1. Volume 17, Issue 9, Pages 2234-2246 (November 2016)
Tailored Tumor Immunogenicity Reveals Regulation of CD4 and CD8 T Cell Responses against Cancer 
Sarah Knocke, Bettina Fleischmann-Mundt, Michael Saborowski, Michael P. Manns, Florian Kühnel, Thomas C. Wirth, Norman Woller 
Cell Reports 
Volume 17, Issue 9, Pages (November 2016)
DOI: /j.celrep
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2. Cell Reports 2016 17, 2234-2246DOI: (10.1016/j.celrep.2016.10.086)
Copyright © 2016 The Author(s) Terms and Conditions

3. Figure 1
Oncogenic Forms of Ras Rapidly Induce T Cell Responses to Model Antigens in H2-b and H2-d Mice
(A) The depicted plasmids were delivered by hydrodynamic injection to C57BL/6 mice. On day 7 post-injection, splenocytes were stimulated with SIINFEKL and analyzed by IFNγ ELISpot analysis. SIYRYYGL peptides served as the control. Results show the absolute number of spot-forming units (SFUs) per 2 × 105 cells. Mean ± SD; 6 mice/group; Student’s t test; ∗p < 0.05.
(B) A similar setup was used to investigate T cell responses induced by different forms of ras, as indicated in the figure (WT, wild-type; rasG12V, oncogenic forms of ras; rasG12V + D38A, kinase-deficient [inactive] form). Mean ± SD; 3-8 mice/group; Student’s t test; ∗p < 0,05; ∗∗p < 0.01; ns > 0.05.
(C) An in vivo cytotoxicity assay was performed in mice upon hydrodynamic injection of Kras(G12V + D38A) + OVA and KrasG12V + OVA. The histograms display representative results for OVA-specific cytotoxicity. Mean ± SD; 5 mice/group; Student’s t test; ∗∗p < 0.01.
(D) In H2-b mice (C57BL/6), an ELISpot was performed using the model antigen dbp with rasG12V and ras(G12V + D38A), respectively. H2-b-restricted GFP-specific peptide was used as the control. Mean ± SD; 5 mice/group; Student’s t test; ∗p < 0.05.
(E) The same setup as in (D) was used with H2-d mice (BALB/c) and H2-d-restricted peptides. Mean ± SD; 5 mice/group; Student’s t test; ∗∗p < 0.01.
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4. Figure 2
Tailored Tumor Immunogenicity Potently Suppresses NrasG12V-Mediated Tumor Development in p19Arf−/− Mice
(A) OVA-specific T cell epitopes for C57BL/6 mice and the Spnb2-R913L epitope were cloned as epitope tags into an NrasG12V expression vector as shown. Transposons encoding full-length (FL) OVA and a 1.6-kb fragment of Spnb2-R913L were used as the controls. On day 24, the induction of T cell responses of tags and proteins upon hydrodynamic injection were compared by ELISpot analysis. Mean ± SD; 5 mice/group; Student’s t test; ns > 0.05.
(B) An epitope tag coding for Spnb2-R913L was added to the O(MII)-NrasG12V transposon to obtain SO(MII)-NrasG12V. Induction of T cell responses against both MHC class I TSAs in individual mice was confirmed by pentamer staining following the application to p19Arf−/− mice, as shown in the dotplots. Tumor suppression by T cell responses after the application of SO(MII)-NrasG12V or NrasG12V alone was assessed by total liver weight and the detection of lymphocytic infiltration in H&E-stained sections. Mean ± SD; 5 mice/group; Student’s t test; ∗∗p < 0.01.
(C) Tumor suppression and induction of T cell responses were monitored in wild-type and p19Arf−/− mice upon application of the indicated constructs. On day 24, mice were sacrificed, liver weight was determined, and OVA- and Spnb2-R913L-specific T cell responses were quantified by pentamer staining. Antibody-mediated depletion of CD8 T cells was performed to investigate the role of CD8 responses for tumor suppression. Mean ± SD; 5 mice/group; Student’s t test; ∗∗p < 0.01; ∗∗∗p < 0.001; ns > 0.05.
(D) An in vivo cytotoxicity assay was performed in p19Arf−/− mice to determine the tumor cytotoxicity induced by the indicated constructs on day 24. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p <
(E) A similar experimental setup as in (C) was performed to validate previous experiments in wild-type mice with and without the additional application of myrAkt1 and TRP53-R246S-sh3′TRP53 expression vectors. Mean ± SD; 5 mice/group; Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
(F) Upon the generation of tumor cell lines HepN and HepSOMIIN from p19Arf−/− mice injected with corresponding transposon vectors, subcutaneous tumors were established in syngeneic mice. Tumor growth is shown for individual mice of each group. Depleting antibodies were applied twice weekly as indicated. SO-specific responses were quantified on day 14 and are shown in the right panel. Mean ± SD; 5 mice/group; Student’s t test; ns > 0.05.
