Mapping endosomal Toll-Like Receptor-driven autoantibody responses
Greg Barton, Professor
Molecular and Cell Biology
Applications for Fall 2026 are closed for this project.
Our immune systems have evolved to balance defense against foreign pathogens with the capacity to damage normal “self” tissues. B cell and antibody function are central to this balance: in nearly all vertebrates, each among billions of B cells co-expresses 1) a highly variable B cell antigen receptor (BCR), and 2) invariant endosomal innate receptors that sense RNA and DNA, namely Toll-Like Receptor 7 (TLR7) and Toll-Like Receptor 9 (TLR9) respectively. The BCR can mediate endocytosis of foreign or self antigens and deliver these to endosomal TLR7/9, enabling rapid antibody responses to viruses and other nucleic acid-containing particles. But because many BCRs in the healthy normal repertoire are capable of recognizing RNA or DNA, the BCR-TLR axis can also trigger systemic autoimmune disease when any component of the system is too responsive. One such case involves rare human allelic variants of UNC93B1, a gene encoding a 12-pass transmembrane protein that regulates the trafficking and signaling of all endosomal TLRs. The Barton lab and others have recently shown that humans and mice with certain missense variants of the UNC93B1 gene develop nucleic acid autoantibodies and systemic autoimmune diseases - including Systemic Lupus Erythematosus, cutaneous lupus and neuroinflammatory disorders - due to hyper-responsive TLR7 signaling.
In this project, we have taken advantage of mouse lines harboring human UNC93B1 knock-in allelic variants to systematically dissect the autoantibody response driven by hypermorphic TLR7 and TLR9 signaling. We used a T7 phage peptide display library representing the entire mouse “self” proteome to immunoprecipitate self-reactive IgG from the serum of our Unc93b1 mutant mice. As predicted, several among the top reactive peptides targeted by Unc93b1 mutant IgG derive from nucleic acid-binding proteins (tRNA synthetases, transcription factors and others), which presumably drive autoreactive B cell activation by simultaneously engaging BCR and TLR7/9 signaling. Unexpectedly though, other top peptides derive from proteins with no known nucleic acid binding, including an endosomal/secreted protease and a cytosolic guanine nucleotide exchange factor among others. We hypothesize that these top antigens elicit autoreactive IgG because they confer a B cell-intrinsic second activation signal in addition to BCR agonism, such as TLR7/9 cleavage in the case of the endosomal protease.
The URAP student will 1) clone, express and purify a panel of these protein autoantigens and test reactivity with Unc mutant mouse serum using assays including ELISA and immuno precipitation; 2) engineer autoantigen:model antigen fusion proteins that will enable BCR-mediated delivery of PhIPseq autoantigens to BCR-transgenic B cells. With additional semesters in the lab, this work might extend to 3) developing assays to validate the folding and function of purified proteins, including protease assays and nucleic acid binding assays.
Role: With training, the apprentice will become fluent with end-to-end completion of gene cloning, protein expression using mammalian suspension cells, protein purification by immobilized metal affinity chromatography, and indirect ELISA using purified recombinant antigens as bait for mouse serum IgG.
Qualifications: Required
-Biology 1A OR MCB50
-Track record of diligence and willingness to maintain detailed and accurate laboratory records
-Track record of curiosity and engagement during coursework and other activities.
-Strong communication skills with ability to 'reason out loud'
Favorable:
-Prior exposure to PCR and other basic molecular biology techniques (restriction enzyme digestion and fragment ligation, plasmid propagation in E. coli, etc)
-Prior experience with mammalian cell culture
-Desire/intention to complete a senior honors thesis
Day-to-day supervisor for this project: John Huizar , Post-Doc
Hours: 12 or more hours
Related website: https://pubmed.ncbi.nlm.nih.gov/38780621/
Related website: https://pubmed.ncbi.nlm.nih.gov/39353255/