Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration

Abstract

Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in naïve and memory cells in circulation. Naïve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration.

Figure 1. Transitions in nuclear plasticity during T-cell development.

a) Representative time series from live-cell imaging of the nuclear boundary in bone marrow, thymocytes and naïve T-cells from H2B-EGFP mice. b) Standard deviation (SD) of mean square fluctuation about the nuclear radius in bone marrow derived cells, thymocytes and naïve T-cells (n = 10 cells each). Error bars are SD. c) Representative images of nuclei stained for DNA pattern with Hoechst 33342 in cells isolated from bone marrow, thymus and spleen. Scale bar 2 µm. d) Quantitative plot scoring for the two different DNA patterns in field images for single positive (SP) and double positive (DP) thymocytes, naïve and memory T-cells (n = 1000 cells each). Error bars are standard error.

Figure 2. Functional correlations between DNA patterns and transcriptional activity in T-cells.

a) Representative images of CD69 staining and DNA and graph showing fraction of activated cells expressing CD69 and having central or peripheral DNA pattern. Error bars are SD. b) Yellow and white arrows point to cells with peripheral and central patterns of DNA, respectively. Naive T-cells were stained for NF-κB, counterstained with Hoechst 33342 and imaged. Representative confocal images from a minimum of 300 cells analyzed. Scale bar 2 µm. The graph shows the fraction of cells with the two different DNA pattern in the three conditions. Data from a minimum of 38 cells are plotted as mean ± SD from two independent experiments. Cells included for analysis were selected as described in text. b).

Figure 3. Higher order chromatin reorganization during in vitro T-cell activation.

a) Representative images of naïve cells and 48 hours post activation (D2) cells stained with Hoechst 33342. Scale bar 2 µm. Quantitative plot scoring for the two different DNA patterns in field images for naïve and activated cells (n = 1000 cells each). b) Nuclear volume (µm³) calculated from 3D reconstruction of confocal z-stack images of nuclei (n = 50 cells each). Inset- nuclear volume (µm³) for naïve cells getting activated from 0–48 hours. Right hand side shows gradual changes in DNA pattern in naïve T-cells at various time points post-activation. Numbers indicate the hours post-activation of cells. Scale bar 5 µm. c) Representative images of activated (D2) cells and activated cells maintained in culture for 5 more days in IL-7 (D7) stained with Hoechst 33342. Scale bar 2 µm. Quantitative plot scoring for the two different DNA patterns in field images for activated and D7 cells (n = 1000 cells). Error bars indicate SE in all panels.

Figure 4. Nuclear deformation response and transmigration properties in T-cells.

a) Schematic of experimental design. Representative images of nuclei stained for DNA pattern with Hoechst 33342 in naïve T-cells without (Control) and with compressive load (Load). Scale bar 5 µm. Graph shows the quantitative plot scoring for the two different DNA patterns in field images in naïve T-cells without (Control) and with compressive load (Load) for increase in aspect ratio (n = 500 cells each). Error bars are SD. b) Representative field-views of cells isolated from spleen (SPL) and lymph node (LN) 15 hr post adoptive transfer of GFP⁺ naïve T-cells. Graph shows mean ± S.D of fraction of GFP⁺ cells with the two different DNA patterns in host spleen and lymph node from three independent experiments. n = 150 per experiment.

Figure 5. Schematic depicting the various stages in T-cell development and the observed differences in the nuclear organization.