Inducible de novo expression of neoantigens in tumor cells and mice

Abstract

Inducible expression of neoantigens in mice would enable the study of endogenous antigen-specific naïve T cell responses in disease and infection, but has been difficult to generate because leaky antigen expression in the thymus results in central T cell tolerance. Here we develop inversion-induced joined neoantigen (NINJA), using RNA splicing, DNA recombination and three levels of regulation to prevent leakiness and allow tight control over neoantigen expression. We apply NINJA to create tumor cell lines with inducible neoantigen expression, which could be used to study antitumor immunity. We also show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cell responses on neoantigen induction in peripheral tissues. NINJA will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, transplantation, autoimmune diseases and cancer.

Extended Data Figure 1 -. Introduction of splice sites into NM

a, Possible splice donor and acceptor sequence candidates in the NM to create Exon 2. b, Design and inversion of Exon 2, with transcription in OFF state resulting in splicing directly from Exon 1 to Exon 3. Insertion of non-compatible Frt sites shown in light and dark blue arrows. c, FLPo recombinase activity results in permanent inversion and transcription of all exons of the NM.

Extended Data Figure 2 -. Development of advanced versions of NM

a, Schematic shows 7 versions of the NM, with modifications made at each step and the fluorescence status at either ON or OFF state. b, Flow cytometry histograms of GFP or YFP fluorescence in each version, with or without FLPo activation. Representative of 3 independent experiments. c, Western blotting for GRP94 (control, top panel) or N-terminal GFP (bottom panel) on lysates from 293T cells transiently transfected with each version of NM, with (top blot) or without (bottom blot) FLPo. Positive control (+) is cell lysate from KP-C4A3D6 after FLPo. Representative of 3 independent experiments.

Extended Data Figure 3 -. Introduction of frameshift into NM

Schematic of NM.3, which shows the two possible in silico insertions of the spliced neoantigen construct from NM.2 in YFP, where in one skipping exon 2 results in a frameshift and premature stop codon. b-c,The transcription product and fluorescence status in the ON or OFF state is shown for the frameshift version (b) or no frameshift version (c). d, Flow cytometry histograms of YFP fluorescence of the constructs from c. The frameshift version is only YFP positive after FLPo exposure. Representative of 3 independent experiments. e, 293T cells transiently transfected with plasmids expressing the indicated version of the NINJA NM were imaged by confocal microscopy after staining with an antibody specific for either the N-term portion of GFP (middle panels), for a conformational epitope of GFP (bottom panels) or with no antibody (top panels). BLUE = DAPI, RED = folded GFP, GREEN = fluorescent GFP. Representative images are shown (n = 3). f, Hydrophobicity score (top graph) in relation to amino acid position along the NM (red/grey/blue rectangle). In version NM.5 (black line) positions GP43-GP59 are predicted to be a transmembrane domain (bottom graph), and this elevated hydrophobicity is abrogated when replaced by a FLAG domain in NM.7 (red line).

Extended Data Figure 4 -. Peptides from splice junction in NM are not presented as antigens

a, The antisense DNA sequence of exon 2 encodes GP34–41 with a preceding amino acid encoded by nCA, which could encode an Ala, Ser, Thr, or Pro residue b, the predicted binding of each peptide (SIINFEKL control, GP34–41, or GP33–41 with K33A, K33S, K33T, or K33P mutations). K33P did not bind to H2-D^b or stimulate T cell activation.

Extended data Figure 5 -. Design and development of RM

a, Flow cytometry plots of GFP expression in transiently transfected 293s with either NM.7 alone or in combination with FLPoER or FLPoER²⁵¹. FLPoER²⁵¹, while leakier, is more responsive to 4-OHT treatment than FLPoER and its activity was not increased by estrogen (E2) treatment. Data reflects n=3 technical replicates per group; 3 independent experiments. (Unpaired two-tailed t tests, *, P = 0.0170, ns, P = 0.896. Measure of centre for no treatment = 15.6(+/−2.8)%, for E2 = 16.1(+/−1.5)%, for 4-OHT = 27.9(+/−1/3%. Error bars = mean with SEM.) b, An early regulatory module design, with pTRE:Lox-STOP-Lox (LSL):FLPoER²⁵¹ 2x CGG insulator, and the NM. We discovered in c, that the NM in this construct was recombined by FLPo activity in E coli, which led to the later inverted design.

