Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus.

The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection.

Mapping immunodominant antigens and H-2-linked antibody responses in mice urogenitally infected with Chlamydia muridarum.

To identify immunodominant antigens and MHC-restricted antibody responses, seven different strains of mice were intravaginally infected with Chlamydia muridarum and compared for antibody responses to 257 C. muridarum proteins. The 7 strains of mice recognized a total of 109 proteins as antigens, of which, 5 antigens (TC0660, TC0727, TC0828, TC0726 & TC0268) were each recognized by 60% or more mice from each mouse strain and thus designated as immunodominant antigens. Furthermore, antibody responses to 19 other antigens displayed strong associations with mouse H-2 haplotypes, including 6 antigens (TC0480, TC0912, TC0229, TCA04, TC0289 & TC0892) whose antibody responses were linked to H-2(b), 8 (TC0035, TC0387, TC0052, TC0781, TC0373, TC0117, TC0066 & TC0396) to H-2(d) and 5 (TC0512, TC0177, TC0589, TC0794 & TC0596) to H-2(k) haplotypes respectively. Interestingly, H-2(b) was negatively associated with antibody responses to most of the antigens that were positively linked to H-2(d) or H-2(k) haplotypes. These results by mapping Chlamydia trachomatis antigens commonly recognized by mice with different strain background and H-2 genes and revealing antigen association with H-2 haplotypes have provided important information for developing chlamydial subunit vaccines and understanding chlamydial pathogenesis.

Immunoproteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia.

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.

Complex assembly, crystallization and preliminary X-ray crystallographic studies of MHC H-2Kd complexed with an HBV-core nonapeptide.

In order to establish a system for structural studies of the murine class I major histocompatibility antigen complex (MHC) H-2Kd, a bacterial expression system and in vitro refolding preparation of the complex of H-2Kd with human beta2m and the immunodominant peptide SYVNTNMGL from hepatitis B virus (HBV) core-protein residues 87-95 was employed. The complex (45 kDa) was crystallized; the crystals belong to space group P222(1), with unit-cell parameters a = 89.082, b = 110.398, c = 47.015 A, alpha = beta = gamma = 90 degrees. The crystals contain one complex per asymmetric unit and diffract X-rays to at least 2.06 A resolution. The structure has been solved by molecular replacement and is the first crystal structure of a peptide-H-2Kd complex.

Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/neu-transgenic mice.

The HER-2/neu (neu-N)-transgenic mice are a clinically relevant model of breast cancer. They are derived from the parental FVB/N mouse strain and are transgenic for the rat form of the proto-oncogene HER-2/neu (neu). In this study, we report the identification of a MHC class I peptide in the neu protein that is recognized by CD8(+) T cells derived from vaccinated FVB/N mice. This 10-mer was recognized by all tumor-specific FVB/N T cells generated regardless of the TCR Vbeta region expressed by the T cell or the method of vaccination used, establishing it as the immunodominant MHC class I epitope in neu. T cells specific for this epitope were able to cure FVB/N mice of transplanted neu-expressing tumor cells, demonstrating that this is a naturally processed peptide. Altered peptide analogs of the epitope were analyzed for immunogenicity. Vaccination with dendritic cells pulsed with a heteroclitic peptide provided FVB/N and neu-N mice with increased protection against tumor challenge as compared with mice immunized with dendritic cells loaded with either wild-type or irrelevant peptide. Discovery of this epitope allows for better characterization of the CD8(+) T cell responses in the neu-N mouse model in which neu-specific tolerance must be overcome to produce effective antitumor immunity.

Viral escape at the molecular level explained by quantitative T-cell receptor/peptide/MHC interactions and the crystal structure of a peptide/MHC complex.

Viral escape, first characterized for the lymphocytic choriomeningitis virus (LCMV) in a mouse transgenic for the P14 T cell-receptor (TCR), can be due to mutations in T-cell epitopes. We have measured the affinity between the H-2D(b) containing the wild-type and two of its "viral escape" epitopes, as well as other altered peptide ligands (APL), by using BIACORE analysis, and solved the crystal structure of H-2D(b) in complex with the wild-type peptide at 2.75 A resolution. We show that viral escape is due to a 50 to 100-fold reduction in the level of affinity between the P14 TCR and the binary complexes of the MHC molecule with the different peptides. Structurally, one of the mutations alters a TCR contact residue, while the effect of the other on the binding of the TCR must be indirect through structural rearrangements. The former is a null ligand, while the latter still leads to some central tolerance. This work defines the structural and energetic threshold for viral escape.

