Using MHC Molecules to Define a Chlamydia T Cell Vaccine.

Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that include tuberculosis, malaria, HIV/AIDS, and others such as Chlamydia trachomatis. For these infections, vaccines that induce cellular immune responses are essential. Major impediments in developing such vaccines include difficulty in identifying relevant T cell antigens and delivering them in ways that elicit protective cellular immunity. Genomics and proteomics now provide tools to allow unbiased empirical identification of candidate T cell antigens. This approach represents an advance on bioinformatic searches for candidate T cell antigens. This chapter discusses an immunoproteomic approach we have used to identify Chlamydia T cell antigens. We further discuss how these T cell antigens can be developed into a human vaccine.

Profiling genome-wide chromatin methylation with engineered posttranslation apparatus within living cells.

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.

Constitutive and inflammatory immunopeptidome of pancreatic ß-cells.

Type 1 diabetes is characterized by the autoimmune destruction of pancreatic ß-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 ß-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant K(d)-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206₋214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by ß-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP206₋214 revealed low-level presentation by K(d) (~25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed K(d)-restricted peptide Janus kinase-1355₋363 (~15,000 copies/cell). We have successfully sequenced peptides from NIT-1 ß-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.

Expression, purification and preliminary X-ray crystallographic analysis of the human major histocompatibility antigen HLA-B*1402 in complex with a viral peptide and with a self-peptide.

The product of the human major histocompatibility (HLA) class I allele HLA-B*1402 only differs from that of allele HLA-B*1403 at amino-acid position 156 of the heavy chain (Leu in HLA-B*1402 and Arg in HLA-B*1403). However, both subtypes are known to be differentially associated with the inflammatory rheumatic disease ankylosing spondylitis (AS) in black populations in Cameroon and Togo. HLA-B*1402 is not associated with AS, in contrast to HLA-B*1403, which is associated with this disease in the Togolese population. The products of these alleles can present peptides with Arg at position 2, a feature shared by a small group of other HLA-B antigens, including HLA-B*2705, the prototypical AS-associated subtype. Complexes of HLA-B*1402 with a viral peptide (RRRWRRLTV, termed pLMP2) and a self-peptide (IRAAPPPLF, termed pCatA) were prepared and were crystallized using polyethylene glycol as precipitant. The complexes crystallized in space groups P2(1) (pLMP2) and P2(1)2(1)2(1) (pCatA) and diffracted synchrotron radiation to 2.55 and 1.86 A resolution, respectively. Unambiguous solutions for both data sets were obtained by molecular replacement using a peptide-complexed HLA-B*2705 molecule (PDB code 1jge) as a search model.

Repercusión del herpes gestationis en los estados materno y fetal / Effect of herpes gestationis on maternal and fetal health

Objetivo: Estudiar la repercusión del herpes gestationis en los estados materno y fetal. Sujetos y métodos: La base de este estudio la constituyen 2 pacientes controladas durante los meses de marzo a octubre de 2003 por el Servicio de Ginecología y Obstetricia del Hospital Universitario Son Dureta de Palma de Mallorca. Ambas fueron fotografiadas en diferentes momentos de su evolución y se les realizaron biopsias de las lesiones que confirmaron el diagnóstico de herpes gestationis. Los controles obstétricos realizados no difieren de los habituales durante una gestación de curso normal. Resultados: La evolución de las pacientes fue favorable tras el uso de corticoterapia. Respecto a las complicaciones fetales descritas, observamos un caso de crecimiento intrauterino retardado y otro de prematuridad. El carácter autoinmune de la enfermedad y su predisposición hereditaria quedan reflejados por el hallazgo de antígenos de histocompatibilidad específicos en una de nuestras pacientes. Conclusiones: El herpes gestationis no es una entidad grave siempre y cuando realicemos un diagnóstico y tratamiento correctos

Objective: To analyze the effect of herpes gestationis on maternal and fetal health. Subjects and methods: Two patients were studied from March to October, 2003 by the Obstetrics and Gynecology Service of Son Dureta University Hospital of Palma, Majorca. Both patients were photographed at various times during the course of the disease. The diagnosis of herpes gestationis was confirmed by biopsy. Their obstetric management did not differ from that in normal pregnancy. Results: Outcome in these patients was favorable after steroid therapy. Regarding fetal complications, low intrauterine growth was observed in one fetus and prematurity in the other. The autoimmune character of the disease and hereditary predisposition were reflected in the finding of specific histocompatibility antigens in one of the two patients. Conclusion: Herpes gestationis is not a serious disease if a correct diagnosis is made and appropriate treatment provided

Single-step purification of human C4b-binding protein (C4BP) by affinity chromatography on a peptide derived from a streptococcal surface protein.

