Class II antigen processing defects in two H2d mouse cell lines are caused by point mutations in the H2-DMa gene.

The molecular nature of the defect in two mouse antigen processing-defective cell lines was examined. Both mutants were derived from the A20 (BALB/c, H2d) B cell line, and both were found to have defects in the H2-DMa gene. Mutant 3A5 exhibits severely reduced amounts of H2-DMa message, and no detectable DMalpha protein. cDNA sequence revealed a C-->T transition at nucleotide 118, introducing a premature stop codon in exon 2 of the H2-DMa gene. In contrast, mutant 2A2 exhibits reduced but detectable levels of H2-DMa message and DMalpha protein only after treatment with IL-4, which induces the expression of both the H2-DMa and the H2-DMb genes in B cells. In this mutant the cDNA sequence revealed a missense mutation in exon 3 resulting in the conversion of a conserved proline residue in the Ig-like domain to serine. Stable transfection with full-length H2-DMa cDNA reconstitutes the antigen processing capacity of both mutants, as demonstrated by the ability to present native antigen to T cell clones, and by restored class II SDS stability.

Proteolysis and class I major histocompatibility complex antigen presentation.

The class I major histocompatibility complex (MHC class I) presents 8-10 residue peptides to cytotoxic T lymphocytes. Most of these antigenic peptides are generated during protein degradation in the cytoplasm and are then transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Several lines of evidence have indicated that the proteasome is the major proteolytic activity responsible for generation of antigenic peptides--probably most conclusive has been the finding that specific inhibitors of the proteasome block antigen presentation. However, other proteases (e.g. the signal peptidase) may also generate some epitopes, particularly those on certain MHC class I alleles. The proteasome is responsible for generating the precise C termini of many presented peptides, and appears to be the only activity in cells that can make this cleavage. In contrast, aminopeptidases in the cytoplasm and endoplasmic reticulum can trim the N terminus of extended peptides to their proper size. Interestingly, the cellular content of proteases involved in the production and destruction of antigenic peptides is modified by interferon-gamma (IFN-gamma) treatment of cells. IFN-gamma induces the expression of three new proteasome beta subunits that are preferentially incorporated into new proteasomes and alter their pattern of peptidase activities. These changes are likely to enhance the yield of peptides with C termini appropriate for MHC binding and have been shown to enhance the presentation of at least some antigens. IFN-gamma also upregulates leucine aminopeptidase, which should promote the removal of N-terminal flanking residues of antigenic peptides. Also, this cytokine downregulates the expression of a metallo-proteinase, thimet oligopeptidase, that actively destroys many antigenic peptides. Thus, IFN-gamma appears to increase the supply of peptides by stimulating their generation and decreasing their destruction. The specificity and content of these various proteases should determine the amount of peptides available for antigen presentation. Also, the efficiency with which a peptide is presented is determined by the protein's half life (e.g. its ubiquitination rate) and the sequences flanking antigenic peptides, which influence the rates of proteolytic cleavage and destruction.

Immune evasion strategies of the herpesviruses.

All viruses can deal with the immune response to some extent, and herpesviruses are exceptionally sophisticated in this ability. Recent work has uncovered some of the mechanisms by which herpesviruses subvert the antigen-presentation systems of their host cells.

Antigen processing and presentation by the class I major histocompatibility complex.

Major histocompatibility complex (MHC) class I molecules bind peptides derived from cellular proteins and display them for surveillance by the immune system. These peptide-binding molecules are composed of a heavy chain, containing an antigen-binding groove, which is tightly associated with a light chain (beta 2-microglobulin). The majority of presented peptides are generated by degradation of proteins in the cytoplasm, in many cases by a large multicatalytic proteolytic particle, the proteasome. Two beta-subunits of the proteasome, LMP2 and LMP7, are inducible by interferon-gamma and alter the catalytic activities of this particle, enhancing the presentation of at least some antigens. After production of the peptide in the cytosol, it is transported across the endoplasmic reticulum (ER) membrane in an ATP-dependent manner by TAP (transporter associated with antigen presentation), a member of the ATP-binding cassette family of transport proteins. There are minor pathways for generating presented peptides directly in the ER, and some evidence suggests that peptides may be further trimmed in this location. The class I heavy chain and beta 2-microglobulin are cotranslationally translocated into the endoplasmic reticulum where their assembly may be facilitated by the sequential association of the heavy chain with chaperone proteins BiP and calnexin. The class I molecule then associates with the lumenal face of TAP where it is retained, presumably awaiting a peptide. After the class I molecule binds a peptide, it is released for exocytosis to the cell surface where cytotoxic T lymphocytes examine it for peptides derived from foreign proteins.