Extended Data Figure 6 -. Schematic diagram of completed NINJA construct

Final version of the NINJA construct, including the final RM and NM.

Extended Data Figure 7 -. Targeting of ES cell clones for NINJA

a, Schematic showing the Rosa 26 targeting construct used for generation of the NINJA mouse. b, Southern blotting confirmation of successful target insertion of NINJA. Single experiment. c, Confirmation of germline transmission in two pups via PCR in the Rosa locus. Representative of >1000 experiments.

Extended Data Figure 8 -. Time course of neoantigen-specific CD8 T cell response in NINJA

Images show local accumulation and expansion of fLuc⁺ P14 T cells adoptively transferred into NINJA mice that were subsequently infected S.C. in the footpad with Ad-FLPo (10⁷ PFU/mouse) and imaged by IVIS at the indicated day after infection. Representative mice are shown (n = 3). Intensity of signal (blue to red) indicates accumulation of T cells.

Extended Data Figure 9 -. Ad-Cre infection does not lead to leaky neoantigen expression

Quantification of endogenous H2Db/GP_33–43-specific Thy1.2⁺CD8⁺ cells from the spleen (gray dots) and draining LNs (white dots) of NINJA mice 8 days after S.C. infection in the footpad with the indicated mixes of Ad-FLPo + Ad-Cre or Ad-FLPo + Ad-GFP (total dose 10⁷ PFU/mouse) was performed by flow cytometry. Representative experiment is shown (n = 5). n.s. = difference not significant by two-tailed unpaired t test for comparisons of (Ad-FLPo + Ad-Cre) vs. (Ad-FLPo + Ad-GFP) responses in the spleen (P = 0.9) and in the draining LNs (P = 0.3). Average values ± SD are shown.

Extended Data Figure 10 -. Analysis of thymic development in NINJA strains

Dot plots from Figure 3b show the average frequency ± SD of each thymocyte population as determined by FC analysis for the indicated mouse strains (each dot represents one mouse, n ≥ 3, 2 experimental repeats). n.s., not significant by two-tailed unpaired t test (0.1 ≤ P ≤ 0.9).

Figure 1 -. Design and development of NINJA targeting construct.

Schematic of NINJA and transfections confirming fluorescence, protein size, T cell response, and D/T drug response. a, Schematic of NINJA targeting construct (NINJA TC) after integration into Rosa26 (R26) locus. NINJA contains regulatory (RM) and neoantigen (NM) modules. Schematic shows modules after Cre or FLPo recombinase and doxycycline/tamoxifen (D/T) exposure. (Red/ white fill arrow pairs = non-compatible loxP sites. Black/white ball and stick lines = splice sites, and black/grey arrows = promoters. Light/dark blue fill arrow pairs = non-compatible FRT sites.) b-c, FLPo exposure is required for GFP expression from NM. 293T cells transfected with indicated constructs and assessed by FC or western blot. b, GFP expression 72 hours post-transfection with NM.7 alone (gray, filled) or NM.7+FLPo (line). % GFP+ is indicated. c, Western blotting for GRP94 (control, top panel) or N-terminal GFP (bottom panel) on cell lysates. Positive control (+) is KP-C4A3D6 after FLPo (see Figure 2). d-e, NM is only immunogenic after FLPo exposure. DC2.4 cells transfected with NM.7 or NM.7+FLPo and cultured +/− naive Cell Trace Violet (CTV)-stained P14 T cells. d, Tumor cells stained with crystal violet, day 4. e, CTV dilution and CD44 expression on CD8+ Va2+ T cells f, NINJA TC expresses GFP after Cre, rtTA, and D/T treatment. 293T cells were transfected with NINJA TC with and without plasmids expressing Cre and rtTA. Cells were then cultured with or without D/T. Histograms show GFP expression from the 293T cells with the indicated conditions. A-C, F Representative of 3 independent experiments, D-E single experiment.

