Jane A. McCutcheon

HLA-B∗0702 antibody epitopes are affected indirectly by distant antigen residues

We examine the effect of mutations in the HLA-B*0702 alpha 1 domain on the binding of several well-characterized monoclonal antibodies. BB7.1 recognizes the alpha-helix, with a special requirement for residue 67. Combined with an established requirement for the alpha 2 alpha-helix, BB7.1 appears to span the B*0702 peptide-binding groove. Alternatively, BB7.1 epitope conformation may be altered by distant B*0702 sites. ME1 and B27M1 recognize connecting loop residues 41 and 43 and alpha 1 alpha-helical residues 67-71. Instead of contacting residue 67-71 side chains directly, however, ME1 appears to recognizes a B*0702 configuration that depends upon the proper interaction of these and other HLA residues. In addition to solvent-accessible residues 41 and 43, the B27M1 epitope depends on solvent-inaccessible residue 32 at the bottom of the peptide-binding groove. MB40.2, known to require residues 169-182 near the alpha 2-alpha 3 junction, also requires the proper combination of distant residues in the alpha 1 beta-strand and alpha-helix. The effect of mutations near the peptide-binding groove suggests that bound peptides may directly or indirectly affect HLA epitopes. These results illustrate that HLA epitope conformation is very sensitive to changes at distant HLA sites and forecast that epitope models based on sequential amino acid residues will often fail to predict HLA epitopes.

Mutagenesis around residue 176 on HLA-B∗0702 characterizes multiple distinct epitopes for anti-HLA antibodies

Preformed antibodies against HLA-A,B,C molecules cause hyperacute rejection of transplanted allogeneic tissues. To understand better the molecular basis of hyperacute rejection, narrowly reactive anti-HLA-B*0702 monoclonal antibodies have been studied. Previous epitope mapping studies of these monoclonal antibodies by mutating B*0702 have conflicted with antibody-blocking studies. To resolve these discrepancies, we mutated B*0702 residues around the antigenically important residue 176, and measured anti-B*0702 antibody binding. Antibody MB40.2 binding is abrogated by mutations at residues 169, 180, and 182, close to residue 176 in the primary structure. However, MB40.2 binding is not affected by 12 other B*0702 mutations close to residue 176 in the tertiary structure. This suggests that MB40.2 may recognize a sequential B*0702 epitope including residues between positions 169-182. Antibody BB7.1 binding requires B*0702 alpha 2-domain residues 166 and 169. Competition for B*0702 residue 169 explains why MB40.2 and BB7.1 crossblock. Because BB7.1 binding also requires B*0702 alpha 1-domain residues, BB7.1 may contact both alpha-helices, straddling the B*0702 peptide-binding groove. Previous results showed that both MB40.2 and MB40.3 binding require B*0702 residues 176/178. However, MB40.3 binding is not affected by any of 15 other mutations near residue 176. This suggests that MB40.3 does not contact residues 176/178; rather, residues 176/178 appear to affect MB40.3 binding by subtly influencing B*0702 conformation. Thus, monoclonal antibodies influenced by a defined B*0702 region around residue 176 appear to recognize three different types of epitopes. This suggests that human alloantibodies also recognize diverse types of HLA epitopes.

Posttranscriptional Regulation of Neuronal Nitric Oxide Synthase Expression by IFN-γ

In this report, the mechanism through which interferon-gamma (IFN-gamma) regulates the expression of nitric oxide synthase (NOS-1) in neurons was examined. We have shown previously that IFN-gamma treatment of cells results in a two log inhibition of vesicular stomatitis virus (VSV) production. This inhibition of VSV replication is dependent both in vitro and in vivo on nitric oxide (NO) production by NOS-1. Furthermore, this effect is associated with the increased expression and activity of NOS-1 following IFN-gamma treatment. In vitro, exposure to IFN-gamma prior to infection with VSV is a prerequisite to establish an effective antiviral state, indicating the necessity for a priming event. Neuroblastoma cells (NB41A3) were treated with IFN-gamma or medium and examined for changes in NOS-1 protein and mRNA expression. NOS-1 protein expression was found to be increased after IFN-gamma treatment, and this was associated with increases in both neosynthesis and NOS-1 protein stability. NOS-1 transcription and mRNA levels were unaffected by IFN-gamma treatment. These data demonstrate that IFN-gamma regulates NOS-1 expression through posttranscriptional and posttranslational mechanisms.

Tobacco Reduces Membrane HLA Class I That Is Restored by Transfection with Transporter Associated with Antigen Processing 1 cDNA

HLA class I molecules are recognized by CTL that eliminate virally infected and malignantly transformed cells presenting foreign peptide—a process termed immunosurveillance. Many tumors have reduced levels of membrane HLA class I. Tumor cells with mutations that reduce HLA class I avoid immunosurveillance and continue to proliferate. As tobacco use can induce tumors, we examined the effect of tobacco extracts on membrane HLA class I. These studies show that culture of cells in media containing tobacco extracts reduces membrane HLA class I, but not other proteins, on primary keratinocytes and other cell types. Culture in tobacco extracts, but not extracts of other substances, reduces TAP1 protein, but does not reduce expression of HLA class I H chain, L chain, or the housekeeping protein β-actin. The reduction of TAP1 protein occurs within 4 h and is dose-dependent. Culture in tobacco extracts reduces TAP1 protein abundance, but not steady-state mRNA abundance. Tobacco-treated cells show defects in HLA class I biosynthesis similar to those found in TAP1-deficient cell lines. Transfection with TAP1 cDNA restores TAP1 protein abundance, HLA class I biosynthesis, and cell surface expression. Combined, these data show that culture in tobacco extracts reduces TAP1 protein abundance and membrane HLA class I levels. Reduction in membrane HLA class I could permit subsequent malignant transformation of cells to be undetected by the immune system.

Suicide gene therapy for premalignant disease: A new strategy for the treatment of intraepithelial neoplasia

The potential of gene therapy to treat premalignant disease or recurrent cancer has not been investigated. The goal of the present investigation was to explore the efficacy of pro-drug-mediated, suicide gene therapy as a strategy to treat incipient neoplasia in stratified squamous epithelium. To test this strategy, a tissue model of premalignancy was generated by mixing normal human keratinocytes (NHK) that express the bacterial cytosine deaminase gene (CD) with premalignant keratinocytes which have been genetically marked with the bacterial gene for β-galactosidase (II-4-β-gal) in skin-like organotypic cultures. Preliminary studies in monolayer cultures demonstrated that CD-transduced NHK (NHK/CD) efficiently expressed the transgene and deaminated the pro-drug 5-fluorocytosine (5FC) to the toxic product 5-fluorouracil (5FU). The capacity of NHK/CD to kill II-4-β-gal cells through bystander effect was assayed in both submerged culture and in the organotypic model of premalignancy. In submerged cultures, it was found that CD-mediated killing of II-4-β-gal cells did not require cell–cell contact and that the LD50 of 5FC for efficient bystander killing of II-4-β-gal was 0.5 mM. When this concentration of pro-drug was used in organotypic cultures, a significant number of dysplastic II-4-β-gal cells were eliminated from the tissue. Bystander killing of II-4-β-gal cells was related to the number of NHK/CD present. These findings demonstrated that potentially malignant keratinocytes could be eliminated from a dysplastic tissue through activation of pro-drug and killing of adjacent cells through the bystander effect. By establishing an in vitro model to eliminate premalignant cells using suicide gene therapy, these studies provide a new approach for the treatment of incipient cancer as it develops, thereby preventing invasive disease.