Fig. 5. CTPS1 inactivation in T lymphocytes leads to impaired expansion and memory T-cell differentiation.

a–e Analyses of Ctps1^wt/flox (Ctps1^wt/flox), Ctps1^flox/flox (Ctps1^flox/flox), CD4-Cre; Ctps1^wt/flox (Ctps1^wt/ko) and CD4-Cre; Ctps1^flox/flox (Ctps1^ko/ko). a Representative dot-plots, of two independent experiments, from FACS analyses of thymic sub-populations stained with CD4 and CD8 double negative CD4 and CD8 (DN) double positive CD4 and CD8 (DP), single positive CD4 (SP4) and single positive CD8 (SP8) cells. DN cells were further labelled with CD44 and CD25 to identify stages of DN differentiation. b RT-PCR analyses of the Ctps1 deletion in total thymus (bulk), sorted cells from DN2, DN3 and DN4 subtypes of DN cells and SP4 and SP8 cells. Corresponding amplified alleles/genotypes are indicated on the right (flox, wt and ko). c–e Analysis of splenic enriched T cells activated by anti-CD3/CD28 plus IL-2 for 3 days. c Representative proliferation dot plot profiles (left panels) from FACS analysis of T cells. Graph bars (right panels) corresponding to proliferation indexes calculated from histogram profiles on CD4⁺ T cells subset (upper panel), and CD8⁺ T cells subset (lower panel). d Immunoblots for CTPS1, CTPS2 and Actin expression in lysates of non-activated or activated T cells. Molecular weights in kDa on the left. e Proportions of T cell subsets from FACS analyses in the cultures after activation. Naive T cells (CD44^-CD62L⁺), central memory (CD44⁺CD62L⁺), effector memory (CD44⁺CD62L^-) are shown in both CD4⁺ and CD8⁺ T cells. Controls correspond to littermates (Ctps1^wt/flox, Ctps1^flox/flox and Ctps1^wt/ko) of CD4-Cre; Ctps1^flox/flox (Ctps1^ko/ko). Blot is representative of four independent experiments. f–h Analyses of splenic enriched T cells analyses from CD8-Cre; Ctps1^wt/flox (Ctps1^wt/ko) and CD8-Cre; Ctps1^flox/flox (Ctps1^ko/ko) activated by anti-CD3/CD28 plus IL-2 for 3 days. f Representative proliferation histogram profiles from FACS analysis of enriched CD8-Cre-Ctps1^ko/ko and Ctps1^wt/ko splenic T CD4⁺ (left panels) or CD8⁺ (right panels) cells labelled with cell trace violet (CTV) and activated. White and red histograms correspond to Ctps1^wt/ko and Ctps1^ko/ko respectively. Grey histograms represent unstimulated cells. Graph bars on the right correspond to proliferation indexes calculated from histogram profiles. g Proportions of CD4⁺ and CD8⁺ T cell subsets from FACS analyses in the cultures after activation. Naive T cells (CD44^-CD62L⁺), central memory (CD44⁺CD62L⁺), effector memory (CD44⁺CD62L^-). Controls correspond to littermates (Ctps1^wt/ko) of CD4-Cre; Ctps1^flox/flox (Ctps1^ko/ko). h Same as (f, right panel) except that CD8⁺ T cells from Ctps1^wt/ko (upper panel) and Ctps1^ko/ko (lower panel) were supplemented (green filled) or not (white or red filled histograms) with cytidine (200 μM). c, e–g Non-parametric Matt-Whitney two-tailed test were used. Data are presented as the mean ± SD of n = 5 (wt/flox), n = 6 (flox/flox), n = 6 (wt/ko) and n = 11 (ko/ko) animals per group (c), n = 11 per group (e), n = 12 animals per group (f) and of n = 9 animals per group (g). Source data are provided in Source Data file.