Positional cloning of polymorphic loci that control autoreactive T cells

Autoimmune diseases are expensive diseases, both financially and socially, because they are in most cases life long and require a long-lasting treatment. They can also be life threatening, as they are among top 10 causes of death in women and therefore, they represent a serious clinical problem and require careful medical management. There is an autoimmune disease for every system or organ and the one discussed mainly in this thesis is rheumatoid arthritis (RA).

RA is the most common autoimmune disease and primarily affects the joints. It is more prevalent in women than man (3:1) and known to have a strong genetic component for its predisposition. Animal models offer an attractive strategy to study the underlying arthritis-associated genes under a controlled environment. Forward genetics is an approach to dissect genes that regulate a desired trait, from phenotype to genotype. However, this method often fails to pinpoint a single gene but identifies sized genetic fragments that harbor several point mutations inside or outside the genes. These single nucleotide polymorphisms or SNPs in short could have a dramatic effect on the transcription or translation of the gene. Once the arthritis-associated SNP is identified and the gene of interest is positionally cloned, the search for a mechanism of action commences.

All the studies included in this thesis have forward genetics as a common denominator except the last manuscript. In Study I, we positionally cloned the arthritis regulating gene in congenic rats that derive from a cross between an arthritis susceptible (DA) and resistant (E3) inbred strains. We found that a SNP in the second exon of clec4b caused the introduction of a stop codon and lead to the abrogation of the coded protein Dcar. Arthritis susceptibility increased drastically because of this loss of function mutation and influenced profoundly T cells which are the major driving force of induced arthritis models in rats. In Study II, a natural polymorphism in the promoter of Vdr was cloned in congenic mice and was found to be overexpressed selectively in T cells without any calcemic abnormalities. Consequently, antigen-specific T cell responses were enhanced, and autoimmunity was worsened. In Study III, we set out to investigate the interaction between the two most significant arthritis QTL in rats that were previously positionally cloned, Ncf1 and Clec4b. We found that Dcar and Ncf1 regulate in concert arthritis severity and their expression on neutrophils influenced their capacity to produce reactive oxygen species. In Study IV, we ventured to answer one of immunology’s longstanding question; are antigen-specific B cells positively selected? We show that indeed collagen type-II (Col2) specific B cells are positively selected and could be detected in transgenic mice, wild type mice and inbred rats. Moreover, we cloned the B cell receptor from human blood donor, expressed the antibody and validated its specificity to Col2. Moreover, we demonstrate that these Col2-specific B cells inhibit arthritis by inducing Col2-specific regulatory T cells.

The data illustrated in this thesis confirm in part, the importance of animal models in deciphering disease regulating genes and highlight the implication of antigen specific B cells in a prominent autoimmune disease.

List of scientific papers

I. Backdahl, L.*, Aoun, M.*, Norin, U., Holmdahl, R. Identification of Clec4b as a novel regulator of bystander activation of auto-reactive T cells and autoimmune disease. PLoS Genet. 16, 1008788-1008808. (2020). *These authors contributed equally.
https://doi.org/10.1371/journal.pgen.1008788

II. Fernandez Lahore, G., Raposo, B., Lagerquist, M., Ohlsson, C., Sabatier, P., Xu, B., Aoun, M., James, J., Cai, X., Zubarev, R. A., Nandakumar, K. S., Holmdahl, R. Vitamin D3 receptor polymorphisms regulate T cells and T cell-dependent inflammatory diseases. Proc Natl Acad Sci. 117, 24986-24997. (2020).
https://doi.org/10.1073/pnas.2001966117

III. Aoun, M., Cai, X., Xu, Bingze., Bonner, M., He, Y., Frenandez Lahore, G., Bäckdahl, L., Holmdahl, R. Glycan activation of Clec4b induces reactive oxygen species and protects against neutrophilia and arthritis. [Manuscript]

IV. Aoun, M.*, Saxena, A.*, Coelho, A., Krämer, A., Sabatier, P., Beusch, C., Lönnblom, E., Nguyen, N., Zhang, J., He, Y., Li, T., He, C., Fernandez Lahore, G., Xu, B., Viljanen, J., Rorbach, J., Kihlberg, J., Kastbom, A., Sjöwall, C., Gjertsson, I., Zubarev, R., Burkhardt, H., Holmdahl, R. Bone marrow-selected antigen specific regulatory B cells. *These authors contributed equally. [Manuscript]