Active maintenance of CD8+ T cell naivety through regulation of global genome architecture

Abstract

The differentiation of naive CD8⁺ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8⁺ T cells. We observe that the architecture of the naive CD8⁺ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8⁺ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8⁺ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.

Keywords: CD8+ T cell; CP: Immunology; CP: Molecular biology; T cell memory; chromatin.

Figure 1.. Analysis of higher-order chromatin structures during CTL differentiation.

Sort purified, naive (CD44^loCD62L^hi) CD45.1⁺ CD8⁺ OT-I CTLs were adoptively transferred into CD45.2⁺ congenic C57BL/6J mice prior to recipients being infected with A/HKx31-OVA. Effector (CD44^hi CD62L^lo) and memory (CD44^hi) OT-Is were isolated and sort purified either 10 or 60 days p.i., respectively, and then subjected to Hi-C. Virus-specific CD8⁺ T cells were compared with sort purified CD4⁺CD8⁺ (double-positive) thymocytes from C57BL/6J mice. (A) Eigenvectors calculated at 1Mb resolution for chromosome 17 of naive CTL, with A and B compartments shown in light blue and dark blue, respectively. Minor changes in A/B compartment structure was observed between differentiation states (dashed boxes), compartment structures and proportion of the genome in each compartment was largely conserved with differentiation. (B) Changes in compartment from A to B and B to A with differentiation did not, on average, coincide with changes in gene transcription. Changes in A/B compartment upon naive (blue) to effector (green) differentiation versus average transcript frequency (log counts per million [cpm]) are shown as an example. (C and D) The number and average size of TADs for each differentiation state. (E) The frequency of shared TAD borders between naive and effector Hi-C data determined at 50 kb, 100 kb, and 150 kb bin sizes (F) Heatmaps showing interaction frequency within 50 Mb windows of chr16, for DP, naive, effector, and memory CTL.

Figure 2.. Distinct higher-order chromatin structures within distinct CD8⁺ T cell populations.

(A) MDS plot showing relationship between Hi-C samples derived from double-positive (CD4⁺CD8⁺) thymocytes, naive, effector, and memory OT-1 CD8⁺ T cells. (B–F) Hi-C data (50 kb bins) normalized using ICED method showing interaction frequency at Sox4, Prickle1, Satb1. Prdm1, and Drmta1 loci, respectively, in naive, effector, and memory OT-1 CD8⁺ T cells. Tracks below memory panels show gene structures, with purple arrow highlighting genes of interest and their direction of transcription. Locus coordinates for (B)–(F) are: Sox4 chr13: 28,125,000–30,325,000; Prickle1 chr15: 93,000,000–95,000,000; Satb1 chr17: 51,200,000–53,000,000,. Prdm1 chr10: 43,500,000–45,500,000, and Dmrta1 chr4:88,000,000–90,000,000. Shown below gene tracks are ATAC-seq normalized read counts for each locus in the naive, effector, and memory states Hi-C correlations are shown as pairwise comparisons of binned interaction frequencies (50 kb) for naive and effector (N-E), and naive and memory (N–M) samples, with dotted line indicating 0 on the y axis. (G–K) Normalized RNA-seq counts for each gene loci listed. (L) Quantification of domain changes identified by pairwise correlation (50 kb) analysis depicted.

Figure 3.. Loss and gain of cis-regulatory interactions underscores CTL differentiation-state-specific gene transcription profiles.

(A) Numbers of cis interactions unique to each differentiation state, determined by pairwise comparisons using multiHiCcompare (50 kb resolution, 0.05 false discovery rate). (B) GSEA analysis comparing genes connected by loops enriched in one condition over another (y axis), against RNA-seq data derived from matching samples. Circle sizes reflect adjusted p values (−log10) and color represents normalized enrichment score (NES), with red indicating enrichment versus the first RNA-seq condition listed in pairwise comparison, and blue indicating enrichment is the second RNA-seq condition listed. (C–E) Examples of loci where loops were lost and gained upon differentiation (blue loops are present in naive over effector or memory; red loops are gained on differentiation). Locus coordinates (base pairs) are shown within the naive versus effector circus plot.

Figure 4.. Hi-C loops border active regions containing differentiation-state-specific enhancers.

