Identification of the nonamer peptide from influenza A matrix protein and the role of pockets of HLA‐A2 in its recognition by cytotoxic T lymphocytes

Abstract

Influenza matrix peptide 58-66 is shown to be the optimal nonamer for binding to HLA-A2 and presentation to cytotoxic T lymphocytes (CTL). If titered out to 2 x 10(-10) - 4 x 10(-10) M in CTL-mediated lysis assays and to 3 x 10(-9) M in an HLA-A2 assembly-stabilization assay in cell lysates. The peptide was shown to make probable contacts with its carboxy terminus close to residue 116 in the floor of the cleft of HLA-A2, close to the F pocket. The side chain of the amino-terminal amino acid was unimportant, but its free amino and carbonyl groups in the A pocket appeared important in optimizing peptide presentation. The B pocket probably accommodates the side chain of residue 2 (isoleucine) and was shown to be critical in peptide presentation.

Dicer-regulated microRNAs 222 and 339 promote resistance of cancer cells to cytotoxic T-lymphocytes by down-regulation of ICAM-1

“…As demonstrated in Fig. 1 (16), demonstrating specificity and activity of the CTL lines. HCT116 (ex5Ϫ/Ϫ) cells were remarkably more susceptible to cytolysis by 2 established CTL lines when compared with wild-type HCT116 cells (Fig.…”

Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

“…The mature human ␤ 2 m sequence was spliced into these constructs in place of the mouse ␤ 2 m sequence. Thus, HLA-I SCTs retained the subunit order and linker lengths [G 4 S] 3 and [G 4 S] 4 found in the original SCT design (10) (Fig. 1).…”

“…Synthetic DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA) encoding peptide antigens were ligated into the SCT vectors at restriction sites specifically designed for expeditious shuttling of peptide sequences into the SCT construct, which for this study included the melanoma G280-9V peptide (27), the human T-lymphotropic virus TAX peptide (3), and the influenza A virus M1 58 -66 peptide (4) for A2, as well as the influenza A virus NP 383-391 peptide (6) for B27. As a control, a cDNA was produced for expression of ␤ 2 m.[G 4 S] 4 .A2, with no covalently linked peptide at the N terminus. A single point mutation (R48Q) generated by site-directed mutagenesis (QuikChange II XL, Stratagene) was required to introduce the 64-3-7 epitope (28 -32) into A2.…”

“…1). Although SCT constructs have been reported for several different mouse and human MHC-I complexes, only the K b /OVA SCT (Ovalbumin 257-264 -[G 4 S] 3 -␤ 2 m-[G 4 S] 4 -K b ) has been rigorously characterized (10,18) in terms of (i) the biochemical integrity of the covalent SCT polypeptide and (ii) refractoriness of the SCT to exogenous peptide binding, which measures the binding strength of SCTencoded peptide. Specifically, in initial studies, the K b /OVA SCT was found to remain biochemically intact when expressed in cells because all three SCT components were linker-attached as detected in Western blots.…”

Large TCR Diversity of Virus-Specific CD8 T Cells Provides the Mechanistic Basis for Massive TCR Renewal after Antigen Exposure

“…Although the mechanisms have not been clearly established (reviewed in Refs. 4,5), the limited pattern of epitopes recognized among the large number of potent immunogenic epitopes encoded by viruses (immunodominant epitopes) is an important factor for the limitation of the TCR repertoire diversity (6)(7)(8)(9). Furthermore, in several situations, certain virus epitopes recruited a low number of clonotypes (reviewed in Ref.…”