Murine class I major histocompatibility complex H-2Dd: expression, refolding and crystallization.

A truncated soluble form of the murine class I major histocompatibility antigen complex H-2Dd was cloned using an Escherichia coli based system. It was expressed, refolded in vitro and crystallized in a complex with murine beta2 microglobulin and the peptide RGPGRAFVTI from the V3-loop of the gp160 HIV-1 protein. Crystals belonging to the space group P212121 with cell dimensions a = 51.3, b = 92.5, c = 108.8 A were obtained using two different crystallization conditions. The crystals contain one complex per asymmetric unit and diffract to at least 2.4 A resolution.

[Extraction and purification of mouse H-2 antigen].

This study was designed to purify the mouse major histocompatibility complex antigen (H-2Ag). The detergent was used to extract the crude H-2Ag, and monoclonal antibodies affinity chromatography was applied to purify H-2 antigen. A 45 kd heavy chain and a 12 kd light chain from the purified proteins were shown by electrophoresis. Besides, the pure H-2 antigen was found to have immunological and biological activity. This method should be useful in purifying large quantities of H-2Ag for the researches in transplantation and functional analysis of H-2 antigen.

Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide.

In this report, we demonstrate stimulation of T cell receptor (TCR) transgenic CD8 T cells by isolated major histocompatibility complex (MHC) class I H-2Ld complexes and antigenic peptide. This is the first demonstration of CD8 T cells activated by MHC and antigenic peptide in the absence of antigen priming. Furthermore, isolated MHC and a potent peptide antigen can stimulate phenotypically naive CD44- T cells to become CTL effectors and to produce interleukin-2 in nanogram per milliliter amounts. These results demonstrate that particular TCR antigen pairs may overcome the need for specialized antigen-presenting cells and have implications for mechanisms of autoimmunity and tolerance induction.

Deglucosylation of N-linked glycans is an important step in the dissociation of calreticulin-class I-TAP complexes.

Recent evidence indicates that newly synthesized major histocompatibility complex (MHC) class I proteins interact with calnexin, a transmembrane endoplasmic reticulum protein specific for certain glycoproteins bearing monoglucosylated glycans. Here, we studied the association of newly synthesized class I proteins with calreticulin, a soluble calnexin-related ER protein, in murine T cells. We found that, unlike calnexin-class I interactions, calreticulin assembly with class I proteins was markedly decreased in the absence of beta 2 microglobulin expression and that calreticulin associated with a subset of class I glycoforms distinct from those assembled with calnexin but similar to those bound to TAP (transporter associated with antigen processing) proteins. Finally, these studies show that deglucosylation of N-linked glycans is important for dissociation of class I proteins from both calreticulin and TAP and that the vast majority of newly synthesized class I proteins associated with calreticulin are simultaneously assembled with TAP. The data demonstrate that calnexin and calreticulin chaperones assemble with distinct MHC class I assembly intermediates in the ER and show that glycan processing is functionally coupled to release of MHC class I proteins from peptide transport molecules.

Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.

We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum.

Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50 kDa (p50) and further distinct 20 and 130 kDa polypeptides were detectable. Class II binding of p50 was characterized in detail. Point mutations within the Ak antigen binding groove destabilized the p50:class II complexes; a mutation outside the groove had no effect. A short segment of p50 was sufficient for association with Ak. The p50 polypeptide was synthesized endogenously, bound to Ak in a pre-Golgi compartment, and was transported to the cell surface in association with Ak. Thus, Ii protects the class II groove from binding endogenous, possibly misfolded polypeptides in the ER. The possibility is discussed that polypeptide binding is an ancestral function of the MHC antigen binding domain.

Structure of the N-linked oligosaccharides of MHC class I molecules from cells deficient in the antigenic peptide transporter. Implications for the site of peptide association.