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5. Figure 3 Interactions between T Cell Responses and Tumor Development
(A) Analysis of tumor-residing lymphocytes by histology and immunohistochemistry. NrasG12V and SO(MII)-NrasG12V tumors were investigated for tumor-residing leukocytes by histology and immunohistochemistry. The graph displays quantifications of mean fluorescence intensity (MFI) of CD45 within the tumor area. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p <
(B) Tumor-infiltrating T cells were analyzed by flow cytometry. The left graph depicts relative changes of CD8 T cells within the total T cell pool (based on CD90+ TILS). Pentamer-positive cells are shown in the right panel. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p <
(C) IFNγ and TNF-α secretion of stimulated tumor-specific T cells was quantified in an intracellular cytokine staining by flow cytometry. The graph shows the ratio of IFNγ and TNF-α double-positive CD8 T cells to total IFNγ-secreting CD8 T cells. Mean ± SD; 5 mice/group; Student’s t test; ns > 0.05.
(D) The ability to mediate tumor suppression in dependence on tumor immunogenicity was investigated upon the application of depicted transposon constructs. On day 24, liver weight was determined and T cell responses were quantified by IFNγ ELISpot. Mean ± SD; 5 mice/group; Student’s t test; ∗p < 0,05; ∗∗p < 0.01; ns > 0.05.
(E) Survival of p19Arf−/− mice injected with NrasG12V and SO(MII)-NrasG12V was followed up for 220 days. Mean ± SD; 10 mice/group; Log-rank (Mantel-Cox) test; ∗∗∗p <
(F) At the end of the survival experiment, T cell responses were quantified by pentamer staining. This figure shows pooled data from two independent experiments. Mean ± SD.
(G) Cytotoxicity and liver tumor burden were inspected in mice with late-occurring tumors (day 130) and compared to mice with no macroscopic tumors of the same group. Mean ± SD.
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6. Figure 4
Interplay of Tumor-Specific CD4 and CD8 T Cells Mediates Tumor Suppression
(A) The role of CD4 T cells in this model was investigated by the application of CD4-depleting antibodies upon the injection of SO(MII)-NrasG12V into p19Arf−/− mice. Liver weight was determined and CD8 T cell responses were quantified by pentamer staining. The NrasG12V group and the isotype-treated SO(MII)-NrasG12V group served as the controls. Mean ± SD; 5 mice/group; Student’s t test; ∗∗p < 0.01; ns > 0.05.
(B) The construct SO-NrasG12V was generated and injected into p19Arf−/− mice, and tumor development was investigated and compared to control groups. Liver weights and results from pentamer staining are shown in the corresponding graphs. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p < 0.001; ns > 0.05.
(C) MHC class II responses specific for both OVA-derived I-Ab epitopes were measured by ELISpot in the NrasG12V and SO(MII)-NrasG12V groups. Full-length OVA (FL-OVA-NrasG12V) served as the positive control. Additionally, peptide-stimulated IFNγ-secreting CD4 T cells were analyzed for the expression of T-Bet. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p < 0.001; ns > 0.05.
(D) The transposon (MII)-NrasG12V was cloned and injected into p19Arf−/− mice. Tumor development was compared to control groups. Quantification of liver weight and CD4-specific tumor immune responses is shown in the graph. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p < 0.001; ns > 0.05.
(E) CD4 IHC from the indicated groups was performed to validate systemic CD4-specific immune responses and to investigate the distribution of CD4 T cells within livers. Clusters of CD4 cells are marked by arrows.
(F) Cytotoxic activity of tumor-induced CD4 T cells was investigated in an in vivo cytotoxicity assay. Therefore, both OVA MHC class II epitopes were used to pulse target cells. These cells were adoptively transferred to p19Arf−/− mice that received injections of NrasG12V and SO(MII)-NrasG12V, respectively. To analyze cytotoxicity, CFSE+ target cells were gated on CD19 by flow cytometry. Analysis of SO-specific cytotoxicity of CD8 T cells served as the control. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p < 0.001; ∗p < 0.05.
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7. Figure 5
Cancer Immunosurveillance Is Not Capable of Eliciting Sufficient T Cell Responses for Tumor Suppression against MHC Class I-Restricted Epitopes Derived from Immunoedited Tumors
(A) To investigate tumor-directed T cell responses to epitopes derived from immunoedited tumors, we generated the construct NL(MII)-NrasG12V that contains epitope tags of the epitopes Ndufs1-V491A and Lama4-G1254V. This construct was injected into p19Arf−/− mice, and tumor development was compared to NrasG12V and the SO(MII)-NrasG12V groups. Liver weights and results from quantified CD8 and CD4 T cell responses by intracellular IFNγ staining are shown in corresponding graphs. Mean ± SD; 5-8 mice/group; Student’s t test; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns > 0.05.
(B) A DC vaccination was performed to assess the induction of T cell responses to Ndufs1-V491A. The dotplot displays a representative result. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p <
(C) The figure shows results of CD4 responses to MHC class II OVA epitopes and the neoantigen Kif18b-K739N in dependence of Spnb2-R913L and NrasG12V. Indicated constructs were injected into WT and p19Arf−/− mice, respectively. On day 7 the immune responses for each antigen were measured by ELISpot. Mean ± SD; 5 mice/group; Student’s t test; ∗∗∗p < 0.001; ∗∗p < 0.01; ns > 0.05.
Cell Reports  , DOI: ( /j.celrep )
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