Many Gram-positive bacteria express surface proteins that bind human plasma proteins. These bacterial proteins, and derivatives of them, are of interest for analysis of bacterial pathogenesis and as immunochemical tools. Well-characterized examples include the IgG-binding reagents staphylococcal protein A and streptococcal protein G, and the recently described streptococcal IgA-binding peptide Sap. Here, we show that a peptide derived from the streptococcal M22 protein can be used for single-step affinity purification of the human complement regulator C4b-binding protein (C4BP). Binding of C4BP was strongly enhanced by dimerization of the peptide via a C-terminal cysteine residue not present in the intact M22 protein. The purified C4BP had the expected binding characteristics, and acted as a cofactor for factor I in the degradation of C4b. Passage of serum through a peptide column under non-saturating conditions resulted in binding of >99.5% of serum C4BP, implying that such a column can be used to deplete serum of C4BP. These data indicate that the C4BP-binding peptide is a versatile tool that can be used for simple and rapid purification of biologically active human C4BP or for removal of C4BP from serum.

Human histocompatibility proteins.

Transplantation antigens (later called histocompatibility proteins) were named by Peter Gorer in the 1930s. After 4 decades of immunological work emphasizing their importance in immunobiology, structural work on these proteins began about 1970. During the first decade, HLA proteins were isolated and then separated into two groups. Biochemical studies established the close structural relationships of these groups (now called class I and class II MHC proteins). These structures both contained four domains, although the domains were linked differently. Two of these domains were immunoglobulin-like. They were the first proteins (aside from immunoglobulins) identified as members of the immunoglobulin superfamily of proteins. The crystallization of these proteins in the second decade led to elucidation of the structures of class I and class II MHC proteins, which has changed the way we think about immunology. These molecules each present peptides (8-9mers in the case of class I and 13-14mers in the case of class II) to initiate the immune response.

Changes in the immunologic phenotype of human malignant glioma cells after passaging in vitro.

Although immunotherapeutic strategies against glioblastomas have been promising both in vitro and in animal models, similar successes have not been realized in human clinical trials. One reason may be that immunotherapeutic strategies are based on prior studies that primarily have used human glioblastoma cell lines passaged in vitro, which may not accurately reflect the in vivo properties of glioblastoma cells. In this report, we used flow cytometry to quantify the expression of immunological cell surface molecules on human glioblastomas directly ex vivo (prior to any in vitro culturing) and after varying passages in vitro. Furthermore, we used ELISA to quantitate cytokine secretion after various passages in vitro. We demonstrate that in vitro culturing of established cell lines led to increases in the cell surface expression of MHC class I and ICAM-1 and secretion of IL-6 and TGF-beta(2). Furthermore, there were significant changes in the expression of MHC class I, MHC class II, B7-2, ICAM-1, and FasL when comparing ex vivo tumor cells to those after a single passage in vitro. After passaging once in vitro, there were also significant changes in the secretion of TGF-beta(2) and IL-10. This report indicates that in vitro culturing leads to significant changes in both cell surface molecules and secreted cytokines, which are known to affect the ability of immune cells to initiate an anti-tumor immune response. These changes in the immunological phenotype of glioblastomas after in vitro culturing may in part explain the limited success of immunotherapeutic strategies against glioblastomas in human clinical trials.

Isolation of the melanoma-associated antigen p23 using antibody phage display.

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.

RT1-U: identification of a novel, active, class Ib alloantigen of the rat MHC.

In common with other mammalian species, the laboratory rat (Rattus norvegicus) expresses MHC class I molecules that have been categorized as either classical (class Ia) or nonclassical (class Ib). This distinction separates the class Ia molecules that play a conventional role in peptide Ag presentation to CD8 T cells from the others, whose function is unconventional or undefined. The class Ia molecules are encoded by the RT1-A region of the rat MHC, while the RT1-C/E/M region encodes up to 60 other class I genes or gene fragments, a number of which are known to be expressed (or to be expressible). Here we report upon novel MHC class Ib genes of the rat that we have expression cloned using new monoclonal alloantibodies and which we term RT1-U. The products detected by these Abs were readily identifiable by two-dimensional analysis of immunoprecipitates and were shown to be distinct from the class Ia products. Cellular studies of these molecules indicate that they function efficiently as targets for cytotoxic killing by appropriately raised polyclonal alloreactive CTL populations. The sequences of these class Ib genes group together in phylogenetic analysis, suggesting a unique locus or family. The combined serological, CTL, and sequence data all indicate that these products are genetically polymorphic.

Two distinct forms of soluble MHC class I molecules synthesized by different mechanisms in normal rat cells in vitro.

