A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development

Abstract

The neonatal thymus generates Foxp3⁺ regulatory T (tT_reg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tT_reg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tT_reg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IA^b-Padi4 with moderate dwell times within a conventional docking orientation are exported as tT_reg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4⁺ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tT_reg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tT_reg selection window.

Figure 1.

T cell receptors expressed on neonate-derived tT_reg cells can recognize steady state, inflammation- and age-dependent self-antigens. (a) IL-2 release and (b) frequency at which 66 C57BL/6-derived tT_reg hybridomas and (c, d) 316 Yae62β⁺ tT_reg hybridomas react with splenocytes isolated from adult naïve mice (red) or mice pretreated with LPS and αCD40 (pink). (e) IL-2 response of B6–50.1C10, (f) 6287, (g) B6–13 and (h) 4699 tT_reg hybridomas cultured with titrating numbers of cDC1, cDC2 and macrophages isolated from naïve mice (filled symbol) or mice pretreated with LPS and αCD40 (open symbol). Data are an example of three independent experiments giving similar results. Error bars show standard deviation. (i) IL-2 release by 80 in vitro splenocyte-reactive tT_reg hybridomas cultured with spleen cells isolated from 2 week old and 8 week old C57BL/6 mice. Results are from two independent experiments with similar results. (j) Quantification of splenocyte-reactive hybridomas that increase IL-2 production in response to adult v. neonatal spleen cells. (k) The frequency of age-dependent (red, filled circles), age-independent in vitro splenocyte-responsive (red, open circles) and below detection limit (blue) TRAV14⁺ (Vα2⁺) tT_reg TCRs carried in the thymic Foxp3⁺ tT_reg pool at 2 weeks of age as compared to 8 weeks of age. Lines represent the data geometric mean. Data are from (i,j) 80 self-reactive and (k) 103 Va2⁺ clonotypes; 28 age-dependent, 12 age-independent and 63 below limit of detection. (i) ****P<0.0001 ratio paired 2-tailed t test, (k) *P<0.05, **P<0.01 one-way ANOVA Tukey multiple comparisons test.

Figure 2.

Identification of self-ligands recognized by neonatal tT_reg TCR using an immunopeptidome library screen. (a) Chart of EC₅₀ values of IL-2 release for tT_reg hybridomas responding to titrating concentrations of library-identified self-peptides. (b-e) IL-2 release of the (b) 4699 tT_reg hybridoma and (c) 6287 4699 tT_reg hybridoma cultured with titrating concentrations of identified Padi4_92–105 or Add2_606–621 presented by IA^b expressing mouse fibroblasts, or (d) with titrating concentrations of C57BL/6 or Padi4^−/− splenocytes, or (e) with titrating concentrations of C57BL/6 or Add2^−/− splenocytes. Data are example of three independent experiments giving similar results. Error bars show standard deviation. (f) IA^b-Padi4 tetramer staining of the 4699 tT_reg hybridoma, and (g) IA^b-Add2 tetramer staining of the 6287 tT_reg hybridoma (black line). Gray negative control stains are the IA^b-Add2 tetramer staining of the 4699 tT_reg hybridoma and the IA^b-Padi4 tetramer staining the 6287 tT_reg hybridoma.

Figure 3.

Development of Padi4_92–105-specific tT_reg cells is restricted to the neonatal thymus. (a, b) Flow cytometry of Vα2⁺ CD4⁺CD8⁻ thymocytes in (a) YAe62β.Foxp3-GFP and (b) Padi4^−/−.YAe62β.Foxp3-GFP mice at different ages; numbers adjacent to outlined areas indicate percent IA^b-Padi4 tetramer⁺ Foxp3-GFP⁻ (left) and Foxp3-GFP⁺ (right). (c-f) Frequency and total cell numbers of IA^b-Padi4⁺ (c, d) CD4⁺CD8⁻ Foxp3⁻ and (e, f) Foxp3-GFP⁺ tT_reg cells in WT and Padi4^−/− YAe62β.Foxp3-GFP mice at different ages. Bars represent the data mean, n = 6, 12, 6, 6, 6, 6 WT mice at 1, 2, 3, 4, and 8 weeks old, n = 7 Padi4^−/− 2 and 6 weeks old. (c-f) NS P>0.05, **P<0.01, ***P<0.01, ****P<0.0001 one-way ANOVA Tukey multiple comparisons test.

Figure 4.

