eTreg and eCD4 subsets.

a-c. Clonality and diversity of lung eTreg and eCD4 subsets in response to distinct antigenic challenges. We calculated the (a) Chao1 estimator, (b) normalized Shannon-Wiener index, and (c) observed diversity for each TCRα repertoire obtained from each CD4+ T-cell subset from each animal (3 < n < 7) with different antigenic challenges. P-values shown as: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, based on parametric t-test for each group versus control. d. Relative overlap between amino acid-defined lung CDR3α repertoires, visualized as a VDJtools MDS plot. Euclidean distance between points reflects the distance between repertoires. Data were normalized to the top 1,000 most frequent clonotypes and weighted by clonotype frequency (F2 metric in VDJtools). Clonotypes were matched on the basis of identical TRAV gene segments and identical CDR3α sequences. The closer two circles are, the higher the overall frequency of shared clonotypes. Node colors correspond to the challenge antigen. Filled circles are lung eTregs, open circles are lung eCD4 cells. e. The same F2 metrics as in (d), shown separately for each challenge and for eTreg-eTreg, eTreg-eCD4, and eCD4-eCD4 repertoire overlap. For both (d) and (e), mice were analyzed in an all-versus-all fashion, irrespective of whether eTreg and eCD4 subsets were obtained from the same or distinct mice.

eTreg TCR repertoire in various tissues following antigen challenge in the lung.

Relative overlap between amino acid-defined eTreg CDR3α repertoires visualized using VDJtools. Data were normalized to the top 1,000 most frequent clonotypes and weighted by clonotype frequency (F2 metric). Clonotypes were matched on the basis of identical TRAV gene segments and identical CDR3α sequences. (a) Dendrogram of Lung eTreg CDR3α repertoires, where branch length shows the distance between repertoires. Heat map at right shows the distribution of selected CDR3α clusters associated with specific challenges. Corresponding CDR3α logos are shown at bottom; clusters also observed in corresponding eCD4 samples are indicated in red. (b–e) MDS plots comparing repertoires between pairs of tissues, where the Euclidean distance between points reflects the distance between repertoires. Overlap is shown for (b) lung versus MLN, (c) lung versus AXLN, (d) lung versus IGLN, and (e) lung versus spleen. The closer two samples are, the higher the overall frequency of shared clonotypes. Node colors correspond to different challenges. Mice were analyzed in an all-versus-all fashion, irrespective of whether tissues were obtained from the same or distinct mice. Circles: lung. Squares: MLN. Triangles: AXLN. Snowflakes: IGLN. Crosses: spleen. (f) Graphs show eTreg F2 repertoire overlap between lung tissue from different animals, and between lungs and other tissues of all animals.

Convergence of eTreg and eCD4 TCR response in distinct tissues.

(a,b) Relative overlap between amino acid-defined lung and MLN (upon lung challenge) and skin (upon skin challenge) eTreg CDR3α repertoires as visualized using VDJtools. Data were normalized to the top 1,000 most frequent clonotypes and weighted by clonotype frequency (F2 metric in VDJtools). Clonotypes were matched on the basis of identical TRAV gene segments and identical CDR3α sequences. (a) Dendrogram branch length shows the distance between repertoires. (b) Euclidean distance between points on the MDS plot reflects the distance between repertoires. The closer two samples are, the higher the overall frequency of shared clonotypes. Mice were analyzed in an all-versus-all fashion, irrespective of whether tissues were obtained from the same or distinct mice. Node colors correspond to d fferent challenges. Circles: lung. Crossed squares: skin. (c) Relative overlap between amino acid-defined lung and MLN (upon lung challenge) and skin (upon skin challenge) eCD4 CDR3α repertoires.

Estimated eCD4 to eTreg conversion.

(a–d) Relative overlap between eTreg and eCD4 nucleotide-defined CDR3α repertoires upon lung (a, b) and skin (c, d) challenges. Overlaps are shown for the same organ (a, c) and spleen (c, d). Data were normalized to the 100 most frequent clonotypes and overlap was weighted by clonotype frequency (F2 metric). Clonotypes were matched on the basis of identical TRAV and TRAJ gene segments and identical TCRα CDR3 nucleotide sequences. Overlap was analyzed separately for each challenge for eTreg and eCD4 subsets obtained from the same mice. The closer two samples are, the higher the F2 metric, which reflects the overall frequency of shared clonotypes. e. Distribution of TCR cluster 408 in eTreg and eCD4 repertoires upon tumor challenge in lung and skin. (f) Treg-iNKT proportion of eTreg cells in lung upon lung challenge. (g) iNKT proportion of eCD4 cells in the lung upon lung challenge. (h) Treg-iNKT proportion of eTreg cells in spleen upon lung challenge. (i) iNKT proportion of eCD4 cells in the spleen upon lung challenge. P-values shown as: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 based on parametric t-test for each group versus control.

Lymphatic tissue-resident eTreg and eCD4 cells.

(a–d) MDS plots showing relative overlap between amino acid-defined eTreg CDR3α repertoires as visualized using VDJtools. Repertoire overlaps are shown for (a) eTreg in MLN, spleen, AxLN, and IgLN; (b) eTreg in MLN, spleen, AxLN, IgLN, and thymus; (c) eCD4 in MLN, spleen, AxLN, and IgLN; and (d) eCD4 in MLN, spleen, AxLN, IgLN, and thymus. Data were normalized to the top 1,000 most frequent clonotypes and weighted by clonotype frequency (F2 metric). Clonotypes were matched on the basis of identical TRAV gene segments and identical CDR3α sequences. Euclidean distance between points reflects the distance between repertoires; the closer two samples are, the higher is the overall frequency of shared clonotypes. Mice were analyzed in an all-versus-all fashion, irrespective of whether the tissues were obtained from the same or distinct mice. Node colors correspond to the different challenges. Squares: MLN. Triangles: AXLN. Snowflakes: IGLN. Crosses: spleen. Stars: thymus.

TCR group behavior in response to different antigenic challenges.

In this TCR homology cluster expression matrix, each column corresponds to a TCR sequence homology cluster (i.e., group of TCR clonotypes having similar CDR3α sequences) and each row corresponds to a particular condition (e.g., tissue type, antigenic challenge, etc). The color of each cell highlights the overall (sum of frequency across all clonotypes) expression of each cluster in a given condition. Rows are sorted by cell type, challenge, organ and cancer type as highlighted by the colored bands at left; columns are sorted according to hierarchical clustering of TCR cluster expression.