In vitro and in vivo differentiated effector CD8 T cells display divergent histone acetylation patterns within the Ifng locus

Abstract

Epigenetic remodeling of genes encoding effector cytokines that permit accessibility to the transcriptional machinery is a central event in the differentiation of naive T cells into effectors that can attack pathogens and tumors. Covalent modifications of histones that cause a loosening of nucleosomal structures occur not only in promoter regions, but also at upstream and downstream enhancer elements that integrate various cellular stimuli to modulate the rate of transcriptional initiation. This knowledge derives mostly from the analysis of in vitro differentiated effector T cells. Here, we compared acetylation of histone H3 (AcH3) at several sites within the Ifng locus in CD8 T cells that underwent effector differentiation in vitro vs. in vivo. While AcH3 was similar at the proximal promoter, it displayed a reciprocal pattern at two well-characterized upstream and downstream sites. These data suggest that certain epigenetic remodeling events may be artifactual consequences of in vitro culturing conditions, and indicate the importance of using in vivo models to study effector cytokine gene remodeling.

Fig. 1

Histone H3 acetylation (AcH3) within the Ifng locus of in vitro differentiated polyclonal effector CD8 T cells. (A) Naive (CD44^low) CD8 T cells from non-transgenic (NT) LN were primed in vitro with anti-CD3 mAb plus cytokines for the indicated times, restimulated with PMA + I and analyzed for total expression of intracellular IFN-γ protein. Data is expressed in arbitrary units (mean ± SEM), n=6. (B) Expression of IFN-γ mRNA in in vitro primed and PMA + I-restimulated CD8 T cells. Data is expressed as the fold-difference between day 1 or 2 and the day 0 (naive) samples, n=4. (C) Non-restimulated in vitro primed CD8 T cells were directly analyzed by ChIP to measure AcH3 at the Ifng proximal promoter, CNS1 and Intron 3. For each site, data is expressed as the fold-difference between day 1 or 2 and the day 0 samples, n=5. Note that the proximal promoter data was used to generate part of a figure presented in a previous study [15]. (D) Diagram of the Ifng gene locus drawn approximately to scale, with the location of the four exons (thick vertical lines identified by roman numerals) as well as CNS1, the proximal promoter and Intron 3 indicated.

Fig. 2

Histone H3 acetylation within the Ifng locus of in vivo differentiated adoptively transferred TCR Tg effector CD8 T cells. One million naive Bcl-2-overexpressing TCR Tg CD8 T cells were adoptively transferred into viral-HA-infected recipients and recovered from spleens on day 4. (A) Intracellular IFN-γ vs. TNF-α expression following restimulation with cognate HA peptide, representative of n=3 per group. (B) AcH3 within the Ifng locus of non-restimulated purified TCR Tg CD8 T cells. For each site, data is expressed as the ratio between effector and corresponding naive (pre-transfer) samples, n=4. The proximal promoter data was presented in a previous study [15].

Fig. 3

Histone H3 acetylation within the Ifng locus of in vitro vs in vivo differentiated wild type TCR Tg effector CD8 T cells. Naive TCR Tg CD8 T cells from three clone 4 Tg mice were enriched (by depleting CD44^high cells) and portions from each were individually cultured for 4 days with HA peptide plus cytokines (in vitro). The remaining cells were pooled with one portion being retained as the naive (day 0) baseline, and the rest were adoptively transferred into three separate viral-HA-infected recipients (1 × 10⁶ TCR Tg CD8 T cells per transfer) and purified from spleens on day 4 (in vivo). (A) Intracellular IFN-γ vs. TNF-α expression following restimulation with PMA + I, representative of n=3 per group. (B) AcH3 within the Ifng locus of non-restimulated purified TCR Tg CD8 T cells. For each site, data is expressed as the ratio between individual in vitro and in vivo primed TCR Tg effector CD8 T cell samples (n=3 per group) and the pooled naive (day 0 pre-transfer) sample.

Fig. 4

Histone H3 acetylation within the Ifng locus of naturally formed polyclonal effector/memory CD8 T cells. (A) CD44 vs intracellular IFN-γ expression in anti-CD3 mAb-stimulated polyclonal CD8 T cells prepared from NT LN, representative of n=3 per group. (B) AcH3 within the Ifng locus of FACS-sorted non-stimulated polyclonal effector/memory CD8 T cells. For each site, data is expressed as the ratio between individual effector/memory (CD44^high) samples (n=3) and the pooled naive (CD44^low) samples.