Figure 2 -. NINJA cell line generates antigen specific T cell response following induction.

KP lung tumor cell line with poised NINJA turns on neontigens in response to D/T in vitro and in vivo.a, Schematic depicting generation of the “poised” KP-C4A3D6 cell line.. b, GFP expression by untreated KP-C4A3D6 (OFF), or KP-C4A3D6 72 hours post-D/T treatment or Ad-FLPo. FC-sorted GFP+ KP-C4A3D6 cells shown as ON. c-f, KP-C4A3D6 cells only elicit tumor-specific CD8 T cell responses in vivo when ON. KP-C4A3D6 OFF and ON cells were intramuscularly transplanted bilaterally into B6 recipients (25–200 × 10³ cells, n = 24). Mice also received naive fLuc+ Thy1.1+ P14 T cells. c, Representative IVIS image shows signal from P14 T cells in ON and OFF tumors in the same mouse on day 11 after transplant. Intensity of signal (blue to red) indicates accumulation of T cells. d, FC dot plots pregated for CD8+ cells show presence of intratumoral Thy1.1+ CD44+ P14 T cells from ON and OFF tumor (numbers are average percent of n = 13 in one experiment, plots are one representative mouse). e, Graph shows the frequency of intratumoral Thy1.1+ CD8+ P14 T cells from paired ON and OFF tumors (connected by line). (*, P = 0.0006 (paired t test, two tailed, n = 11 co-injected animals)). f, Graph shows average tumor measurements of tumors in ON and OFF legs (*, P < 0.0001 in unpaired t test, two tailed; ns, P=0.96, P=0.54. Minimum 3 experimental repeats; error bars are SD. (n = 29, +P14 n = 36 biologically independent animals))). g, In vivo activation of neoantigens triggers tumor-specific CD8 T cell response. KP-C4A3D6 OFF and ON cells were intramuscularly transplanted into B6 recipients that contained naive fLuc+ Thy1.1+ P14 T cells. IVIS images show signal from P14 T cells on indicated days. Intensity of signal (blue to red) indicates accumulation of T cells. Middle panels show treatment with D/T day 6–9 post-tumor inoculation. (2 independent experiments, n = 10/12 treated animals responding).

Figure 2 -. NINJA cell line generates antigen specific T cell response following induction.

KP lung tumor cell line with poised NINJA turns on neontigens in response to D/T in vitro and in vivo.a, Schematic depicting generation of the “poised” KP-C4A3D6 cell line.. b, GFP expression by untreated KP-C4A3D6 (OFF), or KP-C4A3D6 72 hours post-D/T treatment or Ad-FLPo. FC-sorted GFP+ KP-C4A3D6 cells shown as ON. c-f, KP-C4A3D6 cells only elicit tumor-specific CD8 T cell responses in vivo when ON. KP-C4A3D6 OFF and ON cells were intramuscularly transplanted bilaterally into B6 recipients (25–200 × 10³ cells, n = 24). Mice also received naive fLuc+ Thy1.1+ P14 T cells. c, Representative IVIS image shows signal from P14 T cells in ON and OFF tumors in the same mouse on day 11 after transplant. Intensity of signal (blue to red) indicates accumulation of T cells. d, FC dot plots pregated for CD8+ cells show presence of intratumoral Thy1.1+ CD44+ P14 T cells from ON and OFF tumor (numbers are average percent of n = 13 in one experiment, plots are one representative mouse). e, Graph shows the frequency of intratumoral Thy1.1+ CD8+ P14 T cells from paired ON and OFF tumors (connected by line). (*, P = 0.0006 (paired t test, two tailed, n = 11 co-injected animals)). f, Graph shows average tumor measurements of tumors in ON and OFF legs (*, P < 0.0001 in unpaired t test, two tailed; ns, P=0.96, P=0.54. Minimum 3 experimental repeats; error bars are SD. (n = 29, +P14 n = 36 biologically independent animals))). g, In vivo activation of neoantigens triggers tumor-specific CD8 T cell response. KP-C4A3D6 OFF and ON cells were intramuscularly transplanted into B6 recipients that contained naive fLuc+ Thy1.1+ P14 T cells. IVIS images show signal from P14 T cells on indicated days. Intensity of signal (blue to red) indicates accumulation of T cells. Middle panels show treatment with D/T day 6–9 post-tumor inoculation. (2 independent experiments, n = 10/12 treated animals responding).

