T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

Prediction of tinnitus masking benefit within a case series using a spiking neural network model.

Masking has been widely used as a tinnitus therapy, with large individual differences in its effectiveness. The basis of this variation is unknown. We examined individual tinnitus and psychological responses to three masking types, energetic masking (bilateral broadband static or rain noise [BBN]), informational masking (BBN with a notch at tinnitus pitch and 3-dimensional cues) and a masker combining both effects (BBN with spatial cues). Eleven participants with chronic tinnitus were followed for 12 months, each person used each masking approach for 3 months with a 1 month washout-baseline. The Tinnitus Functional Index (TFI), Tinnitus Rating Scales, Positive and Negative Affect Scale and Depression Anxiety Stress Scales, were measured every month of treatment. Electroencephalography (EEG) and psychoacoustic assessment was undertaken at baseline and following 3 months of each masking sound. The computational modeling of EEG data was based on the framework of brain-inspired Spiking Neural Network (SNN) architecture called NeuCube, designed for this study for mapping, learning, visualizing and classifying of brain activity patterns. EEG was related to clinically significant change in the TFI using the SNN model. The SNN framework was able to predict sound therapy responders (93% accuracy) from non-responders (100% accuracy) using baseline EEG recordings. The combination of energetic and informational masking was an effective treatment sound in more individuals than the other sounds used. Although the findings are promising, they are preliminary and require confirmation in independent and larger samples.