Class I molecules are N-linked glycoproteins encoded by the MHC. They carry cytosolic protein-derived peptides to the cell surface, displaying them to enable immune surveillance of cellular processes. Peptides are delivered to class I molecules by the transporter associated with Ag processing (TAP). Peptide association is known to occur before exposure of class I molecules to the medial Golgi-processing enzyme alpha-mannosidase II, but there is limited information regarding the location or timing of peptide binding within the earlier regions of the endoplasmic reticulum (ER)-Golgi pathway. A reported association of newly synthesized class I molecules with the ER chaperonin calnexin raises the possibility of persistence of the monoglycosylated N-linked oligosaccharide (NLO) Glc1Man8GlcNAc2, known to be recognized by this lectin. To explore these matters, we determined the structure of the NLOs on the subset of newly synthesized class I molecules awaiting the loading of peptide. We pulse-labeled murine MHC H-2Db class I molecules in RMA/S cells, which lack one of the TAP subunits, causing the great majority of the molecules to be retained for prolonged periods in an early secretory compartment, awaiting peptide binding. MHC molecules pulse-labeled with [3H]glucosamine were isolated, the NLOs specifically released and structurally analyzed by a variety of techniques. Within the chosen window of biosynthetic time, most Db molecules from parental RMA cells carried mature NLOs of the biantennary complex-type, with one to two sialic acid residues. In RMA/S cells, such chains were in the minority, the majority consisting of the precursor forms Man8GlcNAc2 and Man9GlcNAc2. No glucosylated forms were detected, nor were the later processing intermediates Man5-7GlcNAc2 or GlcNAc1Man4-5GlcNAc2. Thus, most Db molecules in TAP-deficient cells are retained in an early compartment of the secretory pathway, before the point of first access to the Golgi alpha-mannosidase I, which trims alpha 1-2 linked mannose residues, but beyond the point where the alpha 1-3-linked glucose residue is finally removed by the ER glucosidase II. Thus, structural analysis of NLOs on class I molecules within a defined biosynthetic window has established a biochemical measure of the timing of peptide association.

The costimulatory and differentiating activity of soluble class I MHC antigens for an autologous thymocyte population.

Combined cultivation of macrophages with syngeneic thymocytes resulted in accumulation of soluble H-2Kk antigens in culture medium. Incubation of intact autologous thymocytes with these soluble class I MHC molecules was shown to induce functional maturation of thymocytes assayed in local graft-vs-host reaction. Similar thymocyte costimulating activity was detected for the papain-solubilized purified H-2Kk antigens. Soluble class I antigens were shown to costimulate IL2 production by thymocytes in response to submitogenic doses of exogenous IL2 and to increase PHA-induced thymocyte proliferation. Soluble class I molecules were shown to increase the level of expression of function-associated membrane antigens, H-2Kk, CD8 and CD4, and to trigger thymocyte differentiation. The expression of I-Ak antigens remained invariable. It was also shown that soluble autologous class I molecules may function as direct amplifiers of thymocyte proliferation in autologous, but not allogeneic, mixed leukocyte reactions. It is concluded that soluble MHC class I molecules are capable of triggering functional and phenotype differentiation of syngeneic thymocytes and acting as restricted coaccessory molecules when thymocyte activation is induced by a submitogenic dose of different stimuli.

Identification of a recombinational breakpoint at the BAT5 locus in three intra-H-2 recombinant inbred mouse strains.

We have analyzed the S/D region in 14 inbred mouse strains by restriction fragment length polymorphism (RFLP) using human BAT2 and BAT5 genes as probes. In all recombinant strains, the recombinational breakpoint mapped centromeric to Bat-2 (D17H6S51E). In recombinant strains B6.R4, B6.AK1 and B10.BYR1, recombination occurred within or close to the Bat-5 (D17H6S82E) locus. The immunogenicity of B10.BYR1 and B6.R4 bone marrow cell (BMC) grafts differs from that of both sets of parents, as if genes at or just centromeric to Bat-5 are involved. The phenotype of B6.AK1 BMCs (Kb Dk) is similar to that of the H2k parent suggesting no changes occurred. However, RNA blot analysis has shown that the Bat-5 gene is expressed well in bone marrow cells of B10.BR mice but not in B6.AK1 marrow cells. Analysis of a limited number of tumor cells of hemopoietic origin identified a single transcript for Bat-5. Our present data identify a recombinational hot spot at the Bat-5 locus. The expression of Bat-5 or a nearby gene may influence the immunogenicity of BMCs.

Characterization of an incompletely assembled major histocompatibility class I molecule (H-2Kb) associated with unusually long peptides: implications for antigen processing and presentation.