Figure 3 -. Central and peripheral tolerance are bypassed in NINJA mice

a, No neoantigen expression at baseline in NINJA mice. Graph shows the frequency of NINJA-expressing GFP⁺ peripheral blood mononuclear cells (PBMCs) isolated from NINJA, NINJA-F and NINJA-C mice (determined by flow cytometry). Dotted line represents background fluorescence signal from wild-type B6 PBMCs. Representative experiment (n = 3, 5 experimental repeats). Graph shows average values ± SD. * P = 0.02 and ** P = 0.004 by two-tailed unpaired t test for comparison of NINJA vs. NINJA-F or NINJA vs. NINJA-C. b, NINJA mice bypass central tolerance towards neoantigens. Bar graphs show the frequency of each gated thymocyte population determined by FC analysis for the indicated mouse strains (n ≥ 3, 2 experimental repeats). Graph shows average values ± SD. n.s., not significant by two-tailed unpaired t test (0.1 ≤ P ≤ 0.9). Single dot plots are shown in Extended Data Figure 10. c–d, NINJA mice have normal CD8 and CD4 T cell responses to acute LCMV infection. Graphs showing the number of c, H2Db/GP_33–41-specific (top) and H2Db/NP_396–404-specific (bottom) Thy1.2⁺CD8⁺ cells or d, I-A^b/GP_66–77-specific Thy1.2⁺CD4⁺ cells from the spleen on day 8 after LCMV-Armstrong infection. Representative experiment (n = 3, 3 experimental repeats). Dotted lines represent background tetramer staining as determined on splenocytes from untreated B6. Graphs in c-d show average values ± SD. n.s., not significant by two-tailed unpaired t test (0.5 ≤ P ≤ 1). e, T cells are not tolerized by the peripheral environment of NINJA mice. Thy1.1⁺/1.1⁺ P14 T cells were adoptively transferred into B6, NINJA, NINJA-F and NINJA-C mice. 2 weeks later, mice were infected with LCMV-Armstrong and analysis was conducted 8 days after infection. FC plots show the average frequency ± SD of P14 CD8⁺ T cells in the spleen (n = 6, 2 experimental repeats).

Figure 4 -. Expression and presentation of neoantigens after activation of the NM.

a. Activation of neoantigen expression in NINJA BMDCs stimulates neoantigen-specific CD8 and CD4 T cell responses. BMDCs were generated from NINJA mice and infected with Ad-GFP (control, top) or Ad-FLPo (bottom) to turn on the NM directly. BMDCs were used to immunize B6 mice (subcutaneously, S.C.) that contained adoptively transferred Thy1.1⁺/1.2⁺ P14 T cells and Thy1.1⁺/1.1⁺ SMARTA T cells. Dot plots show average frequency ± SD of P14 (left) or SMARTA (right) T cells from the indicated mice from a representative experiment (n = 11, 2 experimental repeats). b. Expansion of neoantigen-specific CD8 T cells is localized to the site of antigen induction. fLuc⁺ Thy1.1+ P14 T cells were adoptively transferred into NINJA mice that were subsequently infected intravenously (I.V.), intramuscularly (I.M.), intratracheally (I.T.) or S.C. (footpad) with Ad-FLPo. IVIS images show the location of P14 T cells 8 days after infection. Intensity of signal (blue to red) indicates accumulation of T cells. Representative mice are shown (n = 6, 2 experimental repeats). c. Neoantigen-specific T cells interact with neoantigen expressing skin cells after Ad-FLPo infection. dsRED⁺ P14 T cells were adoptively transferred into NINJA mice that were subsequently infected with Ad-FLPo S.C. in the footpad (left) or left untreated (right). Confocal microscope images show GFP⁺ NINJA-expressing cells (green), P14 T cells (red) and DAPI (blue) 8 days after infection. Representative images (n = 3). d-e, Transgenic neoantigen-specific CD8 T cells respond to in vivo induction of neoantigens in the footpad. d, Dot plots show accumulation of Thy1.1⁺ P14 T cells in the footpad of NINJA mice that were infected S.C. locally with Ad-FLPo 8 days before analysis. Representative mice are shown (n = 3, 2 experimental repeats). Average frequency and average MFI ± SD are shown. e, Immunohistological sections of footpads from d show signs of immuno-mediated pathology indicated by epidermal/dermal thickening and infiltration by CD3⁺ cells (arrows) in Ad-FLPo-infected samples but not in untreated samples. Representative images (n = 3).

