SATB1 is a key regulator of quiescence in stem-like CD8+ T cells

Cytotoxic T-cell immunity in response to chronic infection and cancer is sustained by a self-renewing population known as the progenitor CD8+ T (TPRO) cells. These TPRO cells exhibit stem-like characteristics, including quiescence, multipotency, and self-renewal, which are the cardinal features of memory T cells. However, the mechanisms that sustain their stem-like properties under chronic antigen stimulation remain unclear. In this study, we identified SATB1 as a shared feature that is specifically enriched in both TPRO and memory CD8+ T cells. While the roles of SATB1 in stem-like CD8+ T cells remain unknown, its function as an epigenetic regulator in promoting quiescence in hematopoietic stem cells led us to hypothesize that SATB1 plays a pivotal role in regulating the stemness of TPRO and memory CD8+ T cells. To test this hypothesis, we employed CRISPR-mediated gene editing to delete the Satb1 gene specifically in CD8+ T cells. During chronic LCMV infection, we found that SATB1-deficient CD8+ T cells failed to maintain the TPRO subset and instead showed an enhanced transition toward terminally differentiated cells. Similarly, SATB1 deficiency during acute viral infection impaired the formation of memory CD8+ T cells. Mechanistically, our multi-omic assays revealed that SATB1 regulates the chromatin accessibility, transcriptional activities, and genomic architecture of stemness-associated genes, such as Tcf7, Bach2, and Myb. Overall, our results underscore the critical role of SATB1 in maintaining the transcriptional and epigenetic features of stem-like CD8+ T cells, shedding light on the previously unappreciated regulatory mechanisms that sustain the stemness of antigen-specific CD8+ T cells. Overall design: Hi-C libraries were prepared using a modified three-enzyme Hi-C (3e Hi-C) protocol103. Wild-type (WT) and CD8CreSatb1fl/fl (KO) mice were infected with LCMV Clone 13 and sacrificed at day 21 post-infection. Antigen-specific CD8? T cells were sorted, and 300,000 to 500,000 cells per sample were collected for Hi-C. Cells were crosslinked with 1% formaldehyde for 10 minutes at 25 °C and quenched with 2.5 M glycine. Fixed cells were lysed in 10 mL of ice-cold lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40) supplemented with protease inhibitors and incubated at 4 °C for 1 hour. Nuclei were pelleted and permeabilized in 0.1% SDS in CutSmart buffer (NEB) at 65 °C for 10 minutes, followed by quenching with Triton X-100 to a final concentration of 1%. Chromatin was digested with the restriction enzymes FatI, CviQI, and BfaI (New England Biolabs) at 37 °C for 20 minutes. Digestion was stopped by two washes with buffer containing 10 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100. DNA ends were filled in using Klenow enzyme in the presence of dCTP, dGTP, dTTP, and biotin-14-dATP to label ligation junctions. Proximity ligation was performed using T4 DNA ligase under dilute conditions to promote intranuclear ligation. After reverse crosslinking, DNA was sheared to 200–700 bp fragments using a Diagenode Bioruptor. Fragmented DNA was end-repaired, and biotinylated ligation products were purified using Dynabeads MyOne Streptavidin C1 beads (Invitrogen). Sequencing libraries were constructed by ligating Illumina-compatible paired-end adapters and performing PCR amplification. Size selection (200–700 bp) was performed using E-gel electrophoresis, and libraries were sequenced on an Illumina NovaSeq 6000 platform with 50 bp paired-end reads.

慢性感染与癌症诱导的细胞毒性T细胞免疫，由一类名为祖细胞样CD8+ T细胞（progenitor CD8+ T cells，TPRO）的自我更新群体所维持。这类TPRO细胞具有干细胞样特性，包括静息状态、多能性与自我更新能力，这些均为记忆性CD8+ T细胞的核心特征。然而，在慢性抗原刺激下维持其干细胞样特性的分子机制仍不明确。本研究中，我们鉴定出SATB1是一类特异性富集于TPRO细胞与记忆性CD8+ T细胞中的共同特征分子。尽管SATB1在干细胞样CD8+ T细胞中的功能尚未阐明，但其作为表观遗传调控因子促进造血干细胞静息的功能，使我们提出假说：SATB1在调控TPRO细胞与记忆性CD8+ T细胞的干细胞干性中发挥关键作用。

为验证该假说，我们采用CRISPR介导的基因编辑技术，特异性敲除CD8+ T细胞中的Satb1基因。在慢性淋巴细胞脉络丛脑膜炎病毒（LCMV）Clone 13感染模型中，我们发现SATB1缺陷的CD8+ T细胞无法维持TPRO细胞亚群，反而向终末分化细胞的转化进程增强。类似地，急性病毒感染过程中SATB1缺陷会损伤记忆性CD8+ T细胞的形成。从机制层面而言，我们的多组学分析揭示，SATB1可调控与干细胞干性相关基因（如Tcf7、Bach2与Myb）的染色质开放性、转录活性以及基因组三维结构。综上，本研究结果证实了SATB1在维持干细胞样CD8+ T细胞的转录与表观遗传特征中的关键作用，为此前未被揭示的、维持抗原特异性CD8+ T细胞干性的调控机制提供了新的研究视角。

整体实验设计：采用改良的三酶法Hi-C（3e Hi-C）protocol103制备Hi-C文库。将野生型（WT）与CD8CreSatb1fl/fl（KO）小鼠感染LCMV Clone 13，于感染后第21天处死小鼠。分选抗原特异性CD8+ T细胞，每份样本收集30万至50万个细胞用于Hi-C实验。细胞于25℃下用1%甲醛交联10分钟，随后用2.5M甘氨酸终止交联反应。固定后的细胞在10mL冰浴裂解液（10mM Tris-HCl，pH 8.0、10mM NaCl、0.2% NP-40，添加蛋白酶抑制剂）中裂解，于4℃孵育1小时。离心收集细胞核后，在含0.1% SDS的CutSmart缓冲液（NEB）中于65℃透化10分钟，随后加入Triton X-100使其终浓度为1%以淬灭SDS。使用限制性内切酶FatI、CviQI与BfaI（New England Biolabs）在37℃消化染色质20分钟，通过两次使用含10mM NaCl、1mM EDTA与0.1% Triton X-100的缓冲液洗涤以终止消化。使用Klenow酶，在dCTP、dGTP、dTTP与生物素-14-dATP存在下填补DNA末端，以标记连接位点。在稀释条件下使用T4 DNA连接酶进行近距离连接反应，以促进核内连接。逆转交联后，使用Diagenode Bioruptor将DNA剪切为200–700bp的片段。对碎片化DNA进行末端修复，再使用Dynabeads MyOne Streptavidin C1磁珠（Invitrogen）纯化生物素标记的连接产物。通过连接Illumina兼容的双端接头并进行PCR扩增构建测序文库。采用E-gel电泳进行片段大小筛选（200–700bp），随后在Illumina NovaSeq 6000平台上进行50bp双端测序。