Memory CD4+ T cells sequentially restructure their 3D genome during stepwise activation.

Background: CD4+ T cells are a highly differentiated cell type that maintain enough transcriptomic plasticity to cycle between activated and memory statuses. How the 1D chromatin state and 3D chromatin architecture support this plasticity is under intensive investigation.

Methods: Here, we wished to test a commercially available Hi-C kit (Arima Genomics Inc.) to establish whether published performance on limiting cell numbers from clonal cell lines copies across to a primary immune cell type. We achieved comparable contact matrices from 50,000, 250,000, and 1,000,000 memory CD4+ T-cell inputs. We generated multiple Hi-C and RNA-seq libraries from the same biological blood donors under three separate conditions: unstimulated fresh , IL-2-only stimulated, and T cell receptor (TCR)+CD28+IL-2-stimulated, conferring increasingly stronger activation signals. We wished to capture the magnitude and progression of 3D chromatin shifts and correlate these to expression changes under the two stimulations.

Results: Although some genome organization changes occurred concomitantly with changes in gene expression, at least as many changes occurred without corresponding changes in expression. Counter to the hypothesis that topologically associated domains (TADs) are largely invariant structures providing a scaffold for dynamic looping contacts between enhancers and promotors, we found that there were at least as many dynamic TAD changes. Stimulation with IL-2 alone triggered many changes in genome organization, and many of these changes were strengthened by additional TCR and CD28 co-receptor stimulation.

Conclusions: This suggests a stepwise process whereby mCD4+ T cells undergo sequential buildup of 3D architecture induced by distinct or combined stimuli likely to "prime" or "deprime" them for expression responses to subsequent TCR-antigen ligation or additional cytokine stimulation.