1. Major histocompatibility complex class I presentation of ovalbumin peptide 257-264 from exogenous sources: protein context influences the degree of TAP-independent presentation
M J Wick, J D Pfeifer Eur J Immunol . 1996 Nov;26(11):2790-9. doi: 10.1002/eji.1830261135.
Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules. To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1-/- mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used. TAP1-/- or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257-264 epitope from ovalbumin [OVA(257-264)], which binds the mouse class I molecule Kb. The source of the OVA(257-264) epitope was either the Crl-OVA(257-264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads. The data reveal that peritoneal macrophages from C57BL/6 and TAP1-/- mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257-264) for recognition by OVA(257-264)/Kb-specific T hybridoma cells, albeit with different relative processing efficiencies. The processing efficiency of TAP1-/- macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257-264) was presented 100 times less efficiently when the source of OVA(257-264) was full-length OVA. Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1-/- macrophages, which also depended upon the source of the OVA (257-264) epitope (Crl-OVA versus full-length OVA). These data suggest that TAP1-/- and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257-264) epitope influences the extent of TAP-independent processing for MHC class I presentation.
2. Antigen-Specific In Vivo Killing Assay
Nada Chaoul, Gilles Dadaglio Methods Mol Biol . 2021;2325:55-64. doi: 10.1007/978-1-0716-1507-2_4.
The in vivo killing assay allows the quantification of the antigen-specific killing capacity of Cytotoxic CD8+T Lymphocytes (CTLs) in mice. CTLs are indeed known for the lysis of cells expressing foreign or modified antigen peptides on their MHC class I molecules. Here we describe the detailed protocol used for the in vivo specific lysis of cells expressing the H-2 Kbimmunodominant CD8+T-cell epitope of the OVA protein: an 8 amino acid peptide corresponding to the 257-264 region of OVA (SIINFEKL).
3. Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors
Ichiro Katayama, Hiroshi Ishikawa, Shusaku Mizukami, Heiichiro Udono, Katsuyuki Yui, Chiaki Kajiwara Cancer Sci . 2008 May;99(5):1008-15. doi: 10.1111/j.1349-7006.2008.00788.x.
Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.
