Paul J. Durda

Role of the Mitogen-Activated Protein Kinase Signaling Pathway in the Regulation of Human Melanocytic Antigen Expression

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1–specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy. (Mol Cancer Res 2006;4(10):779–92)

A Novel Autocrine Pathway of Tumor Escape from Immune Recognition: Melanoma Cell Lines Produce a Soluble Protein That Diminishes Expression of the Gene Encoding the Melanocyte Lineage Melan-A/MART-1 Antigen Through Down-Modulation of Its Promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as “Ag silencing,” is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

The Binding of a Glycoprotein 120 V3 Loop Peptide to HIV-1 Neutralizing Antibodies STRUCTURAL IMPLICATIONS

The structural and antigenic properties of a peptide (“CRK”) derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved “GPGR” residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II β-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK “GPGR” residues to adopt a β-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: “5023A” and “5025A,” for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR β-turn structure.

Enhancement of Human Melanoma Antigen Expression by IFN-β

Although many immunotherapeutic investigations have focused on improving the effector limb of the antitumor response, few studies have addressed preventing the loss of tumor-associated Ag (TAA) expression, associated with immune escape by tumors. We found that TAA loss from human melanomas usually results from reversible gene down-regulation, rather than gene deletion or mutation. Previously, we showed that inhibitors of MAPK-signaling pathways up-regulate TAA expression in melanoma cell lines. We have now identified IFN-β as an additional stimulus to TAA expression, including Melan-A/MART-1, gp100, and MAGE-A1. IFN-β (but neither IFN-α nor IFN-γ) augmented both protein and mRNA expression of melanocytic TAA in 15 melanoma lines (irrespective of initial Ag-expression levels). Treatment of low Ag melanoma lines with IFN-β increased expression of melanocyte-lineage Ags, inducing susceptibility to lysis by specific CTLs. Treatment with IFN-β also enhances expression of class I HLA molecules, thereby inducing both nominal TAA and the presenting HLA molecule. Data from fluorescent cellular reporter systems demonstrated that IFN-β triggers promoter activation, resulting in augmentation of Ag expression. In addition to enhancing TAA expression in melanomas, IFN-β also stimulated expression of the melanocytic Ag gp100 in cells of other neural crest-derived tumor lines (gliomas) and certain unrelated tumors. Because IFN-β is already approved for human clinical use in other contexts, it may prove useful as a cotreatment for augmenting tumor Ag expression during immunotherapy.

The binding of a gp120 V3 loop peptide to HIV-1 neutralizing antibodies: structural implications

The structural and antigenic properties of a peptide (“CRK”) derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved “GPGR” residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II β-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK “GPGR” residues to adopt a β-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: “5023A” and “5025A,” for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR β-turn structure.

NMR study and comparison of the antigenic properties of a peptide recognized by two HIV-1 neutralizing antibodies

Fab–peptide complexes formed between a 15 residue peptide derived from the HIV‐1 gp120 V3 loop and two of its cognate monoclonal antibodies, 5023A and 5025A, were studied using isotope‐edited solution nuclear magnetic resonance (NMR) techniques. Since these antibodies neutralize HIV‐1 virus with different strain specificities, this study was conducted to better understand the nature of these differences. The amide proton and nitrogen NMR resonances of specific residues were used to monitor the backbone of this peptide in these complexes. Three central residues of this peptide (‘RAF’) were found to be strongly affected by binding to both antibodies. Several other peptide residues were affected by binding to antibody 5023A but not 5025A. The antibody epitopes mapped by NMR are similar to those obtained previously via PEPSCAN at higher pH. One main difference between the PEPSCAN and NMR determined epitopes for 5023A involved two glycine residues of the peptide. By NMR, one of these glycines was more dramatically affected by antibody binding than predicted by PEPSCAN, while the other was much less so.

[59] Lyt-1, Lyt-2, and Lyt-3 antigens

Publisher Summary This chapter explores that the Lyt-1, Lyt-2, and Lyt-3 alloantigens resides on the surface of mouse thymocytes and on subpopulations of peripheral lymphocytes. It discusses that the Lyt-2 and Lyt-3 cell surface molecules has been kindled by the finding that they are T cell specific, and they mark certain functional subpopulations of T cells. The Lyt-2 and Lyt-3 alloantigens are present on cytotoxic killer and suppressor T cells but not on T cells which mediate helper function or delayed type hypersensitivity (DTH). The chapter also reviews that the presence of Lyt-2 and Lyt-3 on only certain functional subsets of T cells has raised the possibility that they may participate in the specialized function of the cells that bear them. Close linkage of Lyt-2 and Lyt-3 to the immunoglobulin light chain locus raised the possibility that these molecules might be structurally related to immunoglobulins and might contribute in some way to the specific antigen receptor on these T cells.