Redistribution of critical major histocompatibility complex and T cell receptor-binding functions of residues in an antigenic sequence after biterminal substitution

Residues critical for establishing a trimolecular interaction with a major histocompatibility complex (MHC)-encoded receptor and a T cell antigen receptor (TcR) were determined for an antigenic nonapeptide. The N-terminal residue proved to be involved in binding of the peptide to both receptors and the C-terminal residue was essential for MHC binding. While substitution of either of these critical terminal residues by alanine resulted in an almost complete loss of peptide antigenicity, simultaneous substitution of both created a new functional ligand for the same MHC molecule and the same TcR. Notably, in the biterminally substituted peptide, the core residues took on new roles in the trimolecular interaction in that a residue critical in the authentic nonapeptide for TcR binding became critical for MHC binding and former spacer residues became essential to various degrees for the interaction with either receptor or both. Thus, apparently, the loss of the terminal residues' contribution was at least partially compensated by a redistribution of the roles among the remaining residues. These results reflect a cooperative contribution of all residues of an antigenic peptide to its binding to both receptors and thus challenge a static definition of agretope and epitope as MHC and TcR binding sites

Frequency analysis of cytolytic T lymphocyte precursors (CTL-P) generated in vivo during lethal rabies infection of mice. II. Rabies virus genus specificity of CTL-P

Cytolytic T lymphocyte precursors (CTL-P) were sensitized in vivo by intraplantar infection of C57BL/6 mice with a lethal dose of rabies virus, strain ERA (ERA). As a result of sensitization CTL-P matured to interleukin-receptive CTL-P (IL-CTL-P) that could be expanded in vitro to Thy-1+, Lyt-2+ CTL clones in the presence of IL without subjection to antigen-driven selection. After infection with ERA, IL-CTL-P-derived CTL lysed fibroblasts infected with rabies virus but not those infected with another rhabdovirus, the vesicular stomatitis virus. These CTL, however, did not discriminate between fibroblasts infected with the serologically closely related laboratory strains of classic rabies virus, ERA and HEP-Flury, and the serologically distinct rabies-related African isolate Mokola. This finding implies that in vivo sensitized IL-CTL-P recognize common genus-specific determinants expressed on cells infected with members of the lyssavirus genus

Presentation of CMV immediate-early antigen to cytolytic T lymphocytes is selectively prevented by viral genes expressed in the early phase

The regulation of antigen processing and presentation to MHC class I-restricted cytolytic T lymphocytes was studied in cells infected with murine cytomegalovirus. Recognition by cytolytic T lymphocytes of the phosphoprotein pp89, the immunodominant viral antigen expressed in the immediate-early phase of infection, was selectively prevented during the subsequent expression of viral early genes. The surface expression of MHC class I glycoproteins and their capacity to present externally added pp89-derived antigenic peptides were not affected. Because recognition of several other antigens occurred during the early phase, a general failure in processing and presentation was excluded. Since neither rate of synthesis, amount, stability, nor nuclear transport of pp89 was modified, the failure in recognition indicates a selective interference with pp89 antigen processing and presentation

In vivo application of recombinant interleukin 2 in the immunotherapy of established cytomegalovirus infection

We have shown in a murine model system for cytomegalovirus (CMV) disease in the immunocompromised host that in vivo application of recombinant human IL-2 (rhIL-2) can enhance the antiviral effect of a limited number of CD8+T lymphocytes, not only in prophylaxis, but also in therapy, when virus has already colonized host tissues. The observed net effect of IL-2 was consistent with the assumption of daily effector population doublings. The prospects for IL-2-supported immunotherapy of established CMV infection depend upon the tissues involved in disease. It appears that the prospects for controlling established CMV adrenalitis are less promising than for a therapy of interstitial CMV pneumonia

