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 Autoimmune disorders affect approximately 10% of the global population, presenting symptoms ranging from mild to life-threatening. Current treatments largely involve broad immunosuppression, which can leave patients vulnerable to infections. We introduce an immunomodulatory strategy that targets the immune response to specific antigens while preserving overall immune functionality. 

Our work centers on developing single-domain antibody fragments, known as nanobodies or VHHs, derived from alpacas. These nanobodies specifically target major histocompatibility complex class II (MHCII), allowing them to interact with all professional antigen-presenting cells (APCs). We developed a method for site-specific modification of these VHHs at their C-terminus using various chemo-enzymatic techniques, enabling the attachment of autoimmune disease-associated autoantigens and anti-inflammatory drugs, such as dexamethasone (DEX). We demonstrated that a single dose of a VHHMHCII conjugated to myelin oligodendrocyte glycoprotein (MOG) peptides and DEX (VHHMHCII-MOG-DEX) provides long-lasting protection against experimental autoimmune encephalitis (EAE), a model for multiple sclerosis (MS), and can reverse paralysis in symptomatic mice without compromising their immune defense against pathogens. This approach can be adapted for other autoimmune diseases like type I diabetes and rheumatoid arthritis by simply changing the autoantigens. This strategy is also applicable in suppressing unwanted immune response against viral gene therapy vectors. 

To explore the mechanisms behind inducing antigen-specific tolerance, we employed comprehensive flow cytometry and transcriptomic analyses, which indicated an increase in antigen-specific regulatory T cells and evidence of bystander immune suppression mechanism. Additionally, we utilized positron emission tomography (PET)-based imaging to non-invasively monitor the biodistribution and tolerogenic activity of our engineered nanobodies in real-time. Furthermore, we developed anti-idiotypic nanobodies that recognize specific T-cell receptors (TCRs), such as 2D2 TCR, which interacts with the I-Ab MOG35-55 complex. These nanobodies were used as imaging agents in immuno-PET to detect infiltrating T cells in the spinal cord of symptomatic EAE mice, helping us trace the efficacy of our antigen-specific tolerance approach. 

In conclusion, our engineered nanobodies offer a promising and versatile tool for both the specific modulation and real-time tracking of immune responses in autoimmune diseases, providing targeted treatments and future diagnostic avenues.