Rat soluble MHC class I synthesis was studied at both RNA and protein levels to determine whether multiple forms of soluble MHC class I molecules are produced by different mechanisms. RT-PCR and sequencing of MHC class I transcripts identified an alternatively spliced nonclassical MHC class I gene product, lacking both exon 5 and 6, in both spleen and liver. Immunoprecipitation and SDS-PAGE identified two distinct soluble MHC class I proteins in both splenocyte- and hepatocyte-culture supernatants. The 36Kd classical soluble MHC class I protein (RT1.Aa) was precipitated by both allele-specific (MN4.91.6, R3/13, R2/15S) and pan-reactive (OX18) mAbs. The 39Kd non-RT1.A soluble MHC class I protein was precipitated only by OX18. The production of soluble RT1.Aa was inhibited by a metalloproteinase inhibitor, but not by serine/thiol protease inhibitors. None of these protease inhibitors interfered with the soluble non-RT1.A production, suggesting that it might be derived from an alternatively spliced MHC class I transcript. The soluble non-RT1.A was always associated with beta2m. However, soluble RT1.Aa molecule was cleaved in beta2m-free form and was reassociated with beta2m in culture supernatants. Thus two soluble MHC class I molecules, classical (36Kd RT1.Aa) and nonclassical (the alternatively spliced transcript), were produced from rat cells. Alternative splicing led to the nonclassical soluble MHC class I synthesis. Proteolytic cleavage by metalloproteinase led to the classical soluble MHC class I synthesis.

Structure of the gene encoding the rat T cell ecto-ADP-ribosyltransferase RT6.

Cellular functions, such as the cytolytic potential of CTLs, can be regulated by mono-ADP-ribosylation of target proteins. Recently, the T cell differentiation marker RT6 has been shown to possess mono-ADP-ribosyltransferase activity. Defects in RT6 expression coincide with increased susceptibility in animal models for insulin-dependent diabetes mellitus and other autoimmune diseases. We present an analysis of the rat RT6 gene, providing a basis for studying the regulation of this gene in T cells of normal and diabetes-prone rats. It is the first structural analysis of a mammalian mono-ADP-ribosyltransferase gene. The RT6 gene consists of eight exons spanning approximately 20 kb. The proximal four exons encode 5' untranslated region sequences and are found in multiple alternatively spliced variants. Exon 5 encodes the N-terminal signal sequence. An unusually large exon 7 encodes the entire native polypeptide. The final exon 8 encodes the C-terminal signal sequence for glycosylphosphatidylinositol anchor attachment and the 3' untranslated region. Two independent TATA box-containing promoters associated with exons 1 and 2 were identified, and their activity was verified in transient transfection assays. The distal promoter displays elements contained in the regulatory regions of T cell-specific genes, such as ets and ikaros. Analysis of RT6 transcripts showed that this promoter is the major one in adult rat spleen cells. The 3' end of the gene does not display alternative splicing. However, two polyadenylation signals are found in the 3' untranslated region.

Molecular characterization of mouse T-cell ecto-ADP-ribosyltransferase Rt6: cloning of a second functional gene and identification of the Rt6 gene products.

RT6 is an enzymatically active GPI-anchored membrane protein that was originally discovered in the rat as a peripheral T cell alloantigen. It has attracted interest as an activation antigen and because defective RT6-expression coincides with increased susceptibility for autoimmune type I diabetes in the BB rat. Southern blot analyses indicate that the rat carries a single copy RT6 gene whereas the mouse carries a duplication of the homologous locus. We had previously cloned and sequenced a RT6-homologous cDNA from BALB/c mouse spleen. We now report the cloning and characterization of a second RT6-homologue from BALB/c and 129/Sv mice. The two mouse Rt6 genes (designated Rt6-1 and Rt6-2) encode similar open reading frames that are disrupted by conserved introns. The nucleotide sequences of the Rt6-1 and Rt6-2 coding regions show 87% sequence identity, the deduced amino acid sequences 79% identity. The amino acid sequences reveal significant similarity to recently cloned ADP-ribosylating ectoenzymes from rabbit and human skeletal muscle as well as chicken bone marrow cells. RT-PCR analyses reveal that the two Rt6 genes are differentially expressed in distinct inbred mouse strains and that their transcripts are properly processed. Western blot analyses demonstrate that the respective gene products are released from cells by treatment with PI-PLC. The results further show that both mouse Rt6 genes are translated into GPI-anchored cell surface molecules and that Rt6 gene expression is restricted to peripheral lymphoid tissues.

Myogenin is required for late but not early aspects of myogenesis during mouse development.

Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD-lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts.

Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains.

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.