First Results of High Throughput Pipeline for Serotypic and TCR Clonotypic Specificities in Aplastic Anemia and Large Granular Lymphocyte Leukemia Uncovering Antigenic Peptides Relevant to Disease Etiology

Clinical and experimental evidence indicates that idiopathic aplastic anemia (AA) is mediated by the cellular immune system following, to date, an unrecognized immunogenic trigger. As a result, polyclonal to oligoclonal cytotoxic T-cell responses are recognized in AA. Similarities are observed between AA and large granular lymphocyte leukemia (LGLL), whereby AA/LGLL overlap or highly clonally skewed CTLs are found in up to 15% of cases. However, one can assume that several antigenic epitopes are targeted in AA, whereas in LGLL, a single clone is likely responsive to a specific antigenic peptide.

We hypothesized that the study of peptidomic targets of CTLs of such disease may be very instructive for the understanding of both diseases, whereby the main unresolved conundrum is the nature of the inciting events. To that end, we conducted a high throughput serology assay coupled with deep-TCR sequencing of a large cohort of AA and LGLL to uncover potential antigenic triggers important for the etiopathogenesis of both diseases.

We secured transfusion and treatment-naïve samples of 153 AA and 83 LGLL patients. Clinical and molecular features and autoimmune markers were examined and correlated with experimental results. A high throughput peptide array with 801,064 semi-randomized 12-mer peptides was used to characterize antibody responses. An in-house pipeline was developed to preprocess the background normalized fluorescence data and remove noise. Specifically, peptide sequence filters were applied, taking the maximum signal intensity observed in the controls (n=20) as the lower threshold for the corresponding peptide, thus removing potentially false positive hits. Kruskal-Wallis test removed peptides that did not show statistically significant differences across AA, LGL, and healthy subjects. Lastly, a non-negative matrix factorization was deployed to identify and reduce noise, and the number of components was selected by cross-validation. After preprocessing, 11,253 peptides remained for downstream analysis.

Utilizing the preprocessed and de-noised peptide array data we investigated commonalities between peptides across AA and LGL and identified overlapping peptides as well as unique specificities as putative candidates. When we investigated peptides unique to AA (n=2105), we identified multiple RNA polymerase III subunit (RPC1) peptides as significant hits, including IDEVAVPVHVAK and AEETLEALILKE. These target specificities were absent in the sera of LGLL patients. Interestingly, when we investigated peptides uniquely present in LGLL (n=2165), we again identified a peptide with 100% homology to RPC1, albeit with a different sequence NMAPKKWTRGFV, suggesting that the antigen by which RPC1 is engaged is different in AA and LGLL. Of note is that we could not find any association when substratifying based on differences across STAT3 mutants, neutropenic patients, or those with autoinflammatory conditions.

We then investigated the potential association of RPC1 seropositivity with T-cell responses. We selected samples with seropositivity to corresponding RPC1 peptides and extracted the top 3 expanded clones from each sample. Analysis of expanded T-cell clones in seropositive samples revealed potential epitope matches in the IEDB database. About 50% of CDR3-Vβ sequences matched epitopes from common viruses, including CMV, EBV, Influenza A, and SARS-CoV2, with NLVPMVATV as one of the epitopes recapitulating our previous findings.¹ These findings aligned with the known function of RPC1 in sensing and transcribing viral genomic regions to generate AU-rich transcripts that induce type I interferon response.^2,3 Armed with a potential mechanistic characterization, we are currently generating in-silico validations using predictive modeling between CDR3-Vβ and epitope sequences in specific HLA contexts.¹ Further, we are conducting ELISPOT assays to investigate T-cell responses for the given peptide sequence eliciting antibody responses as experimental validation. Results will be presented during the ASH meeting.

In conclusion, the premise that antibody responses, while not pathogenic, may constitute markers for TCR-mediated reactions opens an opportunity to narrow the search for antigenic peptides recognized by expanded clonotypes in AA and clonal CTLs in LGLL.

Pagliuca:Alexion: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Krishnamurthy:Vibrant Sciences: Current Employment. Krishna:Vibrant Sciences: Current Employment. Jayaraman:Vibrant Sciences: Current Employment.