Recognition by HLA-A2-restricted cytotoxic T lymphocytes of endogenously generated and exogenously provided synthetic peptide analogues of the influenza A virus matrix protein.

Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58-66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58-66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58-66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58-66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58-66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

Presentation of endogenous peptides to MHC class I-restricted cytotoxic T lymphocytes in transport deletion mutant T2 cells.

The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.

The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2.

Influenza virus matrix protein-derived peptides were synthesized based on the amino acid motifs for HLA-A2 bound self peptides. Among these peptides a nonamer (amino acids 58 through 66: G I L G F V F T L) was found to be 100 to 1000 times more effective than the commonly used peptide 57-68 (K G I L G F V F T L T V) in sensitizing HLA-A2+ target cells to lysis by influenza virus specific cytotoxic T lymphocytes. The sensitizing activity of the 12-mer 57-68 was not due to contamination with shorter and more active peptides. Intracellular expression of peptide 58-66 (mediated by a stable expression plasmid with DNA coding for this peptide) also sensitized HLA-A2+ cells to lysis. Peptide 58-66 stimulated human PBMC to generate CTL that recognized peptides 58-66 and 57-68 in association with HLA-A2.

Selective release of a glycosylphosphatidylinositol-anchored antigen from the surface of Schistosoma mansoni.

We have recently demonstrated that a 200-kDa antigen that serves as a target of antibodies acting in synergy with praziquantel is linked to the surface membrane of Schistosoma mansoni by a glycosylphosphatidylinositol (GPI) anchor. In the present study we have examined the potential role of this GPI anchor in the therapeutic action of praziquantel by monitoring the release of surface antigens from living adult schistosomes cultured in the presence or absence of praziquantel and exogenous phospholipases. Phosphatidylinositol-specific phospholipase C (PIPLC) selectively released the 200-kDa antigen from the surface of adult schistosomes, as determined by immunoprecipitation experiments; none of the other GPI-anchored proteins, including alkaline phosphatase and a 22-kDa protein, were released by this enzyme. Anti-cross-reacting determinant antiserum (anti-CRD), which recognizes an epitope on GPI-anchored proteins only after the anchor has been removed by PIPLC, specifically precipitated the 200-kDa antigen, confirming the cleavage of its anchor. When the worms were exposed to both praziquantel and PIPLC, the amount of 200-kDa cleaved from the worms was increased five-fold. The selective release of this antigen was also detected by indirect immunofluorescent labeling of praziquantel-exposed adult worms cultured in the presence of phospholipases. Taken together these observations suggest that modulation of the phospholipase-mediated release of GPI-anchored antigens by praziquantel may contribute to the therapeutic action of the drug.

Identification and characterization of glycosylphosphatidylinositol-linked Schistosoma mansoni adult worm immunogens.

Metabolic radiolabeling of adult worms of Schistosoma mansoni with [3H]myristic acid has revealed that the fatty acid is incorporated into more than 15 proteins. We have shown that two of these proteins, a 200-kDa glycoprotein known to be exposed on the surface of the adult worm following praziquantel treatment and a 22-kDa glycoprotein that shows an enhanced immune reactivity with sera of vaccinated mice, are anchored to the adult worm membrane via a glycosylphosphatidylinositol (GPI) linkage. Both antigens partitioned preferentially into the detergent phase of Triton X-114 and were susceptible, following immunoaffinity purification, to hydrolysis by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis and phospholipase C from Bacillus cereus. Diacylglycerol (DAG) was released following hydrolysis by bacterial PIPLC; however, Trypanosoma brucei GPIPLC failed to release the diacylglycerol from either protein. Treatment with nitrous acid generated phosphatidylinositol (PI) from both proteins, and phospholipase D from rat serum cleaved phosphatidic acid from the 200-kDa protein. Although the functional significance of these GPI-anchored proteins is unknown, their release from the surface of the schistosome may contribute to immune evasion.