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. 2019 Nov 3;9(1):1684713.
doi: 10.1080/2162402X.2019.1684713. eCollection 2020.

Identification of a CD8+ T-cell response to a predicted neoantigen in malignant mesothelioma

Sophie Sneddon  1 , Craig M Rive  1 , Shaokang Ma  1 , Ian M Dick  1 , Richard J N Allcock  2   3 , Scott D Brown  4 , Robert A Holt  4 , Mark Watson  5 , Shay Leary  5 , Y C Gary Lee  1   6 , Bruce W S Robinson  1   6 , Jenette Creaney  1
Affiliations

Identification of a CD8+ T-cell response to a predicted neoantigen in malignant mesothelioma

Sophie Sneddon et al. Oncoimmunology. .

Abstract

Neoantigens present unique and specific targets for personalized cancer immunotherapy strategies. Given the low mutational burden yet immunotherapy responsiveness of malignant mesothelioma (MM) when compared to other carcinogen-induced malignancies, identifying candidate neoantigens and T cells that recognize them has been a challenge. We used pleural effusions to gain access to MM tumor cells as well as immune cells in order to characterize the tumor-immune interface in MM. We characterized the landscape of potential neoantigens from SNVs identified in 27 MM patients and performed whole transcriptome sequencing of cell populations from 18 patient-matched pleural effusions. IFNγ ELISpot was performed to detect a CD8+ T cell responses to predicted neoantigens in one patient. We detected a median of 68 (range 7-258) predicted neoantigens across the samples. Wild-type non-binding to mutant binding predicted neoantigens increased risk of death in a model adjusting for age, sex, smoking status, histology and treatment (HR: 33.22, CI: 2.55-433.02, p = .007). Gene expression analysis indicated a dynamic immune environment within the pleural effusions. TCR clonotypes increased with predicted neoantigen burden. A strong activated CD8+ T-cell response was identified for a predicted neoantigen produced by a spontaneous mutation in the ROBO3 gene. Despite the challenges associated with the identification of bonafide neoantigens, there is growing evidence that these molecular changes can provide an actionable target for personalized therapeutics in difficult to treat cancers. Our findings support the existence of candidate neoantigens in MM despite the low mutation burden of the tumor, and may present improved treatment opportunities for patients.

Keywords: Malignant mesothelioma; neoantigen; pleural effusion; tumor-immune interface.

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Figures

Figure 1.
Figure 1.
Neoantigen landscape of tumor cells from malignant mesothelioma pleural effusions. (a) Neoantigens were predicted for each patient based on their specific HLA type. Overview of the 2236 predicted neoantigens that were observed to bind to MHC class I highlighted by their binding change from the wild-type to the mutant state. (b) Predicted neoantigens identified in each sample colored by their binding change from the wild-type to the mutant state.
Figure 2.
Figure 2.
Assessment of the immune compartment of the pleural effusions from 18 patients. Top panel shows the estimation of the size of each of the cell compartments found in the effusions. Bottom panel shows the deconvolution of the immune compartment of the pleural effusions into immune cell type (a). Kaplan-Meier analysis of M0 macrophage, M1 macrophage and activated NK cell proportions within the pleural effusions. The threshold value denoting low and high groups is the median value as determined by CIBERSORT (b).
Figure 3.
Figure 3.
CDR3 detection in immune cells derived from MM pleural effusions. (a) Histogram shows a Gaussian distribution for CDR3 length. (b) Sequence logo of the 14aa CDR3 sequences show a distinct domination of the CASS and EQYF motifs at each end of the sequence.
Figure 4.
Figure 4.
Description of biomarker levels and neoantigen identification in a single human patient (ID 9567). (a) Longitudinal serum mesothelin levels. Indicated by triangles are the time points for first-line chemotherapy (red -cisplatin and pemetrexed); second line (blue – carboplatin and pemetrexed) and third line (green – vinorelbine). (b) PBMC stimulated for 1 h with mutant peptide pool, then expanded in the presence of IL2 and CD3 for 14 days. T cell reactivity was assessed by ELISpot using the indicated mutant peptide exposed HLA expressing single antigen (SAL) lines. Controls show results for indicated HLA expressing SAL without peptides. Controls for HLA expressing SAL with wild-type peptides were performed for each mutant peptide of interest (Additional File 7 and data not shown). Difference between groups determined using non-parametric tests Kruskal-Wallis with Dunn’s multiple correction or Mann-Whitney as appropriate. *p < .05. SFU, spot forming units. (c) PBMC (200,000 cells per well) stimulated for 1 h with mutant ROBO3 peptide, then expanded in the presence of IL2 and CD3 for 14 days. T-cell reactivity was assessed by ELISpot assay. Negative control was T cells and B49:01SAL without peptide. Positive control was T cells plus αCD3 antibody. (d) CD137 cell surface expression measured using flow cytometry after stimulation of T cells with ROBO3 mutant peptide Wild-type controls are included in additional file 6.
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This work was supported in part by the Housing Industry of Australia Charitable Foundation, the Insurance Commission of Western Australia and the Australian National Health and Medical Research Council (NHMRC) (APP1089404 and APP1107043). SS receives a scholarship from the Ross Divett Foundation. BR is supported by a NHMRC Practitioner fellowship (APP1108638). The funding agencies played no part in study design, data analysis, interpretation of data or manuscript preparation.