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. 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.