Danny J. Schust

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

WHY CERTAIN ANTIBODIES CROSS-REACT WITH HLA-A AND HLA-G : EPITOPE MAPPING OF TWO COMMON MHC CLASS I REAGENTS

Antigen presentation at the maternal-fetal interface has been characterized by a reported lack of classical MHC class I products and the presence of a tissue-restricted, non-classical class I product with limited polymorphism, HLA-G. The lack of HLA-A, -B, and -C products at this interface would allow escape from T-cell mediated attack, while the presence of HLA-G may enable evasion of NK cell-mediated destruction. We provide evidence that in addition to HLA-G, the classical class I product HLA-C is also present in trophoblast. Specifically, cDNA from the trophoblast-derived JEG 3 cell line encodes the HLA-C-locus product, HLA-Cw*0401. This protein, obtained by in vitro transcription/translation, has biochemical characteristics identical to MHC class I products immunoprecipitated directly from the same cells. These findings are in agreement with RNA analysis and immunohistochemistry on both cell lines and primary trophoblast tissues. We report here the preferential reactivity in JEG 3 cells of two widely used monoclonal anti-MHC class I heavy chain antibodies, HC10 and HCA2, with HLA-C and HLA-G, respectively. We have mapped the epitopes recognized by these reagents to distinct areas of the alpha1 domain of the MHC class I heavy chain. HCA2 recognizes the motif xLxTLRGx spanning amino acids 77 84 present in both HLA-A and HLA-G. In contrast, HC10 may recognize a discontinuous epitope, with essential elements of the recognized motif surrounding residue 60 in the alpha1 domain of the class I heavy chain, as shown by truncation analysis. These results adequately explain the immunochemical cross-reactivity of HLA-A and HLA-G.

Human Cytomegalovirus US2 Endoplasmic Reticulum-Lumenal Domain Dictates Association with Major Histocompatibility Complex Class I in a Locus-Specific Manner

ABSTRACT The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2s ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.

Down-regulation of MHC class I antigen presentation by HCMV; lessons for tumor immunology

MHC class I antigen presentation is a tightly regulated process that begins in the cell cytoplasm with the generation of peptide ligands by the proteasome (1, 2). Peptides are translocated from the cytosol into the endoplasmic reticulum (ER) lumen through the MHC-encoded TAP complex. These peptides are loaded onto the class I heterodimer that is comprised of a 43 kDa type I membrane glycoprotein heavy chain and a 12 kDa soluble light chain (p2m). Shortly after synthesis of the class I heavy chain and &m, the complex is properly folded with assistance from ER resident chaperones such as calnexin, calreticulin and the thiol-reductase, ERp57(3). Tapasin, an ER-resident protein that associates with TAP, can interact with peptide-free class I heterodimerkhaperone complex and guide it to the TAP, where it is subsequently loaded with peptide. The generation of the trimeric class I complex allows its exit out of the ER and through the Golgi to the cell surface. The major histocompatibility complex (MHC) class I molecules present peptides derived from microbial and cellular proteins on the cell surface (4). The interaction of the MHC class I molecules with cytotoxic CD8T lymphocytes (CTL) can activate an immune response and target the host cell for lysis, thereby eliminating the infected or abnormal cell. Several viruses and tumor cells can down-regulate the surface expression of class I molecuies at the transcriptional and post-translational levels(5, 6). The prevention of class I surface expression allows these cells to escape CTL detection. The mechanism by which tumors down-regulate class I molecules is not clearly understood. However, viral proteins have been identified which interfere with class I antigen presentation and may provide insight into how class I down-regulation is achieved in tumor cells as well.

Comparison of Ophthalmic Sponges for Measurements of Immune Markers from Cervical Secretions

ABSTRACT Measurements of cervical immunity are important for evaluating immune responses to infections of the cervix and to vaccines for preventing those infections. Three ophthalmic sponges, Weck-Cel, Ultracell, and Merocel, were loaded in vitro with interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, gamma interferon (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), immunoglobulin A (IgA), or IgG, and sponges were extracted and evaluated for total recovery by enzyme-linked immunosorbent assay (ELISA). There was excellent (>75%) recovery for all immune markers from all three devices except for IL-6, which was poorly recovered (<60%) for all sponge types, IFN-γ, which was poorly recovered from both Weck-Cel and Ultracell sponges but was completely recovered from Merocel sponges, and IL-4, which was poorly recovered from Weck-Cel sponges but was completely recovered from Ultracell or Merocel sponges. We then compared the absolute recovery of selected markers (IL-10, IL-12, IgG, and IgA) from cervical secretion specimens collected from women using each type of sponge. There were no significant differences in the recoveries of IL-10, IL-12, and IgG from cervical specimens collected by any type of ophthalmic sponge, but there was reduced IgA recovery from Merocel sponges. However, the variability in these measurements attributable to sponge types (1 to 3%) was much less than was attributable to individuals (45 to 72%), suggesting that differences in sponge type contribute only in a minor way to these measurements. We infer from our data that the three collection devices are adequate for the measurements of IL-1β, IL-2, IL-5, IL-12, IL-15, IL-18, and IgG. Merocel may be a better ophthalmic sponge for the collection of cervical secretions and measurements of IL-4, IL-8, IL-10, GM-CSF, and IFN-γ, but our data from clinical specimens, not in vitro-loaded sponges, suggested the possibility of reduced recovery of IgA. These findings require confirmation.