Background

Major histocompatibility complex (MHC) molecules loaded with peptides are displayed on the cell surface to engage T cells via their expressed TCRs. While all nucleated cells express class I MHC molecules, professional APCs such as dendritic cells, macrophages, and B cells additionally express high level of class II MHC molecules to activate T cells (Wieczorek et al., 2017). Formation of assembled class I and II MHC molecules requires binding of the proteolytically generated peptides with specific positioning of anchor residues. Endogenously synthesized proteins or those acquired exogenously by APCs are processed in distinct subcellular compartments to generate peptides that are presented by class I MHC molecules at the cell surface (Princiotta et al., 2003). Endogenous proteins are primarily processed by proteasomal machinery, and peptides thus generated are translocated from cytosol to endoplasmic reticulum (ER) mainly by the transporter associated with antigen processing (TAP) molecules (Vyas et al., 2008). Nascent class I MHC molecules in the ER associate with calreticulin, tapasin, and ERp57 to generate a peptide loading complex (PLC) that facilitates loading of peptides. Then, the assembled complexes traverse to the cell surface. Therefore, TAP deficiency limits the entry of peptides into ER, leading to a reduced surface expression of class I MHC molecules. The heterotrimeric complex formed by heavy chain (HC) of class I MHC molecule, peptide, and β2 microglobulin (β2m) provides the central anchorage signal to engage specific TCR expressing cytotoxic T lymphocytes (CTLs). Additionally, the co-stimulatory receptor-ligand pair (B7.1/7.2 and CD28) by the engaged APCs and T cells helps to efficiently activate CTLs, which are then enormously expanded in the presence of generated cytokine milieu (De Bruijn et al., 1991; La Gruta et al., 2018). Such cells scan infected or transformed cells for cytolysis. Therefore, identifying pathogen derived peptides constitutes a critical step in understanding T cell dynamics following infection or immunization. To validate the immunogenicity of the predicted peptides, fluorescently labelled H-2K^b/peptide complexes were generated and used to detect PPRV-peptides specific CD8⁺ T cells in immunized or the PPRV-infected mice.

In this protocol, we describe assays to ascertain peptide induced class I MHC stabilization (Wieczorek et al., 2017). A combinatorial approach involves in silico, in vitro and in vivo analysis to predict immunogenic PPRV-peptides that can be displayed by class I MHC molecules of mice (H-2K^b). Finally, a throughput approach was used to enumerate PPRV-specific CD8⁺ T cells in the immunized or infected mice. For identifying such peptides, we collected the complete amino acid sequences of PPRV proteins from online NCBI and Uniprot databases to predict class I MHC restricted epitopes using online tools such as IEDB, SYFPEITHI (Figure 1). High affinity epitopes were manually selected based on their hydrophobic residues at carboxy terminus, as the latter are known to favor peptide-MHC class I interaction (Pettersen et al., 2004; Chen et al., 2015). Furthermore, the selected high affinity peptides were subjected to a blind docking analysis with H-2K^b, whereby the top ranked peptides were selected (Vita et al., 2015 and 2019; Zhou et al., 2018). To study the presentation of such peptides in context to H-2K^b, we used TAP deficient RMA/S cells that inherently express low levels of surface class I MHC molecules. RMA/s cells can synthesize heavy chains of class I MHC molecules and β2m, but fail to efficiently assemble these molecules due to the limited availability of processed peptide in the ER (Esquivel et al., 1992; De Silva et al., 1999). This leads to compromised loading of class I MHC molecules with antigenic peptides. Empty class I MHC molecules can, nonetheless, be transported to the cell surface by vesicular transport from the ER (De Bruijn et al., 1991; Esquivel et al., 1992). Empty class I MHC molecules are poorly stabilized at the cell surface and hence lower expression levels are evident. Exogenously added antigenic peptides to RMA/S cells can be loaded to class I MHC molecules by predominantly two mechanisms. First, the exogenous peptides can bind directly to the empty class I MHC molecules at the cell surface (Schumacher et al., 1990). Second, the peptides are internalized by such cells, loaded onto the peptide binding grove of heavy chain (HC) of class I MHC molecule utilizing the vacuolar pathway to generate stable heterotrimeric complexes involving β2m, and subsequently the complexes are efficiently displayed on the cell surface (Figure 2) (Esquivel et al., 1992; De Silva et al., 1999). Fine details of such processes are still to be better defined. The binding ability of anchor residues of the peptide with MHC molecules dictates the stability of such complexes (Garstka et al., 2015). We showed that stable class I MHC molecules were expressed on the cell surface when immunogenic peptides derived from PPRV were added to RMA/s cells, and such molecules were detected by anti-H-2K^b antibody using flow cytometry (Sharma et al., 2021). We also generated stable H-2K^b monomers using a photocleavable conditional ligand, (FAPG[Anp]YPAL), modified derivative from the nucleoprotein of Sendai E virus, which was described earlier byRodenko et al. (2006). Incubation of predicted PPRV-peptides in high concentration with such monomers, followed by exposure to a higher wavelength UV light (UV₃₆₅), cleaved the conditional ligand and replaced it with the added immunogenic peptides (Rodenko et al., 2006). The exchange by different peptides was performed in separate identifiable wells of a microtiter plate, to unambiguously assess their MHC stabilization.

The described protocol can be employed to identify immunogenic epitopes of diverse antigens of a pathogen, cancers, as well as autoantigens; but the availability of cells deficient in antigen-processing machinery for the concerned species, as well as the generation of recombinant proteins for HC of class I MHC, and β2m would be critical. Furthermore, the final validation has to be performed by enumerating antigen-specific CD8⁺ T cells in the host. Therefore, a combinatorial approach could yield validated information on the immunogenicity of epitopes. Another limitation of such protocols might be the inherent polymorphism in MHC molecules occuring in an outbred population. Nonetheless, essential features of T cell differentiation are better captured if the information on immunogenic epitopes of an antigen is available. Further characterization of differentiating antigen-specific CD8⁺ T cells adds value to such analyses.

Figure 2. A schematic to depict how exogenous peptides help stabilize class I MHC molecule on the surface of TAP deficient RMA/S cells. A. Exogenous peptides are internalized and loaded onto class I MHC molecules in a yet to be clearly defined subcellular compartment. Subsequently, the loaded class I MHC complexes are exported to the cell surface. The surface stabilized class I MHC molecules are then detected by flow cytometry, using an anti-class I MHC (H-2K^b) antibody. B. Alternatively, RMA/s cells can display empty class I MHC molecule on their surface, and their loading with the exogenously added peptides helps to stabilize expression.