We have identified two forms of a major histocompatibility complex (MHC) class I molecule, H-2Kb, distinguishable by specific antibodies through a study of a genetically engineered mouse cell line that overexpresses these molecules. One form, a complex associated with beta 2-microglobulin (native, beta 2m+ class I), is detectable by conformation-dependent antibodies. The other form, which remains after preclearing cell lysates of native class I, is only poorly, if at all, associated with beta 2-microglobulin (beta 2m- class I) and is detectable by an antiserum against the cytoplasmic tail region of H-2K molecules. Both forms are also present in normal cell lines. The affinity-purified native class I molecules bind short peptides (8 or 9 residues) and assemble tightly with beta 2-microglobulin. In striking contrast, the beta 2m- class I molecules bind peptides that are longer (> 15 residues) than those bound to native class I molecules. This finding is consistent with the recent evidence that peptides longer than 8-10 amino acid residues are transported into the endoplasmic reticulum and suggests the possibility of a control step for peptide presentation by MHC in which the incompletely processed peptides bind to the heavy chain and a selected fraction undergoes final processing and presentation on the cell surface.

Class I histocompatibility molecule association with phosphorylated calnexin. Implications for rates of intracellular transport.

Recent studies have shown that the endoplasmic reticulum (ER)-resident protein, calnexin, associates with class I major histocompatibility complex (MHC) molecules early in their biosynthesis. It has been suggested that calnexin participates in the assembly of class I MHC molecules or in the retention within the ER of unassembled class I molecules. We have examined the role of phosphorylation of calnexin in its association with mouse class I MHC molecules. We show that phosphocalnexin associates with H-2Ld and H-2Db molecules but not with H-2Kb and H-2Dd molecules, although calnexin-H-2Kb association can be demonstrated. These observations are interesting in light of the fact that H-2Kb and H-2Dd molecules are transported out of the ER more rapidly than are H-2Ld and H-2Db molecules. H-2Ld and H-2Db molecules differ in amino acid sequence only in their membrane-distal alpha 1 and alpha 2 domains. Nevertheless, the affinity of phosphocalnexin for H-2Ld is greater than its affinity for H-2Db. Furthermore, H-2Db becomes endoglycosidase H-resistant more slowly in cells in which it associates with phosphocalnexin than in cells in which it does not. Ca2+ ionophore A23187 prevents association of phosphocalnexin with H-2Ld molecules in vivo but does not cause the disruption of phosphocalnexin-H-2Ld complexes after they have formed. A23187 does not prevent assembly of H-2Ld-beta 2-microglobulin (beta 2-m) heterodimers. Furthermore, phosphocalnexin is found associated with H-2Ld molecules regardless of their state of assembly with beta 2-m and antigenic peptide. These results suggest that phosphocalnexin association with class I MHC molecules does not play a role in assembly of the class I MHC-beta 2-m-peptide complex nor in preventing release of unassembled class I molecules from the ER but may otherwise influence their rate of transport through the ER.

In vivo regulation of the assembly and intracellular transport of class I major histocompatibility complex molecules.

Using H-2Kb-transfected Balb/c 3T3 cells which generate "empty" H-2Kb molecules devoid of antigenic peptides, we show that peptide availability determines the stability of class I molecules and dictates the overall intracellular transport rate of the class I complexes. Our data also indicate that chaperonin-like proteins are involved in class I assembly. Using Drosophila cells transfected with H-2Kb and murine beta 2-microglobulin, we show that one possible candidate, calnexin, associates with class I molecules prior to peptide acquisition. These data suggest that both peptide supply and assembly proteins dictate cell surface expression of class I major histocompatibility complex molecules and ultimately influence T cell recognition. The role of beta 2-microglobulin in class I assembly is also discussed.

Rapid, activity-guided isolation of sex-limited protein (Slp) from mouse serum by fractionated precipitation and high-performance liquid chromatography.

We have recently demonstrated that sex-limited protein (Slp) plays a key role in an EDTA-resistant mouse complement activation pathway. A rapid procedure, utilizing classical chromatography methods on an FPLC system, was developed for the isolation of functionally active Slp. The method is based on the fractionated precipitation of serum by polyethylene glycol 6000, followed by heparin Sepharose Cl-6B affinity chromatography, Mono Q anion exchange and Superose 12 gel filtration. The isolation of Slp was monitored by a hemolytic assay. The procedure resulted in the purification of Slp, which by SDS-PAGE gave a single band of M(r) 2000,000 under non-reducing conditions, and under reducing conditions three bands corresponding to M(rs) of 105,000, 76,000 and 37,000.