Fig 2: Tagln2-/- DCs did not optimally support T cell activation in vitro. a, c Differentiation (a), activation (b), and cytokine secretion (c) of Tagln2-/- BM cells. WT or Tagln2-/- BM cells were cultured with GM-CSF, and then, CD11c+ cell populations were examined at the indicated days (a). Differentiated CD11c+ cells were further activated with LPS or pOVA (323–339), plus OTII T cells. Activation markers (b) and cytokine production (c) were determined. d–f The cells from a were co-incubated with OTII CD4+ T cells in the presence of different doses of pOVA (323–339, 10-3–101 µg/mL), and then, T cell activation (CD69 and CD25 and IL-2, IL-4, and IFN-?) was determined by flow cytometry and ELISA. g Schematic diagram of the experimental setup for G and H. Representative histogram showing the in vitro proliferation of OVA (257–264)-specific CD8+ T cells isolated from C57BL/6 mice administered with pOVA (257–264)-pulsed WT DCs or Tagln2-/- DCs. Isolated CD3+ T cells were stained with CTV and co-incubated with pOVA (257–264)-pulsed WT DCs in vitro for 4 days. h From the supernatant of experiment g, IL-2 or IFN-? production was determined at 24 h by ELISA. All data represent the mean of three experiments ± SEM. *P < 0.05; **P < 0.01

Fig 3: Activation markers CD69 and CD44 are increased under metabolic stress on hepatic CD4 T cells, but not CD8 T cells, while this activation is inhibited in PD1+ CD4 T cells. (A) CD25 expression on blood and liver CD8 T cells under SC or WD for 24 weeks. (n = 5). (B) CD69 expression on blood and on hepatic CD8 T cells after 24 weeks treatment with SC or WD. (n = 5), (***p < 0.001). (C) CD69 expression on blood and hepatic CD4 T cells after 24 weeks SC and WD feeding. (n = 5, *p < 0.05). (D) CD44 expression on blood and intrahepatic CD4 T cells under SC or WD treatment for 24 weeks. (n = 5, **p < 0.01 and ***p < 0.001). (E) CD25 expression on blood and hepatic CD4 T cells under SC or WD. (n = 5), (*p < 0.05 and ***p < 0.001). (F) CD44 and CD69 expression on PD1+ or PD1- CD4 T cells after SC or WD feeding. (n = 5), (*p < 0.05; **p < 0.01; and ***p < 0.001).

Fig 4: Super-enhancers prime T cell activation genes.(A) Gene Ontology (GO) analysis of genes nearest to the ~450 super-enhancers shared by treated and untreated conditions show enrichment for T cell activation genes. P-values shown are Benjamini-Hochberg adjusted p-values. (B) H3K27Ac marks a large super-enhancer around the lineage-determining transcription factor Ets1 in both the No Peptide and 1 µM PCC conditions. The super-enhancer spans the ~600 kbp region shown. (C) The ~400 kbp super-enhancer region encompassing Cd28, Ctla4, and Icos is marked by H3K27Ac in both treated and untreated conditions. (D) Despite being heavily marked by H3K27Ac in both untreated and treated conditions, shared super-enhancers near activation signature genes show a significant gain in H3K27Ac tags in response to stimulation as compared to the shared super-enhancers not proximal to activation signature genes. In other words, basally primed super-enhancers near activation signature genes see significant increases in activity upon stimulation, correlating with increased gene expression at the activation signature genes. (E) Some regions of H3K27Ac deposition required TCR stimulation to pass the super-enhancer threshold, as can be seen here at the ~60 kbp region encompassing BATF, an AP-1 family member. While H3K27Ac is clearly present under basal conditions, there is a substantial increase in enhancer activity upon treatment with 10 µM PCC. (F) Il2ra (CD25) shows increased enhancer activity and formation of a super-enhancer in the treated condition. (G) Similarly, the region surrounding Tbx21 (Tbet) shows substantial increases in activity subsequent to stimulation, resulting in the formation of a super-enhancer. (p-values based on Student’s t test; *p<0.05, **p<0.01, ***p<0.001).DOI: http://dx.doi.org/10.7554/eLife.10134.015

Fig 5: Primed enhancers are pre-existing, but gain activation markers with treatment.(A) Comparing primed enhancers marked by H3K4me2 peaks reveals strong correlation between untreated and treated samples. Normalized tag counts in the No Peptide condition are plotted against those in a 1 µM PCC condition, with red dots coloring those that are more than two-fold up-regulated in the 1 µM PCC condition. The up-regulated enhancers are both few in number and low in tag count. (B) De novo motif finding identifies lineage-determining transcription factor (LDTF) motifs among primed enhancers shared by the five conditions. An ETS motif is most prominent, and a RUNX motif is likewise highly enriched over the randomly selected background. Both ETS and RUNX factors play important roles in T cell development. (C) Among primed enhancers shared by all five conditions, including the untreated condition, pro-inflammatory transcription factor motifs are enriched. An IRF family motif, AP-1 family motif (represented by BATF), and NF-?B motif (represented by REL) are all significantly enriched among shared enhancers marked by H3K4me2. (D). Comparing H3K27Ac tag counts at enhancers in No Peptide as compared to 1 µM PCC treatment reveals that many enhancers see increasing H3K27Ac deposition upon stimulation. Points in red indicate greater than two-fold increase in tags upon treatment. (E) Enhancers that are more active upon stimulation, as determined by greater than two-fold H3K27Ac tags in 1 µM PCC treatment as compared to No Peptide, are enriched for pro-inflammatory transcription factor motifs. BATF, an AP-1 family member, and NF-?B are most prominent. (F) Enhancers that are more active with stimulation are enriched near activation signature genes, as can be seen with this enhancer upstream of the activation signature gene Il2ra (CD25). (G) Enhancers upstream of the activation signature gene Cd69 show an increase in H3K27Ac deposition upon treatment with 1 µM PCC. (H) Genome-wide, deposition of H3K27Ac, a marker of transcription factor activity, reflects increasing TCR signal strength at the binding sites of AP-1 family members, including BATF.DOI: http://dx.doi.org/10.7554/eLife.10134.012