A mouse model for the study of anti-tumor T cell responses in Kras-driven lung adenocarcinoma

Abstract

Kras-driven lung adenocarcinoma (LUAD) is the most common lung cancer. A significant fraction of patients with Kras-driven LUAD respond to immunotherapy, but mechanistic studies of immune responses against LUAD have been limited because of a lack of immunotherapy-responsive models. We report the development of the immunogenic KP × NINJA (inversion inducible joined neoantigen) (KP-NINJA) LUAD model. This model allows temporal uncoupling of antigen and tumor induction, which allows one to wait until after infection-induced inflammation has subsided to induce neoantigen expression by tumors. Neoantigen expression is restricted to EPCAM+ cells in the lung and expression of neoantigen was more consistent between tumors than when neoantigens were encoded on lentiviruses. Moreover, tumors were infiltrated by tumor-specific CD8 T cells. Finally, LUAD cell lines derived from KP-NINJA mice were immunogenic and responded to immune checkpoint therapy (anti-PD1 and anti-CTLA4), providing means for future studies into the immunobiology of therapeutic responses in LUAD.

Graphical abstract

Figure 1

KP-NINJA lung tumor neoantigen induction model (A) Schematic of NINJA and KP allele induction activity. Kras^{Lox-Stop-Lox (LSL)G12D} is activated and p53^flox/flox is deleted after Cre recombinase activity, and the FLPoER in NINJA flips to being in-frame for transcription. After doxycycline and tamoxifen (Dox/Tam) administration, Dox binds the lung-specific CCSP-rtTA to allow the Tet-controlled FLPoER promoter to express, and Tam binds FLPoER to allow nuclear entry and recombination and expression of the NINJA neoantigen. (B) KP-NINJA mouse tumor and antigen induction protocol timeline, with Adeno- or Lenti-Cre induction at day 0, followed by antigen induction by Dox/Tam at days 7–10. Inflammation approximation is based on data presented in Table 1.

Figure 2

Neoantigen induction in KP-NINJA occurs in lung epithelial cells after overt inflammation is resolved (A) Infected (GFP+) cells 4–7 days after Lenti- or Adeno-CMV-GFP infection in lung, showing CD45+ and EPCAM+ populations. Percentages are average ± standard deviation; Lenti-CMV-GFP, n = 8 mice; Ad-CMV-GFP, n = 2 mice, from two independent repeats. (B) Infected (previously gated Tom+) lung cells gated for CD45 and EPCAM 4–7 days p.i. (high dose, 10⁹ PFU; percentages are average ± standard deviation; Ad-SPC-Cre, n = 4 mice; Ad-CMV-Cre, n = 5 mice, from two independent repeats). (C) Selected cytokines from Luminex cytokine panel, assessed for concentration in lung homogenate at 24 h, 7 days, or 14 days p.i. with 2.5 × 10⁷ PFU Adeno-SPC-Cre. n = 3 mice/time point. ∗p < 0.05, unpaired t test versus uninfected. (D) Expression of tomato and NINJA in T cell-depleted KP-NINJA/tomato (NINJA/Tom) lungs or KP-tomato control mouse (Tom) 21 days p.i., with mid-dose 10⁸ PFU Adeno-SPC-Cre and Dox/Tam administration. Percentages shown are out of all infected cells (Tom+) average ± standard deviation, n = 7. Arrows point to expression of EPCAM+ and CD45+ on cells from NINJA/Tom gates in the top right.

Figure 3

Antigen expression in KP-NINJA tumors has more uniform expression versus Lenti-neoantigen tumors (A) Representative H&E images of lung tumors 25 weeks p.i. Left panel: Adeno-SPC-Cre in KP-NINJA; representative of n = 15 tumors imaged (34/34 mice had tumors by necropsy, 100% penetrance). Right panel: LucOS-Cre LV (Lenti-ubc-LucOS-pgk-Cre) in KP; representative of n = 16 tumors imaged (20×). Scale bar, 200 μm. (B) Immunofluorescence image of KP-NINJA lung tumor. Green, endogenous NINJA-GFP (10×). (C) Schematic of cell line generation from primary lung tumors. All analysis occurred after a minimum of six passages in culture. (D) Fold change relative to background of neoantigen-luciferase intensity, in LucOS-Cre LV cell lines derived from 20 week p.i. KP lung tumors. (E) KP-NINJA cell lines. Left panel: fold change in NINJA-GFP expression is measured by geometric mean of GFP+/GFP– MFI. Right panel: percent of cells in each cell line positive for neoantigen expression by flow cytometry. Cre LV (Lenti-pgk-Cre) in KP-NINJA+ Dox/Tam lines from 8 week p.i. tumors. (F) MHC-I and MHC-II expression on a KP-NINJA cell line from (E) after overnight stimulation with IFNγ. A single representative experiment shown. (G) Cre-GFP33 LV cell lines. Left panel: fold change in GFP expression by MFI. Right panel: percentage of cells in each cell line positive for neoantigen expression by flow cytometry. Cre-GFP33 LV (Lenti-CMV-Cre-sv40-GP33) in KP lines from 8 week p.i. tumors.