Figure 5 -. In vivo endogenous GP33-specific responses in NINJA mice after local infection with Ad-FLPo

a-c, NINJA mice were left untreated or infected S.C. in the footpad with Ad-FLPo or Ad-GFP to analyze the development of endogenous neoantigen-specific effector CD8 T cell responses following local infection. a, FC plots show average frequency ± SD of endogenous activated (CD44⁺) H2Db/GP_33–43-specific Thy1.2⁺CD8⁺ cells from the local draining LNs 8 days after infection. Representative dot plots are shown (n = 3, 3 experimental repeats). b, Histograms show Granzyme B expression (top) and FC plots show IFNγ and TNFα production after ex vivo antigen-specific restimulation (bottom) by H2Db/GP_33–43-specific Thy1.2⁺CD8⁺ cells from a. Representative dot plots are shown (n = 3, 3 experimental repeats). Average frequencies and MFI ± SD are indicated. c, Histological sections of footpad show epidermal and dermal thickening and infiltration by endogenous CD3⁺ cells in NINJA mice infected S.C. with Ad-FLPo (top and bottom left) as compared to untreated NINJA mice (top and bottom right). Representative images (n = 3). d, FC plots show average frequency ± SD of endogenous CD44⁺ H2Db/GP_33–43-specific Thy1.2⁺CD8⁺ cells from the local draining LNs 8 or 35 days after S.C. infection in the footpad with Ad-FLPo. Representative dot plots are shown (n = 4, 2 experimental repeats). MFI = mean fluorescence intensity; d = day.

Figure 6 -. In vivo genetic tissue-specific activation of endogenous GP33-specific T cells

a, Temporally delayed antigen induction allows for neoantigen-specific CD8 T cell responses. FC plots show the average frequency ± SD of endogenous activated (CD44⁺) H2Db/GP_33–43-specific Thy1.2⁺CD8⁺ cells from local draining LNs of NINJA mice 8 days after S.C. infection in the footpad with Ad-Cre alone (negative control) or Ad-Cre administered either together with D/T or 14 days prior to treatment with D/T. Representative dot plots (n = 4, 2 experimental repeats). b, NINJA-C-ER mice have normal CD8 and CD4 T cell responses to acute LCMV infection. Graphs show the number of H2Db/GP_33–43-specific and H2Db/NP_396–404-specific Thy1.2⁺CD8⁺ splenocytes in the indicated strains 8 days after infection with LCMV-Armstrong (n = 5). Average values ± SD are shown. n.s., not significant by two tailed unpaired t test (P = 0.6 for comparisons of H2Db/GP_33–41-specific responses; P = 0.4 for comparisons of H2Db/NP_396–404-specific responses). c, fLuc⁺ P14 T cells were adoptively transferred into NINJA-C-ER mice that were subsequently treated systemically with D and topically with T (painted on an area of the skin corresponding to the red square) or left untreated. IVIS images show local skin accumulation of P14 T cells only in the mice receiving D/T. Representative mice are shown (n = 3, 3 experimental repeats). d, Endogenous H2Db/GP_33–43-specific Thy1.2⁺CD8⁺ cells expand following local induction of neoantigen expression in the skin. Dot plots show average frequency ± SD of H2Db/GP_33–43-specific cells (pre-gated on Thy1.2⁺CD8⁺ cells) in the local draining LNs of NINJA-C-ER mice 6 days after completing treatment with systemic D and topic T on a selected area of the skin. Representative mice are shown (n = 5). D = doxycycline; T = 4-OHT.