Fig. 4: HPSE expression supports FOXP3⁺ Treg homeostasis in vitro and in vivo.

a Western blot analysis of human HPSE overexpression and mouse HPSE basal expression in CTLL-2 cells transfected with HPSE construct (HPSE^over), compared to untransfected CTLL-2 cells (WT). b Proliferation of WT CTLL-2 cells (WT, blue) and HPSE-overexpressing CTLL-2 cells (HPSE^over, green) in response to heparin-coated beads that were pre-incubated with IL-2 (closed symbols) or T51P-IL-2 (T51P, open symbols). The concentration of IL-2 or T51P-IL-2 at which the beads were pre-incubated is depicted on the x-axis. Proliferation rate is depicted as equivalent dose of soluble IL-2 or T51P-IL-2 at which a similar proliferative response is elicited as by the beads. Shown is a representative of 3 independent experiments (mean +/− SEM of triplicate samples). P values of variation due to the genotype of the cells are shown, determined with two-way ANOVA with Tukey’s multiple comparison correction. c, d Viability of WT and HPSE^-/- FOXP3⁺ Treg (c) and CD25⁺/FOXP3^- Tconv (d) among CD4⁺ T cells cultured in the presence of heparin-coated beads pre-incubated with IL-2. Viability was measured by flow cytometry 24 h after start of culture. Shown are representatives of 4 independent experiments (mean + SEM of triplicate samples); P values depict variation due to the genotype (WT vs. HPSE^-/-), determined by two-way ANOVA. e, Quantification of STAT5 phosphorylation by flow cytometry in WT and HPSE^-/- FOXP3⁺ Treg after stimulation with IL-2 that was sequestered by plate-bound HS. Shown are mean +/− SEM of technical triplicates of a representative of 4 experiments. P values determined by two-way ANOVA with Sidak’s multiple comparison correction. f Percentage of pSTAT5⁺ cells among CD4⁺/FOXP3⁺ Treg isolated from naïve WT and HPSE^-/- spleen tissue. Shown are mean + SEM, n = 6-8 animals per group. P value determined by unpaired two-tailed t-test. g Representative flow cytometry plots depicting FOXP3⁺ Treg frequencies among CD4⁺ T cells in spleen tissue of WT and HPSE^-/- mice. h Quantification of FOXP3⁺ Treg frequencies among CD4⁺ T cells in lymphoid (left panel) and non-lymphoid (right panel) tissues of WT and HPSE^-/- mice. BM, bone marrow; LI, large intestine. Show are mean + SEM, n = 3-6 animals per group. P values determined by unpaired two-tailed t-test with Holm-Sidak multiple comparison correction. i Linear regression analysis of Foxp3⁺ Treg frequencies among CD4⁺ T cells in the spleens (left panel) and inguinal lymph nodes (right panel) of WT and HPSE^-/- mice during aging. P value shown comparing slopes, n = 44, 38 (WT spleen, LN), and 61, 60 (HPSE^-/- spleen, LN) individual mice. j Schematic overview of competitive bone marrow transplantation of WT and HPSE^-/- donors. k Representative flow cytometry plots depicting FOXP3⁺ Treg frequencies among transferred CD4⁺ T cells recovered from spleen and LN tissue of mice engrafted with bone marrow from WT and HPSE^-/- mice. n = 1 representative mouse for each group. l Frequencies of FOXP3⁺ cells among WT and HPSE^-/- bone marrow-derived CD4⁺ T cells in lymphoid tissues of irradiated recipient mice. n = 3 (spleen, LN) and 2 (thymus) individual mice. m Schematic overview of competitive Treg transfer of WT and HPSE^-/- donor mice. n Representative flow cytometry plots depicting CD45.1⁺ and CD45.2⁺ cell frequencies among transferred Treg recovered from spleen and LN tissue of recipient mice. n = 1 representative mouse. o Frequencies of CD45.1⁺ WT and CD45.2⁺ HPSE^-/- among Treg in lymphoid tissues of recipient mice. n = 5 individual mice per group. l, o Shown are mean + SEM, P values determined by two-way ANOVA with Sidak’s multiple comparison correction.