Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells

Abstract

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.

Identification of naturally processed peptides bound to the class I MHC molecule H‐2Kd of normal and TAP‐deficient cells

“…Leucines or isoleucines were found in majority at P9 (>88%), while a minor fraction contained valine (*6%) (Ile/Leu: 53/60; Val: 4/60). This is in accord with the early analysis by Rammensee [5,9] and the subsequent studies of others [43][44][45][46][47][48]. P2 and P9 interact with pockets B and F in the crystal structure obtained by Mitaksov and Fremont (manuscript submitted).…”

“…To understand the nature of peptides displayed by a particular MHC allele, it is important to identify the preferred peptide-binding motifs that allow interaction with the peptide-binding groove of the MHC molecule. Two broad approaches have been employed in this pursuit: synthesis of peptide libraries that contain motifs important for binding to MHC molecules (for example see [1][2][3][4]), and direct isolation and identification of naturally processed peptides selected by MHC molecules present on the cell surface of APC (examples in [5][6][7][8][9][10]). Although the second approach is more demanding, it has several advantages, most important of which is that the identified peptides are the physiological naturally presented peptides.…”

“…Starting with the pioneering work of Rammensee and colleagues [5,9], Kd has been widely studied, and Kd-specific immune responses described in a number of experimental systems. These include CD8 T cell responses: (1) against viral and bacterial pathogens [6,[19][20][21][22]; (2) in the NOD mouse model of diabetic autoimmunity [23][24][25][26][27]; and, (3), to tumor antigens [28][29][30][31].…”

Minimal Information About an Immuno‐Peptidomics Experiment (MIAIPE)

“…The Rammensee lab reported the first application of mass spectrometry and Edman degradation for the identifications of pMHC,23 while in 1992, Hunt et al 24. published the first LC‐MS/MS approach for the identification of eluted pMHC.…”

Intrabody-mediated phenotypic knockout of major histocompatibility complex class I expression in human and monkey cell lines and in primary human keratinocytes

“…19,20 Assembly of the heavy chain, ␤2-microglobulin, and peptide is required for normal MHC class I surface expression, 21 and the bound peptide contributes to the antigenic determinants recognized by the TCR. 22,23 Because MHCI antigens serve as targets for allograft rejection, 24,25 the present study was undertaken to determine if an ER-directed anti-human MHCI single-chain intrabody that is directed against a monomorphic, conformational epitope on the MHCI heavy chain 26,27 could block transport of MHCI molecules from the ER to the cell surface of cells. We present data demonstrating that an ER-directed sFvhMHCI intrabody is able to effectively block MHCI cell surface expression on both monkey and human cell lines with different HLA-A,B,C haplotypes.…”