Figure 4

KP-NINJA tumors generate tumor neoantigen-specific T cell responses (A) Proportion of CD3 infiltration in tumors 8 weeks p.i. by IHC, blindly scored in KP Cre LV (KP) versus KP Cre-GFP33 LV (KP + GFP33) versus KP-NINJA Cre LV (KP-NINJA) for <10%, 10%–50%, or >50% tumor infiltration (n = 10 KP-NINJA, n = 12 KP + GFP33, n = 10 KP + Cre). (B) Immunofluorescence image of a KP-NINJA tumor. Green, endogenous NINJA-GFP; blue, B220 (B cells); red, CD3 (T cells) (40×, 4 × 4 tiled, 20 weeks p.i.). (C) Proportion of tumors scored in KP-NINJA that were associated with TLS; T cells only (5/60; 8.3%), T and B cells (26/60; 43.3%), or none (29/60; 48.3%). (D) Representative flow cytometry plots of Thy1.2+ CD8+ T cells gated for antigen specificity (GP33+) in tumor-bearing (KP-NINJA) or non-tumor-bearing (P-NINJA) lung. Note, i.v. CD45– indicates tissue infiltration. Percentages are averages ± standard deviation of n = 3 mice (P-NINJA) or n = 20 mice (KP-NINJA). (E) Quantification of lung-infiltrating antigen-specific T cells in (D) (%GP33+ i.v. CD45– Thy1.2+ CD8+) 8 weeks p.i. with Ad-SPC-Cre. The dotted line represents a control C57BL/6J mouse. Red dots represent mice where %GP33+ was not higher than the FMO control (3/20). ∗p < 0.05 in unpaired t test. (F) Quantification of total endogenous T cell infiltration in tumor-bearing or non-tumor-bearing lung in (D) (i.v. CD45– Th1.2+ CD8+). The dotted line represents a control C57BL/6J mouse. Red dots are the same mice as shown in (E). ∗p < 0.05 in unpaired t test.

Figure 5

KP-NINJA tumor cell lines are immunogenic and respond to checkpoint therapy (A) Expression of NINJA-GFP in one of the KP-NINJA primary cell lines (16394, also known as KPN1). Left two panels: KPN1 before and after sorting for GFP+. Right panel: after one passage through C57BL/6J mice and rederivation of the cell line as KPN1.1. (B) Mean intramuscular growth of 200K cells of four transplanted primary KP-NINJA lung cell lines in NSG mice (16394, 16459, and 16579 all n = 3 mice; 16456 n = 5; error bars are SD). (C) Mean subcutaneous growth of 500K KPN1.1 in Rag1^−/−, wild-type C57BL/6J, or NINJA-F (antigen-tolerant) mice. Representative of two independent experiments (RAG n = 5 mice, NINJA-F n = 8, C57BL/6J n = 10; the same C57BL/6J untreated control) is shown in (D). (D) Mean subcutaneous growth of 500K KPN1.1 in wild-type C57BL/6J with or without CD4 or CD8 depletion (200 μg/mouse/antibody were administered i.p. every 3 days, from day −3 until study endpoints, indicated on the x axis). aCD8 and aCD4 n = 5 mice each; C57BL/6J untreated control is also shown in (C). (E) Representative flow cytometry plots of CD8+ CD4− tumor-infiltrating T cells from KPN1.1 subcutaneous transplant tumors, gated for antigen specificity (GP33+). Percentages shown are averages ± standard deviation; n = 3 mice for tumors and n = 5 for lymph nodes (LNs). (F) PD1 expression on T cells from the GP33+ Thy1.2+ gate in (E). Percentages shown are averages ± standard deviation; n = 3 mice for tumors and n = 5 for LNs. (G) Mean subcutaneous growth of 500K KPN1.1 in wild-type C57BL/6J mice, with or without anti-PD1 200 μg and anti-CTLA4 200 μg dosing. Orange arrows indicate treatment; days 3, 6, and 9. Anti-PD1 + anti-CTLA4 n = 10 mice, C57BL/6J n = 10, from two independent experiments. (H) Individual tumor curves from (G). In (C), (D), (G), and (H), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; two-way ANOVA.