Fig 2: Basal expression of SLC7A5/SLC3A2 in the erythroid lineage(A) The panel represents the flow cytometry analysis showing the different splenic cell populations in basal conditions based on the surface expression of CD71 and Ter119 in control mice. SLC3A2 expression is also represented by a color code: yellow-orange representing the strongest expression and dark green-blue the weakest or absence of expression.(B) Histograms showing SLC3A2 expression in the different splenic CD71HI/Ter119NEG/LOW, CD71HI/Ter119HI, CD71NEG/Ter119HI and CD71NEG/LOW/Ter119NEG cell populations in control (in blue) and ErGFPcre Slc7a5LoxP/LoxP mice (in green). The IgG control (in gray) is included. A representative experiment out of 23 is shown.

Fig 3: SLC7A5/SLC3A2 expression in circulating CD71HI/Ter119HI reticulocytes under anemic conditions and in response to rhEPO(A) Representative flow cytometry analysis showing circulating CD71HI/Ter119HI reticulocytes in PHZ-treated and control mice, as well as in mice subjected to phlebotomy. For PHZ a representative experiment out of 20 is shown. For phlebotomy, a representative experiment out of 8 is shown.(B) Representative histograms of SLC3A2 expression in circulating CD71HI/Ter119HI reticulocytes of untreated control mice (in blue), mice treated with PHZ or mice subjected to phlebotomy (in red), as well as ErGFPcre Slc7a5LoxP/LoxP mice treated with PHZ or subjected to phlebotomy (in green). The IgG-APC control is represented in gray. Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71HI/Ter119HI reticulocytes from control untreated mice (n = 33), control mice treated with PHZ (n = 20), ErGFPcre Slc7a5LoxP/+ untreated mice (n = 11), ErGFPcre Slc7a5LoxP/+ mice treated with PHZ (n = 10), ErGFPcre Slc7a5LoxP/LoxP untreated mice (n = 16) and ErGFPcre Slc7a5LoxP/LoxP mice treated with PHZ (n = 14). Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71HI/Ter119HI reticulocytes from control mice (n = 8), control mice subjected to phlebotomy (n = 8), ErGFPcre Slc7a5LoxP/+ untreated mice (n = 4), ErGFPcre Slc7a5LoxP/+ mice subjected to phlebotomy (n = 4), ErGFPcre Slc7a5LoxP/LoxP untreated mice (n = 3) and ErGFPcre Slc7a5LoxP/LoxP mice subjected to phlebotomy (n = 3).(C) Western blot analyses of SLC7A5 relative to β-actin protein levels in circulating erythroid cells of control and ErGFPcre Slc7a5LoxP/LoxP PHZ-treated mice.(D) L-phenylalanine uptake in isolated CD71HI/Ter119HI circulating reticulocytes from PHZ-treated control or ErGFPcre Slc7a5LoxP/LoxP mice (n = 5).(E) Representative flow cytometry analysis of CD71 expression versus GYPA in healthy and anemic subjects (left panels). Histograms of SLC3A2 expression in circulating CD71HI/GYPAHI and CD71NEG/GYPAHI populations of control (blue) and anemic (red) subjects (right panels). A representative experiment out of 3 anemic subjects and 3 control subjects is shown.(F) Representative histograms of SLC3A2 expression in circulating CD71HI/Ter119HI reticulocytes of untreated control mice (blue) or mice treated with rhEPO (200U) (red). The IgG-APC control is represented in gray. Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71HI/Ter119HI reticulocytes from untreated (n = 5) or rhEPO-treated control mice (n = 5), as well as from untreated (n = 3) or rhEPO-treated ErGFPcre Slc7a5LoxP/LoxP mice (n = 5).Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test when comparing more than two groups or two-tailed Student’s t test with Welch’s correction when comparing two groups (∗∗p < 0.01; ∗∗∗p < 0.005).

Fig 4: SLC7A5/SLC3A2 expression in splenic erythroblasts during erythropoietic stress(A) Representative flow cytometry analysis showing the different splenic cell populations in control and PHZ-treated mice based on the surface expression of CD71 and Ter119: CD71HI/Ter119NEG/LOW, CD71HI/Ter119HI, and CD71NEG/LOW/Ter119NEG.(B) Relative Slc7a5 mRNA expression in the spleen of control (n = 8) and ErGFPcre Slc7a5LoxP/LoxP (n = 3) mice in baseline conditions as well as control (n = 6) and ErGFPcre Slc7a5LoxP/LoxP (n = 5) upon PHZ treatment. Data are shown as mean ± SEM. Statistical analysis was performed using a two-tailed Student’s t test with Welch’s correction to compare the groups as indicated in the figure (∗p < 0.05).(C) Histograms showing SLC3A2 expression in each of the populations indicated in untreated control mice (in blue) and control mice treated with PHZ (in red) are shown in left panels. In the right panels, the same histograms of the left panels are shown, but overlaying the histogram of SLC3A2 expression of ErGFPcre Slc7a5LoxP/LoxP (in green) mice treated with PHZ. The IgG-APC control is presented in gray. For A and C a representative experiment out of 9 is shown.(D) Representative flow cytometry analysis of DRAQ5 expression versus forward scatter (FSC) of the splenic CD71HI/Ter119HI erythroid population of PHZ-treated control mice. Two CD71HI/Ter119HI populations differing in DRAQ5 signal can be distinguished, DRAQ5HI and DRAQ5LOW. The dashed line represents unlabeled cells.(E) Representative histograms of SLC3A2 expression in CD71HI/Ter119HI/DRAQ5HI and CD71HI/Ter119HI/DRAQ5LOW populations in control mice treated with PHZ (in red) and ErGFPcre Slc7a5LoxP/LoxP (in green) mice treated with PHZ are shown. The negative signal of the IgG-PyC control is shown in gray. For D and E a representative experiment out of 7 is shown.

Fig 5: Along with proliferation of Tfh-like Tet2/3 DKO ex-Treg cells, plasma cells were diffusely expanded in spleen from DKO-severe mice.A. Flow cytometry analysis of TCRβ− B220− cells (gated on live cells) in pooled spleen and peripheral LNs (cervical and inguinal) of from 13–15 weeks old Foxp3Cre WT and Foxp3-Cre Tet2/3fl/fl mice. B. Flow cytometry analysis of neutrophils (CD11b+ Gr-1+ cells) or plasma cells (CD98+ CD138+ cells) (gated on TCRβ− B220− cells) in pooled spleen and peripheral LNs (cervical and inguinal) of from 13–15 weeks old Foxp3Cre WT and Foxp3-Cre Tet2/3fl/fl mice. C. Quantification of the frequency of CD11b+ Gr-1+ cells in TCRβ− B220− cells and absolute number. D. Quantification of the frequency of CD98+ CD138+ cells in TCRβ− B220− cells and absolute number. Error bars show mean ± SEM. from at least three independent experiments. Statistical analysis was performed using one-way ANOVA (*P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001). E. Immunostaining of CD3ε, CD8α, B220, ICOS, FOXP3 and CD4 in spleen from 14-week-old Foxp3Cre WT and Foxp3-Cre Tet2/3fl/fl mice. Scale bar; top: 1mm, middle: 100μm, bottom: 25μm. F. Immunostaining of IRF4, B220, CD3ε in spleen from 14-week-old Foxp3Cre WT and Foxp3-Cre Tet2/3fl/fl mice. Scale bar; top: 1mm, bottom: 100μm. G. The graphs show the cell numbers of CD4+ CD3ε+ ICOS+ FOXP3− cells or IRF4+ B220− CD3ε− F480− cells counted in the stained sections from spleen.