Delivery of a foreign gene to sympathetic preganglionic neurons using recombinant herpes simplex virus.

Two recombinant herpes simplex type 1 viruses expressing beta-galactosidase (encoded by the Escherichia coli lacZ gene) inserted into the unique long 41 (encoding virus host shutoff) or unique short 5 (encoding glycoprotein J) open reading frames were generated. Purified recombinants or wild-type herpes simplex type 1 were injected into the left adrenal gland of hamsters. Three days later, virus-infected neurons were detected in spinal cord sections from all infected hamsters. Neurons were visualized with beta-galactosidase histochemistry in spinal cord sections from hamsters infected with either of the recombinants but not with the wild-type virus. Wild-type virus could only be detected with immunocytochemistry. Insertional mutagenesis into the unique long 41 or unique short 5 regions of the herpes simplex genome by lacZ did not disrupt the neurotropic properties of the virus. Both recombinant viruses labelled the central nervous system sympathoadrenal preganglionic neurons as well as brainstem neurons. Because the virus host shutoff recombinant more readily crossed synapses to reach the brainstem compared to the glycoprotein J recombinant, the presence of glycoprotein J may facilitate cell to cell transmission in vivo. Both recombinants may be useful for the study of synaptic organization of neural circuits. Our recombinant viruses were less lytic yet neurovirulent after mutation of either glycoprotein J or virus host shutoff of herpes simplex virus type 1 wild-type. These recombinant viruses express the bacterial beta-galactosidase which is readily detectable using simple histochemistry. Inoculation of the adrenal gland or kidney with these viruses led to clear labelling of spinal cord cells. These viruses may be useful markers of specific neural circuits.

A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes.

Herpes simplex virus (HSV) infection of human fibroblasts rapidly renders the cells resistant to lysis by HSV-specific CD8+ cytotoxic T lymphocytes (CTLs), which normally recognize cell surface major histocompatibility complex (MHC) class I proteins presenting viral peptides. Within 3 hr of infection with HSV, MHC class I protein complexes are retained in the endoplasmic reticulum (ER)/cis Golgi and show properties of complexes lacking antigenic peptide. The HSV immediate-early protein ICP47 is both necessary and sufficient to block transport of class I proteins and to inhibit lysis by CD8+ CTLs. The target for ICP47 is not known, but since ICP47 does not associate with membranes, it appears that ICP47 inhibits the production or stabilization of antigenic peptides or their translocation into the ER/cis Golgi. Thus, by expressing ICP47, HSV can evade detection by CD8+ T lymphocytes, perhaps explaining the predominance of CD4+ rather than CD8+ HSV-specific CTLs in vivo.

Direct contact with herpes simplex virus-infected cells results in inhibition of lymphokine-activated killer cells because of cell-to-cell spread of virus.

Natural killer (NK) and lymphokine-activated killer (LAK) cells are disarmed after contact with herpes simplex virus (HSV)-infected cells. Cells infected with HSV-1 mutants that lack glycoproteins essential for viral entry into cells (gB, gD, gK, gH, and gL) did not inhibit LAK cells; cells infected with HSV-1 mutants that lack glycoproteins not required for virus entry into cells (gE, gI, gG, and gJ) inhibited lysis. LAK cells became infected after contact with target cells infected with wild-type HSV-1 but not with a gD-HSV-1, which cannot spread from cell to cell. Because LAK cells were inhibited only by very high concentrations of cell-free preparations of HSV and because neutralizing antibodies did not prevent infection of LAK cells in contact with infected cells, infection of LAK cells is probably greatly enhanced by the apposition of the effector and target cell membranes during target recognition. Disarming of immune effector cells by infection may be a general strategy for immune evasion by HSV.

Evaluation of a subunit vaccine for bovine adenovirus type 3.

The hexon subunit of bovine adenovirus serotype 3 (BAV-3) was purified by use of anion-exchange chromatography. A vaccine composed of the purified hexon in immune-stimulating complexes was administered and induced high titer of virus-neutralizing antibody in rabbits and calves.