Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen

Our plain-English summary of Strong spontaneous tumor neoantigen responses induced by a natural human carcinogen, by J. Creaney, S. Ma, S.A. Sneddon, et al (Oncoimmunology, 2015).

The study

• Authors: J. Creaney, S. Ma, S.A. Sneddon, M.R. Tourigny, I.M. Dick, J.S. Leon, A. Khong, S.A. Fisher, R.A. Lake, W.J. Lesterhuis, A.K. Nowak, S. Leary, M.W. Watson, B.W. Robinson
• Journal: Oncoimmunology
• Published: 2015;4(7):e1011492
• Cited by: 25 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

A key to improving cancer immunotherapy will be the identification of tumor-specific "neoantigens" that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice. A strong endogenous immune response was demonstrated to one of the candidate neoantigens, Uqcrc2; this response was detected in the draining lymph node and spleen. Antigen reactive cells were not detected in non-tumor bearing mice. The magnitude of the response to the Uqcrc2 neoantigen was similar to that of the strong influenza hemagglutinin antigen, a model tumor neoantigen. This work confirms that the approach of RNAseq plus peptide prediction and ELISPOT testing is sufficient to identify natural tumor neoantigens.

In plain English

The authors examined two mouse mesothelioma cell lines: AB1, created in earlier work by injecting crocidolite asbestos into the abdominal cavity of BALB/c mice, and AB1-HA, made from AB1 by adding an influenza protein gene (hemagglutinin, HA). Sequencing found 460 and 276 single-letter genetic changes in AB1 and AB1-HA. Of the resulting mutations, 20 protein fragments predicted to bind BALB/c immune (MHC class I) molecules were selected for testing. The authors grew tumors under the skin of female BALB/c mice and tested immune cells from nearby lymph nodes and spleen. One candidate, mutated Uqcrc2, drew a clear response. In the lymph nodes of 9 mice, the median was 14.38 spot-forming units per 10 to the 5th cells against the mutant fragment versus 0.17 against the normal one (P less than 0.01). Spleen responses were lower but still significantly higher for the mutant fragment. In draining lymph nodes of mice with AB1-HA tumors, Uqcrc2 and HA responses did not differ significantly. The responses did not eliminate the tumors. The authors concluded the model showed strong reactivity to a single mutated protein, Uqcrc2.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment (brief report): genetic sequencing of mouse mesothelioma cell lines, computer prediction of immune targets, and immune testing in tumor-bearing mice
• Who was studied: Female BALB/c mice aged 6 to 8 weeks, with tumors grown from the AB1 or AB1-HA mesothelioma cell lines; work done at the University of Western Australia. Group sizes reported: 9 mice (lymph node test), 7 mice (lymph node versus spleen), and 12 or 15 mice with AB1-HA tumors (the text and the figure legend differ). Study dates not given.
• Exposure: The mice in this study were not given asbestos. They were injected under the skin with tumor cells from the AB1 line, or from AB1-HA, which was made from AB1 by adding the influenza HA gene. AB1 had previously been generated by injecting crocidolite asbestos intraperitoneally into BALB/c mice.
• Main results:
  • Sequencing identified 460 somatic single nucleotide variants in AB1 and 276 in AB1-HA.
  • Computer prediction found 30 mutated peptides with the potential to bind MHC class I of BALB/c mice. Of these, 20 were selected for further study.
  • Of the 20 candidate mutated peptides, Sanger sequencing confirmed 18. The other 2 could not be confirmed because no clean PCR product was obtained.
  • Tumor-draining lymph nodes of AB1 tumor-bearing mice (9 mice): the response to mutant Uqcrc2 was a median of 14.38 (IQR 9.32 to 29.42) spot-forming units per 10 to the 5th cells, versus 0.17 (IQR 0 to 1.42) against the normal (wild-type) peptide, P less than 0.01. Only this 1 of the 5 peptides in pool B drew a response.
  • Spleen: the response to mutant Uqcrc2 was 4.5 (IQR 0.34 to 7.5) spot-forming units per 10 to the 5th cells, versus 0.34 (IQR 0 to 1.12) against the wild-type peptide, P less than 0.05. Total responses were lower in the spleen than in the lymph nodes.
  • Draining lymph nodes of AB1-HA tumor-bearing mice: the median response to mutant Uqcrc2 was 15.38 (IQR 4.90 to 58.50) spot-forming units per 10 to the 5th cells, compared with 5.04 (IQR 0.40 to 63.17) for the HA CL4 peptide.
  • In the draining lymph nodes of AB1-HA tumor-bearing mice, there was no significant difference between the responses to Uqcrc2 and to HA.
  • The responses to Uqcrc2 and HA did not eradicate the tumors in tumor-bearing mice.
• The authors' conclusion: The authors concluded that, in this animal model, there was a strong immune reactivity to a single mutated protein, Uqcrc2. The response was greater in the tumor-draining lymph node than in the spleen but detectable in both, and it was not out-competed by a second strong target (HA) in the same tumor.
• Limitations the authors state:
  • The authors state they do not yet know whether the Uqcrc2 mutation has any relevance in this model beyond the immune response.

Topics

Medical terms in this study, explained in our Lexicon: Immunotherapy, Mesothelioma, Reactive Cells.

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