The cytolytic T lymphocyte response to the murine cytomegalovirus

Limiting dilution (LD) analysis with two modifications, the expansion and the restimulation LD assay, led to the detection and quantification of two distinct in vivo maturation stages within the lineage of virus- specific self-restricted CTL after infection of mice with the murine cytomegalovirus (MCMV). A low frequency set, representing an average of 15% of the specifically activated CTL-P in a draining lymph node, generated virus-specific lytic activity in the absence of antigen, solely under expansion conditions provided by growth and differentiation interleukins. These cells were considered to be active and were denoted antigen-independent or interleukin-receptive CTL-P (IL- CTL-P). A high frequency set required additional antigen in vitro to generate functionally active clones, and therefore the cells were termed antigen-dependent. Both sets are present in vivo simultaneously at the peak of the acute immune response and represent antigen- activated cells because their existence strictly depends on a preceding priming event. IL-CTL-P disappear quickly after acute infection and are absent during the memory state. It is proposed that the isolation of IL- CTL-P could serve to detect viral antigen expression during persistent and/or recurrent herpes virus infections

Host immune response to cytomegalovirus

To confirm that immediate-early (IE) genes of murine cytomegalovirus (MCMV) give rise to antigens recognized by specific cytolytic T lymphocytes (CTL), a 10.8-kilobase fragment of MCMV DNA which is abundantly transcribed at IE times was transfected into L cells expressing the Ld class I major histocompatibility glycoprotein. The viral genome fragment contains sequences of the three IE transcription units of MCMV: ie1, ie2, and ie3. In the transfected cell lines, only the predominant 2.75-kilobase transcript of ie1 and its translation product pp89 could be detected. The transfectants were analyzed for membrane expression of an IE antigen by employing clone IE1, an IE-specific CTL clone, as the probe. Only cells that expressed both the MCMV IE gene(s) and the Ld gene were recognized by the CTL clone

Studies on the Morphogenesis of Murine Cytomegalovirus

Two modes of assembly of murine cytomegalovirus (MCMV) were observed in cultured mouse embryo fïbroblasts, generating two morphologically different types of viral particles: monocapsid virions and multicapsid virions. The assembly of nucleocapsids appeared to be the same for both types of morphogenesis. Three successive stages of intranuclear capsid formation could be distinguished: capsids with electron-lucent cores, coreless capsids, and capsids with dense cores. Some of the capsids were enveloped at the inner nuclear membrane to form monocapsid virions, which were first detectable in the perinuclear cisterna. Other capsids left the nucleus via nuclear pores and usually entered cytoplasmic capsid aggregates that received an envelope by budding into extended cytoplasmic vacuoles, thereby forming multicapsid virions. Since the formation of multicapsid virions is not restricted to cell culture conditions and also occurs in vivo in immunosuppressed mice, multicapsid virions may play a role in the pathogenesis of cytomegalovirus infection

Rescue of myeloid lineage-committed preprogenitor cells from cytomegalovirus-infected bone marrow stroma

The effect of murine cytomegalovirus on myelopoiesis was studied in long-term bone marrow culture to find an in vitro correlate for the lethal virus interference with bone marrow reconstitution (W. Mutter, M. J. Reddehase, F. W. Busch, H.-J. Bühring, and U. H. Koszinowski, J. Exp. Med. 167:1645-1658, 1988). The in vitro generation of granulocyte-monocyte progenitors (CFU-GM) discontinued after infection of the stromal cell layer, whereas the proliferation and differentiation of CFU-GM to granulocyte-monocyte colonies remained unaffected. A protocol was established to probe the functional integrity of earlier hematopoietic cells. Pre-CFU-GM (the progenitors of the CFU-GM) could be recovered from an infected bone marrow donor culture by transfer onto an inductive recipient stromal cell layer. Thus, at least in vitro, infection of bone marrow stroma appears to be the only cause of the defect in myelopoiesis

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein

Computer simulation of the conformations of short antigenic peptides (&lo residues) either free or bound to their receptor, the major histocompatibility complex (MHC)- encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energyminimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall a-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i+3 hydrogen bonds at their Nterminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H- 2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energyminimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template- derived optimal parameters. The bound peptide retains mainly its a-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts a-helical conformation for the bound peptid