细胞移植受体小鼠

B. P14 TCR transgenic mice expressing a TCR specific for the LCMV Db gp33-41 peptide