Padi4-specific tT_reg cells seed the peripheral repertoire during the neonatal window and respond to inflammation. (a, b) Flow cytometry of splenic Vα2⁺ CD4⁺ T cells in (a) YAe62β.Foxp3-GFP mice and (b) Padi4^−/−.YAe62β.Foxp3-GFP mice at different ages (weeks). (c-f) Frequency and (d) total numbers of IA^b-Padi4 tetramer^pos Vα2⁺ CD4⁺ Foxp3-GFP^pos tT_reg cells and (e, f) Vα2⁺ CD4⁺ Foxp3-GFP^neg CD4 T_conv cells in YAe62β.Foxp3-GFP and Padi4^−/−.YAe62β.Foxp3-GFP mice at different ages. (g) Flow cytometry analyzing the expression of PD-1, CD44, GITR, CD25, CTLA4 and Nrp1 on IA^b-Padi4 tetramer^pos Vα2⁺CD4⁺Foxp3-GFP^pos tT_reg cells isolated from 2 week old YAe62β.Foxp3-GFP mice (black line). Gray histograms are IA^b-Padi4 tetramer^neg Vα2⁺CD4⁺Foxp3-GFP^pos tT_reg cells in the same mice. (h-l) Flow cytometry of Vα2⁺ CD4⁺ T cells isolated from 8 week old YAe62β.Foxp3-GFP mice following i.p. injection of (h) PBS, (i) LPS + αCD40 and (j) rVaccina virus 5 days prior. Quantification of IA^b-Padi4 tetramer^pos (k) CD4⁺Foxp3-GFP^pos tT_reg cells and (l) Foxp3-GFP^neg CD4 T_conv cells present in YAe62β.Foxp3-GFP mice following i.p. injection of PBS, LPS+αCD40 or rVac virus. (a g) Data are derived from 3 independent experiments giving similar results, bars represent the data mean, n = 6, 9, 6, 6, 6, 6 WT mice at 1, 2, 3, 4, and 8 weeks old, n = 6 and 8 Padi4^−/− 2 and 6 weeks old. (h-l) Data pooled from 3 independent experiments with similar results, with 9, 5 and 6 mice per group, bars represent the data mean. Significance identified using a one-way ANOVA Tukey multiple comparisons test, and (k,l) non-parametric Dunn’s multiple comparison test. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 5.

Padi4-specific thymocytes are subject to temporally regulated, stage-specific changes in negative selection. Flow cytometry analyses of (a) neonatal and (b) adult Vα2⁺ DP and CD4SP thymocytes isolated from Padi4^+/+.YAe62β.Foxp3-GFP (top row) and Padi4^−/−.YAe62β.Foxp3-GFP (bottom row) mice for Foxp3 expression and IA^b-Padi4 tetramer binding. Subsets shown are pre-selection DP (CD4⁺ CD8⁺ TCRβ^int CCR7^neg), post-selection DP (CD4⁺CD8⁺ TCRβ^hi CCR7^pos), and TCRβ^hi CCR7^pos CD4SP thymocytes pre-gated on CD69⁺ MHC-I^lo (SM), CD69⁺ MHC-I^hi (M1) and CD69^lo MHC-I^hi (M2). (c-g) Quantification of the frequency of IA^b-Padi4 tetramer^pos (c,f) Foxp3^neg, (d,g) Foxp3^pos and (e,h) total (Foxp3^neg + Foxp3⁺) thymocytes within each thymic subset. (c-h) Data are derived from 3 independent experiments giving similar results, bars represent the data mean, n = 6 WT and Padi4^−/− mice at 2 and 6 weeks old. Significance identified using a one-way ANOVA Sidak’s multiple comparison test. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 6.

Limiting Negative Selection restores development of Padi4-specific tT_reg cells in the adult thymus. (a-j) Eliminating Padi4 expression in BM derived cells increases the frequency of Padi4-specific TCRβ^hi CCR7⁺ CD4SP thymocytes and tT_reg cells. Flow cytometry analyses of thymocytes isolated from mixed WT and Padi4^−/− (KO) YAe62β bone marrow chimeric mice. (a, b) Total Vα2⁺ thymocytes analyzed for IA^b-Padi4 tetramer binding and (a) TCRβ and (b) CCR7 expression. (c) Flow cytometry analyses of total Vα2⁺ IA^b-Padi4 tetramer^pos thymocytes for CD4 and CD8 expression. (d-h) Quantification of Vα2⁺ IA^b-Padi4 tetramer^pos (d) TCRβ^int (e) TCRβ^hi (f) CCR7^neg (g) CCR7^pos and (h) CD4SP thymocytes. (i) Vα2⁺ CD4SP thymocytes analyzed for IA^b-Padi4 tetramer binding and Foxp3 expression, and (j) quantification of IA^b-Padi4 tetramer^pos Foxp3-GFP⁺ CD4SP thymocytes. Data is from two independent experiments giving similar results, n = 5 mice per group, bars represent the data mean. (k,l) IL-2 response of the (k) 4699 and (l) 4738 tT_reg hybridomas cultured with titrating numbers of CD11c⁺ Xcr1^neg cDC (filled circle), CD11c⁺ Xcr1^pos cDC (open circle), CD11c^lo B220⁺ pDC (open squares) and CD19⁺ B220⁺ B cells (open triangle) isolated from thymus of adult C57BL/6 mice. Data is from two independent experiments giving similar results. Significance identified using a one-way ANOVA Tukey multiple comparisons test, ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 7.

