Impact of Temperature on the Antigen Presentation pathway in VHSV-infected Rainbow Trout (𝘖𝘯𝘤𝘰𝘳𝘩𝘺𝘯𝘤𝘩𝘶𝘴 𝘮𝘺𝘬𝘪𝘴𝘴)

Abstract

Sudden water temperature shifts are increasing in frequency and altering aquatic ecosystems, where small water bodies are especially vulnerable. These rapid temperature drops can severely impact fish immune system, and can also influence host-pathogen interactions, particularly during viral infections. Rainbow trout (Oncorhynchus mykiss) is economically important in aquaculture and is highly susceptible to disease outbreaks such as viral hemorrhagic septicemia virus (VHSV). A key process during antiviral immunity is the antigen presentation by the major histocompatibility complex class I receptor (MHC-I) and its chaperone β2- microglobulin (β2m). These proteins form a trimeric complex with viral peptides that can be presented to and recognized by CD8+ T cytotoxic cells, which activate adaptive immunity. Although transcriptional responses of antigen presentation molecules to low temperatures during VHSV infection have been reported, little is known about the folding and stability of MHC-I complexes, the antigen processing and presentation pathways, and T cell activation under thermal stress. This thesis investigates how suboptimal temperatures modulate the antigen presentation pathway in rainbow trout infected with VHSV, integrating bioinformatic analyses, in vitro refolding of the MHC-I complexes, in vitro cellular responses and T cell activation using a novel antigen presentation assay. Initially, the thermostability of the rainbow trout MHC-I UBA molecule complexed with β2m and VHSV-derived peptides was evaluated. Peptide candidates derived from VHSV nucleoprotein (Protein N), and glycoprotein (Protein G) were selected using a combination of bioinformatic tools and then, the predicted structure of the MHC-I/β2m/VHSV-peptide complex vi was analyzed using AlphaFold2. Temperature influenced the structural behavior of the MHC-I complexes according to a computational simulation program revealing potential molecular mechanisms of thermal stress on the antigen presentation pathway. To validate these in silico analysis, MHC-I and β2m recombinant proteins were successfully purified using E. coli expression systems, and in vitro refolding with the selected VHSV peptides was optimized. These results provided experimental evidence of rainbow trout MHC-I complexes refolding with VHSV peptides for the first time. Differential scanning fluorimetry confirmed that the stability of these complexes was sensitive to temperature and dependent on both peptide binding and biochemical characteristics. Continuing with the molecular characterization of this pathway, the functional effects of temperature on antigen processing, trafficking and presentation was assessed using a controlled in vitro system with VHSV-infected rainbow trout cell lines; RTGill-W1, RTS11, and RTGut-GC. These cells were infected and exposed to suboptimal temperatures, 4°C and 14°C, for nine days and, 20°C-exposed cells were used as control group . Flow cytometry analysis showed that the cell surface expression of MHC-I/β2m was reduced during infection at 14°C, compared to other temperatures. In addition, these results correlated with the release of β2m to the extracellular space. On the contrary, proteasomal activity and the secretion of the antiviral cytokine IFN-I were not impaired at suboptimal temperatures, highlighting the specificity of temperature-sensitive antigen presentation disruption. Finally, an antigen presentation assay using dorsal fin cells as antigen presenting cells was developed and validated to analyze the impact of temperature in antigen recognition and T cell activation. To perform this, primary fin cell cultures were exposed to heat-killed VHSV and co- vii cultured with VHSV-sensitized splenocytes. Cytotoxicity was evaluated with the release of lactate dehydrogenase (LDH), resulting in an antigen-specific and MHC-I-dependent cytotoxic response from the activated splenocytes. In addition, this cytotoxic response was negatively affected by lower temperatures, once again, supporting the idea that suboptimal temperatures impair the antigen presentation pathways at different levels during a viral infection in rainbow trout. Altogether, this thesis offers a comprehensive and integrated approach into understanding how temperature regulates antigen presentation during viral infections in rainbow trout. It highlights not only the thermal sensitivity of antigen presentation and viral antigen processing but also contributes to the immense knowledge gap about fish immunity. Importantly, these findings provide in silico and in vitro methodologies for studying antigen presentation and T cell cytotoxicity, with direct application for vaccine design and maintaining fish health, especially during thermal stress.