Identification of potentially self-reactive T cell receptors and their candidate epitopes in a mouse model of rheumatoid arthritis

2021 Fiscal Year Research-status Report

• Project/Area Number: 20K16286
• Research Institution: National Institutes of Biomedical Innovation, Health and Nutrition
• Principal Investigator: LlamasCovarrubias MaraAnais 国立研究開発法人医薬基盤・健康・栄養研究所, 医薬基盤研究所 ワクチン・アジュバント研究センター, 研究員 (00867715)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: Autoimmunity / T cell receptor / Rheumatoid arthritis

Outline of Annual Research Achievements

In order to study self-reactive T cell receptor (TCR) and their likely target antigens we performed single cell repertoire and gene expression analysis on regulatory (Treg) and conventional (Tconv) cells from mice with impaired TCR signaling (ZAC) and WT mice. Based on gene expression profiles, we previously identified a group of Th17 cells in ZAC mice spleens which is absent in WT. Additionally, Th17 cells were detected in joints and lymph nodes (LN) of ZAC mice. From these, we selected the best 4 candidate TCRs and 3 corresponding controls to be experimentally tested against self-antigens. Plasmids containing the alpha and beta chains have been purchased for all the sequences. The methodology for expressing and testing candidate TCR reactivity in TCR negative T cells has been standardized. Additionally, a list of potential epitope candidates was obtained by bioinformatic analysis of peptides recovered from MHC-II complexes in BALB/c mice. Computational models of TCR and pMHC interactions are currently being produced. We expect that experimental validation with broad antigenic stimuli and computational modelling together will shed light into the hypothesis that weakened TCR signaling leads to self-reactive Tconv cells, which in turn, causes RA-like autoimmune disease.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

We have previously identified clusters of Th17 cells in arthritic mice tissues. From them, we selected 25 self-reactive candidate TCRs which were further filtered to the top 4 candidates for experimental validation. Top candidate TCRs were selected based on: clonal expansion in joints, co-occurrence in other tissues and co-occurrence in different mice. Additionally, 3 WT TCR sequences sharing gene usage with candidate TCRs were selected as control TCRs. All sequences have been purchased integrated into plasmids. The methodology for cloning, expressing and testing candidate TCRs in TCR negative T cells has been standardized. We plan to conduct antigen stimulation tests with candidate TCRs by using tissue lysates as stimuli next.
Additionally, an extensive list of peptides recovered from pMHC-II complexes in BALB/c mice was obtained from published literature (Sofron et al., 2016). This list has been filtered and 532 unique peptide consensus sequences were obtained. Pathway analysis was conducted and peptides from proteins relevant in joint tissue composition/function have been selected. Computational modeling of TCR-pMHC interactions is being performed but due to the limited availability of molecular structural templates, the accuracy of these models is also limited, and interpretation is being taken with care. We expect that the experimental and computational approaches will be complementary in testing our working hypothesis.
A paper in collaboration with Dr. Atsushi Tanaka and Prof. Shimon Sakaguchi has been submitted but it is still under revision/rebuttal process.

Strategy for Future Research Activity

Next, we will produce T cell hybridomas of candidate and control TCRs that will be screened for tissue self-reactivity with tissue lysates. In parallel, we expect to be able to rank epitope candidates according to the computational models results even though the current limitations in the computational methodology. This will help to shed light into the self-reactivity of expanded TCRs identified in ZAC arthritic mice and propose a number of candidate peptide epitopes, probably including some previously unrecognized ones.

Causes of Carryover

The budget of this year was used in the purchase of the selected TCR sequences synthesis and cloning service, and an amount of it will be carried over for next year. This budget will be used for purchasing laboratory consumables for the antigen challenge experiments (Article costs)