Fig 2: Direct antigen presentation by leukemia cells is required for in vivo antigen recognition by TCRTg101(A) TCR2C or TCRTg101 (1 × 105) were CTV-labeled and cultured in vitro with C1498.SIY cells at various ratios for 72 h, at which point TCR2C and TCRTg101 proliferation, as measured by CTV dilution, was assessed by flow cytometry. TCR2C and TCRTg101 stimulated with anti-CD3 and anti-CD28 antibodies or left unstimulated served as positive and negative controls for proliferation, respectively.(B) CTV dilution profiles of TCR2C or TCRTg101 cultured for 72 h with CD8a+ DCs, CD8 a- DCs, or macrophages isolated from spleens of naive C57BL/6 mice or C57BL/6 mice challenged 3 h earlier with C1498.SIY cells i.v.(C–F) CTV-labeled TCRTg101 (CD45.1.2) were adoptively transferred into CD11c-DTR bone marrow chimeric mice (CD45.2) that received DT or PBS prior to and following i.v. C1498 cell challenge.(C) Representative FACS plot showing frequencies of CD11c+ cells in spleens of CD11c-DTR bone marrow chimeras after DT or PBS treatment. Gating was performed on live CD3-CD19-CD11c+ cells.(D) Representative FACS plots showing CTV dilution of TCRTg101 (TCRb+CD8+CD45.1.2 cells) in livers of naive or C1498 cell-challenged CD11c-DTR bone marrow chimeric mice (treated with DT or PBS) at day 12.(E and F) Quantitative data from (D) are shown.(G) CTV dilution profiles of TCR2C and TCRTg101 cultured with BMDCs pulsed with C1498.SIY or C1498 cell lysates, respectively. CTV dilution profile of TCRTg101 cultured with live C1498 cells was included as a positive control (lower right). Representative FACS plots are shown.(H and I) 2 × 106 CTV-labeled TCRTg101 (CD45.2) were transferred i.v. into B6.SJL (CD45.1) mice followed by an i.v. challenge with 106 C1498 or C1498.H2-Kb-/- cells the following day. On day 18, TCRTg101 proliferation was assessed.(H) Representative FACS plots showing CTV dilution and CD44 expression on TCRTg101 in livers of mice previously challenged with C1498 or C1498.H2-Kb-/- cells.(I) Quantitative data from (H) are shown.(J–L) 2 × 106 CTV-labeled TCRTg101 (CD45.1.2) were transferred i.v. into B6.SJL (CD45.1) mice followed by a s.c. challenge with 106 C1498 or C1498.H2-Kb-/- cells the following day. 14 days later, TCRTg101 frequencies and numbers were assessed in tdLNs and tumors.(J) Representative FACS plots showing TCRTg101 frequencies in tdLNs of mice with s.c. C1498 or Kb-/- C1498 tumors.(K and L) Quantitative data showing frequencies (K) and numbers (L) of TCRTg101 in tdLNs of mice with s.c C1498 or Kb-/- C1498 tumors.(M) Representative FACS plots showing TCRTg101 frequencies among total CD8+ T cell populations in s.c. C1498 or Kb-/- C1498 tumors.(N and O) Quantitative data showing TCRTg101 frequencies (N) and numbers/mg of tumor tissue (O).Data are representative (I) of or pooled (E–F, K–L, and M–O) from 2 independent experiments with 3 mice/group and presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

Fig 3: 4-NQO-induced lesions are not significantly impacted by the endogenous T cell response or treatment with checkpoint blockade monotherapies(A and B) 6- to 8-week-old littermates of indicated genotype were exposed to 4-NQO drinking water for 20 weeks, tongues were subsequently excised, and histopathology was quantified as in Figure 2A. n = 20 mice (Tcrb+/-Tcrd+/-), n = 11 mice (Tcrb+/-Tcrd-/-), n = 18 mice (Tcrb-/-Tcrd+/-), and n = 7 mice (Tcrb-/-Tcrd-/-).(A) Tumor incidence, where each mouse was scored once based on most severe histology grade observed. Summary plot of pooled data shows the percentage of mice scored as indicated histology grade. Each bar represents the cumulative data for mice of the indicated genotype.(B) Tumor burden. Summary plots of pooled data show the percentage of tongue perimeter defined as hyperkeratosis (left plot), dysplasia (middle plot), or SCC (right plot). Each symbol represents an individual tongue from a single mouse of the indicated genotype. Median is indicated.(C–E) 6- to 8-week-old female C57BL/6 mice were exposed to 4-NQO drinking water for 20 weeks. During the last 4 weeks of 4-NQO exposure, mice were treated intraperitoneally twice weekly with 100 µg anti-PD-L1 or isotype control monoclonal antibody. Tongues were excised at endpoint, and histopathology and IHC staining was performed and quantified as in Figures 2A and 2D. n = 10 mice per group.(C) Tumor incidence, where each mouse was scored once based on most severe histology grade observed. Summary plot of pooled data shows the percentage of mice scored as indicated histology grade. Each bar represents the cumulative data for mice that received indicated antibody treatment.(D) Tumor burden. Summary plot of pooled data shows the percentage of tongue perimeter defined as the indicated histology grade. Each symbol represents an individual tongue from a single mouse that received the indicated antibody treatment. Median is indicated.(E) Summary plot of pooled data from CD3 IHC density analysis, showing the number of CD3+ cells per mm2 for each lesion. Each symbol represents an individual lesion. Median is indicated. n = 6–52 lesions from 10 mice per group.Data are pooled from multiple independent experiments. Fisher’s test (A and C); one-way ANOVA, comparing all pairs in column (B); two-tailed nonparametric Mann Whitney test (D and E). NS, not significant. See also Figure S2.

Fig 4: 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.