Dampening of death pathways by schnurri-2 is essential for T-cell development

Abstract

Generation of a diverse and self-tolerant T-cell repertoire requires appropriate interpretation of T-cell antigen receptor (TCR) signals by CD4(+ ) CD8(+) double-positive thymocytes. Thymocyte cell fate is dictated by the nature of TCR-major-histocompatibility-complex (MHC)-peptide interactions, with signals of higher strength leading to death (negative selection) and signals of intermediate strength leading to differentiation (positive selection). Molecules that regulate T-cell development by modulating TCR signal strength have been described but components that specifically define the boundaries between positive and negative selection remain unknown. Here we show in mice that repression of TCR-induced death pathways is critical for proper interpretation of positive selecting signals in vivo, and identify schnurri-2 (Shn2; also known as Hivep2) as a crucial death dampener. Our results indicate that Shn2(-/-) double-positive thymocytes inappropriately undergo negative selection in response to positive selecting signals, thus leading to disrupted T-cell development. Shn2(-/-) double-positive thymocytes are more sensitive to TCR-induced death in vitro and die in response to positive selection interactions in vivo. However, Shn2-deficient thymocytes can be positively selected when TCR-induced death is genetically ablated. Shn2 levels increase after TCR stimulation, indicating that integration of multiple TCR-MHC-peptide interactions may fine-tune the death threshold. Mechanistically, Shn2 functions downstream of TCR proximal signalling compenents to dampen Bax activation and the mitochondrial death pathway. Our findings uncover a critical regulator of T-cell development that controls the balance between death and differentiation.

Figure 1. Increased TCR-induced death in Shn2^−/− thymocytes

a, Representative plots of total thymocytes (mean ± standard devation, n>10). b, Immunoblot of phosphorylated and total ERK1/2 in DP thymocytes after CD3 crosslinking. c, Immunoblot of NFATc1 in DP thymocytes after PMA/Ion or CsA treatment for 30 min. d, DP thymocytes stimulated with platebound αCD3/αCD28 (d) orαCD3 for 20 hours (f), or left unstimulated for indicated times (e). g, DP thymocytes stimulated with platebound αCD3/αCD28 for 20 hours. Percent live DP determined by JC1/2 or FAM-VAD-FMK staining. h, Immunoblot of total Bax in DP thymocytes after 4 hour PMA/Ion stimulation and immunoprecipitation with 6A7 antibody.

Figure 2. Bim deficiency rescues positive selection in Shn2^−/− mice

a, Representative plots of total thymocytes (mean ± standard devation, n>10). b, Total numbers of CD4SP and CD8SP cells from indicated genotypes were calculated to include only mature TCR^hi cells. Each dot represents a mouse, bar represents the mean, number indicates fold change ***p<0.0001.

Figure 3. Conversion of positive selection to death in vivo

a, Representative plots of total thymocytes (n>10). b, Total thymocyte cell numbers. Percent of thymocytes (with CD4SP cells excluded) staining with FAM-VAD-FMK reagent (c) and AnnexinV reagent (d). e, Representative plots of total thymocytes (n=8). f, Total thymocyte cell numbers. Representative plots of thymus (g) and lymph node (h) of Shn2^−/−Bim^−/−DO11⁺ mice (mouse 3 is shown) with total clonotypic CD4 cell numbers from each mouse shown in the table. For all plots, numbers indicate percent of cells within each gate. For all graphs, each dot represents a mouse, bar represents the mean: ***p<0.0001, **p<0.001.

Figure 4. Shn2 dampens TCR-induced death in vivo

a, Representative plots of total thymocytes from bone marrow chimeras. Numbers indicate percent of cells within each gate. b, Total thymocyte cell numbers. Each dot represents a mouse, bar represents the mean. c, DP thymocytes stimulated by M12 cells pulsed with OVA peptide for 20 hours. d, DP thymocytes were stimulated by PI39 cells pulsed with PCC peptide for 20 hours. Quantitative PCR analysis of Shn2 expression by DP thymocytes after 3 hour stimulation with PMA and/or Ionomycin (e), Ionomycin (f), or platebound αCD3/αCD28 (g). Cells were pretreated with inhibitors for 30 min as indicated.