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. 2016 Dec;4(12):1007-1015.
doi: 10.1158/2326-6066.CIR-16-0156. Epub 2016 Oct 31.

Endogenous Neoantigen-Specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach

Tanner M Johanns  1   2 , Jeffrey P Ward  1   2   3   4 , Christopher A Miller  5   6 , Courtney Wilson  2   3 , Dale K Kobayashi  2   7 , Diane Bender  2 , Yujie Fu  2   7 , Anton Alexandrov  3 , Elaine R Mardis  5   6 , Maxim N Artyomov  3 , Robert D Schreiber  2   3   4 , Gavin P Dunn  8   7   4
Affiliations

Endogenous Neoantigen-Specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach

Tanner M Johanns et al. Cancer Immunol Res. 2016 Dec.

Abstract

The "cancer immunogenomics" paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific "neoantigens" in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 nonsynonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2Kb and H-2Db candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFNγ ELISPOT, we confirmed H-2Db-restricted Imp3D81N (GL261) and Odc1Q129L (SMA-560) along with H-2Kb-restricted E2f8K272R (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3D81N and Odc1Q129L were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell-activating immunotherapeutic approaches in preclinical models of glioblastoma. Cancer Immunol Res; 4(12); 1007-15. ©2016 AACR.

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Conflict of interest statement

The current Editor-in-Chief of Cancer Immunology Research (Robert D. Schreiber) is an author on this article. Otherwise, no potential conflicts of interest exist.

Figures

Fig. 1
Fig. 1
Mutational burden of GL261 and SMA-560. Schematic of workflow for GL261 and SMA-560 neoantigen discovery. Numbers to right of bar graph indicate number of mutations identified following application of indicated filter on left side of bar graph.
Fig. 2
Fig. 2
Neoantigen landscape of GL261 and SMA-560. Manhattan plot of mean binding affinity (1/ic50) of putative candidate GL261-derived neoantigens for H-2Db (a) and H-2Kb (b). Manhattan plot of mean binding affinity (1/ic50) of putative candidate SMA-560-derived neoantigens for H-2Db (c) and H-2Kb (d). Labeled are the six highest predicted binding affinity candidate neoantigens. Numbers in parentheses represent calculated ic50 (nM) for each candidate neoantigen.
Fig. 3
Fig. 3
Identification of neoantigen-reactive TIL in GL261 and SMA-560. a) Representative images from IFNγ ELISPOT of GL261 TIL stimulated with H-2Db–restricted candidate neoantigens. TIL were isolated from established subcutaneously implanted GL261 at day 21 and incubated for 4 days in IL-2 (50 U/mL). Cultured TIL (25,000 TIL/well) were then incubated overnight with the indicated peptide (10 µM) and assessed for IFNγ production the following day by ELISPOT. b) Bar graph quantifying number of IFNγ spots per well. c) Representative wells from IFNγ ELISPOT of SMA-560 TIL stimulated with H-2Db–restricted candidate neoantigens. d) Bar graph quantifying number of IFNγ spots per well. e) Representative wells from IFNγ ELISPOT of SMA-560 TIL stimulated with H-2Kb–restricted candidate neoantigens. f) Bar graph quantifying number of IFNγ spots per well. Presented data depict pooled results from at least 3 experiments with 2–3 mice per experiment.
Fig. 4
Fig. 4
Detection of neoantigen-specific CD8 T cells by tetramer within TIL and draining lymph node. TIL isolated from subcutaneously implanted (top left graph) or intracranially implanted (top right graph) GL261 (top row) and SMA-560 (bottom row) tumors were cultured for 4 days in IL-2 (50 U/mL) prior to tetramer staining. Indicated peptides were loaded into H-2Db–restricted tetramers and dual labeled with PE and APC fluorochromes. Tetramer positive cells are identified as double positive populations. Draining cervical lymph nodes were surgically removed at time of sacrifice and stained directly with indicated tetramers (bottom left graph). Representative FACS plots of at least three experiments containing pooled TIL from 2–5 mice with similar results are shown. Number in each FACS plot represents average percentage of tetramer positive cells within CD8+ population of cells.
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