Fig 1.
Gene expression characteristic of CD71+ erythroid cells.
(A) Hierarchical clustering on Euclidian distances showing different gene expression profile in CD71+ erythroid cells. (B) PCA on the Euclidian distances between CD71+ erythroid cells in different ages. (C) Expression of genes associated with inhibitory receptors/ligands is shown using heatmap. (D) Representative plots showing expression of VISTA, (E) Lgals9, and (F) Lgals1 among CD71+ erythroid cells compared to corresponding isotype controls. The underlying data can be found in S1 Data. Ankrd44, ankyrin repeat domain 44 gene; CD59a, glycoprotein-A–associated gene; CD71, cell-surface transferrin receptor; CD274, Program death ligand-1; Gata1, erythroid transcription factor; Ig, immunoglobulin; ITIM, immunoreceptor tyrosine-based inhibition motif; Lag3, lymphocyte activation 3; Lgals1, galectin-1 gene; Lgals3, galectin-3 gene; Lgals9, galectin-9 gene; PC, principal component; PCA, principal component analysis; Pdcd1Ig2, Programmed Cell Death 1 Ligand 2 gene; Ppbp, pro-platelet basic protein gene; Tigit, T cell immunoreceptor with Ig and ITIM domains; VISTA, V-domain Ig Suppressor of T Cell Activation; Vsir, VISTA gene.
Fig 2.
CD71+ erythroid cells express VISTA.
(A) Representative dot plots showing VISTA expression by CD71+ erythroid cells from a day 3 newborn mouse. (B) Percentages of CD71+VISTA+ erythroid cells among splenocytes of BALB/c, (C) C57BL/6, and (D) F1 mice are shown at different ages, respectively. (E) Representative histogram showing VISTA expression (red line) or isotype control (black line) and (F) the MFI of VISTA among CD71+ erythroid cells in BALB/c mice. (G) Representative histogram showing VISTA expression (red line) or isotype control (black line) and (H) the MFI of VISTA among CD71+ erythroid cells in B57BL/6 mice. (I) Representative histogram showing VISTA expression (red line) or isotype control (black line) and (J) the MFI of VISTA among CD71+ erythroid cells in F1 mice. (K) RT-PCR of VISTA gene expression among neonatal CD71+ erythroid cells in BALB/c mice at different ages. (L) Representative histogram of VISTA expression among neonatal CD71+ erythroid cells originated from either spleen or BM. (M) The MFI for VISTA among neonatal CD71+ erythroid cells from spleen versus BM of neonatal mice. Each point represents data from an individual mouse, representative of at least 2–3 independent experiments. Bar, mean ± one standard error. The underlying data can be found in S1 Data. BM, bone marrow; CD71, cell-surface transferrin receptor; F1, filial 1 hybrid mice; Ig, immunoglobulin; Iso., isotype; MFI, mean fluorescence intensity; RT-PCR, reverse transcription PCR; TER119, glycophorin-A–associated protein; VISTA, V-domain Ig Suppressor of T Cell Activation.
Fig 3.
CD71+VISTA+ cells produce more TGF-β compared to CD71+VISTA− cells.
(A) Expression of genes associated with TGF-β is shown using heatmap. (B) TGF-β gene expression in isolated CD71+ erythroid cells from BALB/c mice at different ages using qPCR. (C) Representative flow cytometry dot plot showing TGF-β secretion by CD71+ erythroid cells. (D) Gating strategy and the representative dot plots/histogram showing TGF-β+ cells among CD71+VISTA+ cells compared to CD71+VISTA− cells in 6-day-old BALB/c mouse. (E) Percentages of TGF-β+ cells among CD71+VISTA+ cells compared to CD71+VISTA− cells in 6-day-old neonatal BALB/c mice or (F) C57BL/6 mice and (G) F1 mice. (H) Expression of TLR4, (I) TLR2, and (J) TLR1 genes among CD71+ erythroid cells from 3-day-old mice. (K) Representative flow cytometry zebra plots showing expression of TLR1 among spleen and liver CD71+ erythroid cells in a 3-day-old neonatal mouse. (L) Percentages of TLR1 surface expression among CD71+ erythroid cells from the liver, BM, spleen, and thymus of 3-day-old BALB/c mice. (M) Representative dot plots showing TGF-β+ cells among neonatal CD71+ erythroid cells before and after stimulation with Pam3CSK4 or HL Lm. (N) Representative flow cytometry histogram showing TGF-β expression by Lgals9+ versus VISTA+ CD71+ erythroid cells. Data are representative of at least three independent experiments. Bar, mean ± one standard error. The underlying data can be found in S1 Data. BM, bone marrow; CD71, cell-surface transferrin receptor; HK, heat killed; Ig, immunoglobulin; Lm, Listeria monocytogenes; Pam3CSK4, N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys) 4; qPCR, quantitative PCR; Stim., stimulated; TER119, glycophorin-A–associated protein; TGF-β, transforming growth factor beta; TLR, Toll-like receptor; Unstim., unstimulated; VISTA, V-domain Ig Suppressor of T Cell Activation.
