Fig 1: A fraction of tongue-associated Treg cell clones exhibit clonal expansion and reactivity to regional antigens(A and B) 6- to 8-week-old Foxp3GFP TCRßtg+ mice were placed on 4-NQO drinking water for 8 weeks, followed by 24 weeks of normal drinking water. Treg cells were subsequently isolated from tongues, single-cell sorted by fluorescence-activated cell sorting (FACS), and TCRa chains were amplified by nested PCR and subjected to Sanger sequencing (see STAR Methods). n = 5 mice.(A) Heatmap depicting all CDR3a sequences identified among all single-cell sorted Treg cells. Each row represents a unique CDR3a sequence. (Left) The number of occurrences each CDR3a sequence appears in tongue-associated Treg datasets from individual 4-NQO-treated mice is shown. Each column represents an individual mouse, with the number of cells sequenced per mouse noted in parentheses. The last column represents the cumulative data. Color denotes the total number each CDR3a sequence appears in dataset. (Right) The frequency each CDR3a sequence appears in published datasets of CD4+ Foxp3+ Treg cells or CD4+ Foxp3neg Tconv cells isolated from pooled secondary lymphoid organs (SLOs) of untreated, non-tumor-bearing Foxp3GFP TCRßtg+ mice is shown.59 Each column represents dataset from an individual mouse. Color denotes the frequency of each CDR3a sequence in dataset.(B) Select CDR3a sequences from (A), depicting those recurrent among at least three 4-NQO-treated mice. Underlined amino acid sequence refers to the 3-letter ID used to reference each TCR clone in (C) and (D).(C and D) 104 Thy1.1+ CD4+ TCR retrogenic (TCRrg) T cells were isolated from pooled SLOs of primary retrogenic mice and co-transferred with 106 RBC-lysed splenocytes from CD45.1/.1 mice intravenously into Tcrb-/- secondary recipients (see STAR Methods). 3 weeks after transfer, the fate of transferred T cells was assessed in SLOs.(C) Representative flow cytometric analysis of CD4+ T cells isolated from indicated organs of secondary recipients that received RTSrg donor T cells. The top plots depict Thy1.1 versus CD45.1 expression by CD4+ cells, whereas the middle and bottom plots depict Egr2 versus CD69 expression by donor Thy1.1+ RTSrg or CD45.1+ polyclonal CD4+ T cells, respectively. The frequency of cells within the indicated gates is denoted.(D) Summary plots of pooled data from (C), showing the frequency of Thy1.1+ CD4+ TCRrg T cells among total CD4+ T cells (top) and the frequency of Thy1.1+ CD4+ TCRrg T cells expressing Egr2 and CD69 (bottom), isolated from indicated organs of secondary recipient mice. TCR clone names depicted refer to the 3rd–5th amino acids of the CDR3a sequence, underlined in (B). Mean is indicated. Dotted line denotes y axis = 1 (top) or y axis = 0 (bottom). n = 4–7 mice (RTS TCRrg), n = 2 mice (DPY TCRrg), n = 9 mice (PHI TCRrg), and n = 3 mice (KGN TCRrg).Data are pooled from multiple independent experiments. aLN, axial lymph node; bLN, brachial lymph node; cLN, cervical lymph node; mLN, mesenteric lymph node; pLN, periaortic lymph node; Spl, spleen. One-way ANOVA with Tukey’s post-test analysis, comparing all pairs in column (D; adjusted p values from Tukey’s post-test are depicted). *p < 0.0.5, **p < 0.01, and ***p < 0.001. See also Tables S1 and S2.