Padi4-specific neonatal tT_reg cells express TCRs with modest dwell times. (a) IA^b-Padi4 tetramer^pos Vα2⁺ Foxp3-GFP^neg CD4SP thymocytes isolated from YAe62β.Foxp3-GFP were sorted, and (b) TCRs cloned and re-expressed in T cell hybridomas. (c) IL-2 response of Padi4-reactive T cell hybridomas cultured with IA^b-expressing fibroblasts and titrating concentration of soluble Padi4_92–105 peptide; see key for name of individual TCRs. (d-f) Relative distribution of Padi4-reactive TCR clonotypes carried in the (d) thymic Foxp3-GFP^neg CD4SP, (e) splenic CD4⁺ Foxp3-GFP^pos tT_reg and (f) splenic CD4⁺ Foxp3-GFP^neg T_conv cell repertoires of 2-week old YAe62β.Foxp3-GFP mice. Colored pie slices represent individual TCRs described in (c). (g) Frequency distribution of individual Padi4-reactive T cell clonotypes carried in splenic CD4⁺ tT_reg versus CD4⁺ T_conv cell repertoires of 2-week old YAe62β.Foxp3-GFP mice. Data are derived from 4 independent experiments, (d-f) represented as mean fraction of repertoire, (g) bars represent the data geometric mean. (h-k) Centered second order non-linear regression analysis of 11 Padi4- and Add2-specific TCRs comparing the tT_reg/T_conv cell frequency bias versus the TCR:self-pMHC (h) on-rate, k_on (i) half-life, t_1/2 and (j) equilibrium affinity, K_D, and (k) the clonal frequency in Foxp3^neg CD4SP thymocytes. Correlation plots are based on probable cure fit using Akaike’s Information Criteria, Prism 7.04. R² values represent goodness of fit analysis. (l-n) Time scale of soluble (l) 5287, 6235, 5290, (m) 4699, 4783, 6239, and (n) 6236, 6256, 6237, 6238 TCRs disassociating from immobilized IA^b-Padi4 measured by surface plasmon resonance. Sensograms are background subtracted from each TCR interacting with a non-cognate ligand. Data are derived from 4 biological replicates with similar results. (g) Significance identified using a Kruskal-Wallis test and Dunn’s multiple comparisons test, ***P<0.001, ****P<0.0001.

Figure 8.

TCRs that promote neonatal negative selection, tT_reg differentiation or CD4 T_conv cell development use conventional docking orientations on IA^b-Padi4. (a-c) Ribbon diagrams of (a) 6235 (pdb: 6MNO), (b) 4699 (pdb:6MKD) and (c) 6256 (pdb: 6MNM) TCRs binding IA^b-Padi4. The 6235 TCR is colored red (TCRβ) and pink (TCRα); the 4699 TCR is colored dark green (TCRβ) and light green (TCRα); The 6256 TCR is colored dark blue (TCRβ) and light blue (TCRα). IA^b-Padi4 is colored cyan (IA^bα chain), yellow (peptide), and magenta (IA^bβ chain). (d-f) Projections of the (d) 6235, (e) 4699 and (f) 6256 TCRs bound to IA^b-Padi4. The peptide residues are outlined in black. (g-i) The amount of buried surface area (BSA) of the (g) 6235:IA^b-Padi4, (h) 4699:IA^b-Padi4 and (i) 6256:IA^b-Padi4 complexes contributed by TCRα and TCRβ loops, and the peptide or MHC chains. Figures were made with PyMol. (j) Overlay of 6235, 5287, 4699, 4378, 6256 and 6236 TCRs binding IA^b-Padi4. Dashed lines indicate the crossing and incident angles. (k) Plot of the 6235, 5287, 4699, 4378, 6256 and 6236 TCR:IA^b-Padi4 crossing and incident angles, compared to 129 human and mouse MHC-I and MHC-II TCR:pMHC complexes, including two iT_reg reverse orientation (triangles). (l-q) Van der Waals interactions between the (l) 6235, (m) 4699 and (n) 6256 TCRα chains with the Padi4 P2Y residue, and (o-q) the presence, absence and distance of a hydrogen bond (red dash line) between the CDR3α 94 residue and the Padi4 P2Y hydroxyl moiety. (r, s) Time scale of soluble 6235 (red), 4699 (green) and 6256 (blue) TCRs disassociating from immobilized (r) IA^b-Padi4 and (s) IA^b-Padi4 p2F measured by surface plasmon resonance. Sensograms are background subtracted from each TCR interacting with a non-cognate IA^b-Add2 ligand. SPR data are examples of 4 independent experiments, giving similar results.