Fig 4.
CD71+VISTA+ cells enhance iTreg development in newborn mice.
(A) Representative flow cytometry dot plots showing CD4+FOXP3+ Tregs among neonatal splenocytes. (B) Percentages of Tregs among neonatal splenocytes in BALB/c mice at different ages. (C) Representative flow cytometry dot plots showing percentages of iTregs in the presence of either CD71+VISTA+ or CD71+VISTA− erythroid cells, TGF-β blocker, and re. TGF-β. (D) Cumulative data showing fold change in CD4+FOXP+ Tregs in the presence of either CD71+VISTA+ or CD71+VISTA− erythroid cells. (E) The expression of TIGIT, PDL-1, VISTA, Lgals9, CD73, CD39, and Helios (red lines) on the iTreg were determined by flow cytometry (isotype controls, black line). (F) The expression of Helios on the iTregs (red line) was compared to the spleen Tregs (blue line). (G and H) Assessing the suppressive function of iTregs in vitro. Naïve CD4+ T cells were cocultured with CD71+VISTA+ erythroid cells to promote iTregs following activation with anti-CD3/CD28 antibodies. The iTregs were enriched along total spleen Tregs and cocultured with Teff as shown in the ratios. (I and J) Phospho-Akt (K and L) and phospho-mTOR at 18 h after coculture of CD71+ erythroid cells with naïve CD4+ T cells, following stimulation with anti-CD3/CD28 antibodies (gray line isotype control, red line CD4:CD71, and blue line naïve CD4+ T cells). Asterisks are used to indicate significance (p < 0.0001). Data are representative of at least two to three independent experiments. Bar, mean ± one standard error. The underlying data can be found in S1 Data. BALB/c, mouse strain; CD71, cell-surface transferrin receptor; CFSE, Carboxyfluorescein succinimidyl ester; FOXP3, forkhead box P3; Ig, immunoglobulin; iTreg, induced Treg; Lgals9, galectin-9; MFI, mean fluorescence intensity; phospho-Akt, phosphorylated-Akt; PDL-1, program death ligand-1; phospho-mTOR, phosphorylated-mTOR; re. TGF-β, recombinant TGF-β; Teff, effector T cell; TGF-β, transforming growth factor beta; TIGIT, T cell immunoreceptor with Ig and ITIM domains; Treg, regulatory T cell; VISTA, V-domain Ig Suppressor of T Cell Activation.
Fig 5.
Comparison of CD71+ erythroid cells in VISTA KO to their counterparts in WT mice.
(A). Representative plots showing frequency of CD71+ erythroid cells in a WT versus VISTA KO newborn mouse (day 9). (B) Cumulative data comparing the percentages of CD71+ erythroid cells in days 6 and 9 of WT versus KO mice. (C) Representative flow plots showing TGF-β production by a WT versus KO newborn mouse. (D) Cumulative data showing percentages of TGF-β–producing cells among CD71+ erythroid cells in WT and VISTA KO mice. (E) Representative flow cytometry dot plots showing percentages of iTregs in the presence of naïve T cells; CD71+VISTA KO, CD71+VISTA−, or CD71+VISTA+ erythroid cells; and re. TGF-β. (F) Cumulative data showing percentages of change in CD4+FOXP+ Tregs in the presence of either CD71+VISTA KO, CD71+VISTA+, or CD71+VISTA− erythroid cells and re. TGF-B. (G) Representative flow plots showing percentages of Tregs in WT and VISTA KO newborn mice (day 9). (H) Cumulative data showing percentages of Tregs in WT versus VISTA KO day 9 mice. (I) Representative plots showing expression of GARP on CD71+ erythroid cells in WT and VISTA KO day 9 mice. (J) Cumulative data showing percentages of CD71+GARP+ cells in WT versus VISTA KO day 9 mice. (K) Representative flow plots showing CD71+TER119− cells in WT versus VISTA KO newborns, and representative plot showing percent CD3+ T cells among CD71+TER119− cells in VISTA KO mouse. (M) Cumulative data showing percentages of CD71+TER119− cells in WT and VISTA KO mice (6 and 9 days old). Each point represents data from an individual mouse, representative of at least two independent experiments. Bar, mean ± one standard error. The underlying data can be found in S1 Data. CD71, cell-surface transferrin receptor; GARP, glycoprotein A repetitions dominant; Ig, immunoglobulin; iTreg, induced Treg; KO, knock-out; re. TGF-β, recombinant TGF-β; TER119, glycophorin A-associated protein, an erythroid-specific antigen expressed on erythrocytes; TGF-β, transforming growth factor beta; Treg, regulatory T cell; VISTA, V-domain Ig Suppressor of T Cell Activation; WT, wild type.
Fig 6.
Newly generated CD71+ erythroid cells compensate for the loss of their counterparts.
