J Bastin

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.

Abstract Two monoclonal anti-human platelet antibodies have been used in an immunofluorescent assay to study megakaryocyte maturation. The two antibodies were specific for platelets and megakaryocytes. AN 51 recognizes an antigen on platelet glycoprotein lb; the antigen detected by J 15 is on the glycoprotein IIb/IIIa complex, since it does not bind to platelets from patients with Glanzmann's thrombasthenia. AN 51 did not stain small cells of normal bone marrow, but it labeled most of the megakaryocytes and all of the platelets. In megakaryocyte colonies, megakaryocytes that had reached full maturity (at day 12 of culture) were stained specifically. AN 51 also labeled micromegakaryocytes (small mature megakaryocytes) in fetal or neonatal cultures and in leukemias, but could not be applied to identification of most leukemic promegakaryoblasts. In contrast, J 15 labeled all megakaryocytes in bone marrow as well as some rare small cells. These cells appear to represent an early cell of the megakaryocyte lineage, since their number was increased in autoimmune thrombocytopenia and they were present as early as the sixth day of culture of megakaryocyte colonies. The size of these cells progressively increased as the culture aged, probably due to endoreplication. In the bone marrow of three patients with leukemias in which promegakaryoblasts were identified ultrastructurally by the presence of platelet peroxidase, J 15 labeled blast cells. Thus, glycoprotein lb is progressively expressed during megakaryocyte maturation, but its expression is independent of megakaryocyte ploidy, while the glycoprotein IIb/IIIa complex is an early antigenic marker of megakaryocyte maturation that may be useful in isolation of megakaryocyte precursors and diagnosis of megakaryoblastic leukemia.

The conserved epitopes of influenza nucleoprotein (NP) recognized by class I MHC-restricted CTL from CBA (H-2k) and C57BL/10 (H-2b) mice have been defined in vitro with synthetic peptides 50-63 and 365-379, respectively. Two Db-restricted clones were described that recognize different epitopes on peptide 365-379. Finally, the recognition of complete NP was shown to be approximately 200-fold less efficient than peptide in the cytotoxicity assay. These phenomena are closely related to results with class II-restricted T cells and they strengthen the hypothesis that influenza proteins are degraded in the infected cell before recognition by class I-restricted CTL.

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.