(A) ATAC-seq signal (log2) indicating open chromatin within and surrounding loops that are present in naive but not effector CTLs, or vice versa. Loops are scaled to occupy 100 kb, and ATAC-seq signal is shown for 100 kb up and downstream of the loop borders. (B) Enrichment of active and poised (H3K4me1⁺ H3K4me2⁺) transcriptional enhancers that occur in naive CD8⁺ T cells but not effector T cells within loops that occur in naive but not effector CD8⁺ T cells (upper panel) and vice versa (lower panel). Enhancers present in both naive and effector T cells (constitutive) are shown for both comparisons. (C and D) Cis interactions connect gene regulatory elements. Circos plots show the gene neighborhood of Satb1 (C) and Klrg1 (D) in naive and effector OT-1 CTLs, respectively. Tracks in order from outside to the center are genes, H3K4me1 (blue), H3K4me2 (light green), H3K4me3 (dark green), ATAC-seq (red), Hi-C interactions (naive over effector CTLs [B] and effector over naive CTLs [C]) shown as ribbons. Locus coordinates are given on the bottom left of the plot. (E) Summary of regions connected by loops present in naive but not effector T cells, or vice versa. (F) Enrichment of transcription factor binding at TEs unique to naive or effector, that were connected by loops enriched in naive over effector, or vice versa. Enrichment was performed using curated transcription factor CHIP-seq data through the CistromeDB Toolbox.

Figure 5.. BACH2 enforces a naive chromatin architecture.

(A) MDS plot showing relationship between naive Bach2^−/− and naive Satb1^m1Anu/m1Anu CD8⁺ T cells (described in Figure 7), Hi-C samples, and naive, effector, and memory OT-1 CD8⁺ T cells. (B) Loss and gain of cis interaction in naive Bach2^−/− CD8⁺ T cells in comparison with WT naive- and virus-specific OT-1 CD8⁺ T cells. (C) GSEA analysis comparing genes connected by loops gained in naive Bach2^−/− CD8⁺ T cells relative to naive WT CD8⁺ T cells against RNA-seq data derived from naive and effector CTLs samples. p values and normalized enrichment score (NES) are shown. (D and E) Examples of changes in looping architecture in naive Bach2^−/− CD8⁺ T cells relative to naive WT CD8⁺ T cells (blue loops are enriched in WT naive over naive Bach2^−/− CD8⁺ T cells and red loops are enriched in naive Bach2^−/− CD8⁺ T cells over WT). (F) Loops that occur in naive Bach2^−/− CD8⁺ T cells but not WT naive CD8⁺ T cells are enriched for active and poised (H3K4me1⁺ H3K4me2⁺) transcriptional enhancers that occur in effector CD8⁺ T cells but not naive T cells. Constitutive enhancers are defined as present in both effector and naive.

Figure 6.. SATB1 maintains CD8⁺ T cell naive chromatin architecture.

(A) Loss and gain of cis interactions in naive Satb1^m1Anu/m1Anu CD8⁺ T cells in comparison with WT naive OT-1 T cells. (B) GSEA analysis comparing genes connected by loops gained in naive Satb1^m1Anu/m1Anu CD8⁺ T cells relative to naive OT-1 T cells against RNA-seq data derived from naive and effector OT-1s. Normalized enrichment score (NES) is shown. (C) Loops lost at the type 1 chemokine locus in naive Satb1^m1Anu/m1Anu CD8⁺ T cells relative to naive OT-1 cells. (D) Hi-C contact maps showing the Ccl5 encoding locus in naive OT-1, Satb1^m1Anu/m1Anu naive, and effector OT-1 CTLs. (E) Loops lost at the Satb1 locus in naive Bach2^−/− CD8⁺ T cells relative to naive OT-1 cells.

Figure 7.. Altered chemokine expression in mice following deletion of cis interacting elements mapped by Hi-C.

(A) Identification of interactions between the Ccl5 gene promoter and previously identified transcriptional enhancers at −5 kb and −20 kb from the Ccl5 transcription start site. Data are presented as a virtual 4C plot, showing naive and effector Hi-C data, with the arrow indicating a zone of increased interaction in effector CTLs. (B) Chromatin accessibility data (mapped by FAIRE) in effector CTL, showing the positioning of CRISPR deletions made in separate mouse lines to remove the −5 and −20 transcriptional enhancers. (C) Wild-type and enhancer deletion mice were infected intranasally with 10⁴ pfu A/HKx31influenza virus, and lymphocytes were collected from the bronchiolar lavage (BAL) fluid on d10 for analysis by flow cytometry to assay CCL4 and CCL5 expression in CD8⁺ and CD4⁺ T cells. (D) Reduced H3K27Ac at the Ccl5 locus in in vitro cultured enhancer deletion effector CTLs. Naive CTL from WT (blue) and −5 (red) and −20 (green) enhancer deletion mice were stimulated with plate-bound αCD3 and αCD28 and cultured for 5 days before ChIP assays were performed to measure histone acetylation at the promoter and enhancers of Ccl5. Data are pooled from three independent cultures, and error bars are SEM. Data are expressed relative to a total input, and statistical significance was determined using a two-tailed t test.