(A) Representative flow dot plots illustrating the frequency of CD71+ erythroid cells in the spleen of isotype (Rat-IgG) versus anti-CD71–treated newborn mice. (B) The percentage of CD71+ erythroid cells in the spleen of anti-CD71–treated compared to control groups in 9-day-old BALB/c mice. (C) Representative dot plots illustrating the percentages of CD4+FOXP3+ in the spleen of isotype versus anti-CD71–treated mouse. (D) Percentage of CD4+FOXP3+ in the spleen of anti-CD71–treated compared to control groups in 9-day-old BALB/c mice. (E) Gene expression of TGF-β–associated genes, galectins, and PDL-1 (CD274) in enriched CD71+ erythroid cells from anti-CD71–treated versus controls. (F) Representative histogram of VISTA expression on CD71+ erythroid cells of control versus anti-CD71–treated mouse. (G) Percentages of CD71+VISTA+ erythroid cells among splenocytes of control and anti-CD71–treated BALB/c mice. (H) The MFI of VISTA on CD71+ erythroid cells from the spleen of control versus anti-CD71–treated mice. (I) RNAseq data showing expression of TGF-β1 and (J) TGF-β2 mRNA in CD71+ erythroid cells obtained from control or anti-CD71–treated mice. (K) PCR data showing expression of TLR2, and (L) TLR4 genes in CD71+ erythroid cells obtained from control or treated with anti-CD71 antibody. (M) The representative zebra plots showing surface expression of TLR2 on CD71+ erythroid cells. (N) Cumulative data showing percentages of TLR+CD71+ erythroid cells in control versus anti-CD71–treated group. (O) Representative zebra plots showing percentage of TGF-β+ cells among CD71+ erythroid cells from controls or treated mice with anti-CD71 antibody. (P) Cumulative data showing percentages of TGF-β expressing CD71+ erythroid cells in rat-IgG–versus anti-CD71–treated mice. (Q) The expression of Id1, (R) Id2, (S) PDL-1, (T) Lgals3, and (U) Lgals1 mRNA levels are shown in CD71+ erythroid cells from either control or anti-CD71–treated mice using RNAseq. Each point represents data from an individual mouse, representative of at least three independent experiments. Bar, mean ± one standard error. The underlying data can be found in S1 Data. BALB/c, mouse strain; CD71, cell-surface transferrin receptor; FOXP3, forkhead box P3; Gal, Galectin; Gata1, erythroid transcription factor; ID, inhibitor of DNA binding; Ig, immunoglobulin; IgG, immunoglobulin G; Iso., isotype; Lgals, Galectin genes; MFI, mean fluorescence intensity; mRNA, messenger RNA; PDL-1, program death ligand-1; RNAseq, RNA sequencing; TGF-β, transforming growth factor beta; TLR, Toll-like receptor; VISTA, V-domain Ig Suppressor of T Cell Activation.
Fig 7.
The cross-talk between Tregs and CD71+ erythroid cells.
(A) Representative flow cytometry dot plot of Tregs in neonatal FOXP3-DTR mice either control or treated with DT. (B) Representative dot plots showing percent CD71+ erythroid cells in control versus DT-treated FOXP3-DTR mouse. (C) Percentages of CD71+ erythroid cells in control versus DT-treated mice. (D) Representative dot plots showing percent CD71+ erythroid cells in control versus DT-treated WT mice. (E) Percentages of CD71+ erythroid cells in control versus DT-treated WT mice. (F) Representative histogram plots showing expression of VISTA on CD71+ erythroid cells in control versus DT-treated mice. (G) Cumulative data showing MFI of VISTA on CD71+ erythroid cells in control versus DT-treated mice. (H) Representative histogram plots showing expression of CD71 on CD71+ erythroid cells in the presence and absence of Tregs. (I) Cumulative data showing MFI of CD71 on CD71+ erythroid cells alone or when cocultured with Tregs in vitro. (J) Representative histogram plots showing Ki67 expression on CD71+ erythroid cells in the presence and absence of Tregs. (K) Cumulative data showing MFI of Ki67 on CD71+ erythroid cells alone or upon coculture with Tregs in vitro. (L) Representative dot plots showing expression of VISTA on human cord blood and placenta CD71+ erythroid cells. (M) Cumulative data showing percentages of VISTA+ cells among human cord blood and placenta CD71+ erythroid cells. (N) Data showing PD-1H gene expression in enriched cord blood and placenta CD71+ erythroid cells. (O) Data showing TGF-β gene expression in human cord blood and placenta CD71+ erythroid cells. The underlying data can be found in S1 Data. CD71, cell-surface transferrin receptor; DT, diphtheria toxin; DTR, DT receptor; FOXP3, forkhead box P3; FOXP3-DTR, FOXP3 mice expressing DTR in Treg cells; Ig, immunoglobulin; Iso., isotype; Ki67, antigen KI67; MFI, mean fluorescence intensity; PD-1H, Programmed Death-1 Homologue-1; TER119, glycophorin A-associated protein, an erythroid-specific antigen expressed on erythrocytes; TGF-β, transforming growth factor beta; Treg, regulatory T cell; VISTA, V-domain Ig Suppressor of T Cell Activation; w, with; WT, wild type; w/o, without.