T cell recognition of an HLA-A2-restricted epitope derived from a cleaved signal sequence.

An alternative pathway for class I-restricted antigen presentation has been suggested on the basis of peptides bound to HLA-A2 molecules in cells lacking the transporter for antigen presentation (TAP). Most of these peptides were derived from signal sequences for translocation into the endoplasmic reticulum (ER). However, it is not known whether these peptides can be presented to T cells. The hydrophobic nature of an HLA-A2-restricted T cell epitope (M1 58-66) was exploited to test whether it could be presented to T cells when derived from a signal sequence. Replacing the signal sequence of the influenza virus hemagglutinin molecule H3 with an artificial sequence containing that HLA-A2-restricted T cell epitope resulted in efficient translocation of H3 molecules into the ER and transport to the cell surface. This signal sequence-derived epitope was presented to HLA-A2-restricted T cells. Involvement of cytosolic processing for this presentation is very unlikely, because (a) presentation occurred in cells lacking TAP; (b) expression of H3 molecules with the artificial signal sequence did not produce a detectable cytosolic form of H3; and (c) presentation of the same epitope expressed in cytosolic forms of antigen required TAP. Thus, a peptide derived from a signal sequence cleaved in the ER can provide an epitope for HLA-A2-restricted T cell recognition.

Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis.

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical HLA class I molecule, the gene for which is transcribed in most tissues. It has recently been reported that this molecule binds peptides derived from the signal sequence of HLA class I proteins; however, no function for HLA-E has yet been described. We show that natural killer (NK) cells can recognize target cells expressing HLA-E molecules on the cell surface and this interaction results in inhibition of the lytic process. Furthermore, HLA-E recognition is mediated primarily through the CD94/NKG2-A heterodimer, as CD94-specific, but not killer cell inhibitory receptor (KIR)-specific mAbs block HLA-E-mediated protection of target cells. Cell surface HLA-E could be increased by incubation with synthetic peptides corresponding to residues 3-11 from the signal sequences of a number of HLA class I molecules; however, only peptides which contained a Met at position 2 were capable of conferring resistance to NK-mediated lysis, whereas those having Thr at position 2 had no effect. Interestingly, HLA class I molecules previously correlated with CD94/NKG2 recognition all have Met at residue 4 of the signal sequence (position 2 of the HLA-E binding peptide), whereas those which have been reported not to interact with CD94/NKG2 have Thr at this position. Thus, these data show a function for HLA-E and suggest an alternative explanation for the apparent broad reactivity of CD94/NKG2 with HLA class I molecules; that CD94/NKG2 interacts with HLA-E complexed with signal sequence peptides derived from "protective" HLA class I alleles rather than directly interacting with classical HLA class I proteins.

Strictly transporter of antigen presentation (TAP)-dependent presentation of an immunodominant cytotoxic T lymphocyte epitope in the signal sequence of a virus protein.

Peptides presented by major histocompatibility complex (MHC) class I molecules are derived from intracellularly synthesized proteins. Cytosolic proteins are fragmented into peptides, which are subsequently transported via the transporter of antigen presentation (TAP) into the endoplasmic reticulum (ER), where they bind to MHC class I molecules. We have investigated the requirements for MHC class I presentation of the immunodominant gp33 cytotoxic T lymphocyte epitope of the lymphocytic choriomeningitis virus. This epitope is located within the leader peptide of the virus glycoprotein. Such an epitope is expected to be presented in a TAP-independent manner, since it is released into the ER by signal peptidase. Taking advantage of TAP1-/- mice, however, we show both in vitro and in vivo that, after virus infection, the presentation of the gp33 epitope is strictly dependent on a functional TAP heterodimer. The results are discussed with respect to peptide trimming processes in the ER.

Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen.

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.

In vitro translation and assembly of a complete T cell receptor-CD3 complex.

The T cell receptor for antigen (TCR) is a multisubunit complex that consists of at least seven polypeptides: the clonotypic, disulfide-linked alpha/beta heterodimer that is noncovalently associated with the invariant polypeptides of the CD3 complex (CD3-gamma, -delta, -epsilon) and zeta, a disulfide-linked homodimer. We achieved the complete assembly of the human TCR in an in vitro transcription/translation system supplemented with dog pancreas microsomes by simultaneous translation of the messenger RNAs encoding the TCR-alpha, -beta and CD3-gamma, -delta, -epsilon, and -zeta subunits. CD3-epsilon, one of the subunits that initiates the assembly of the TCR in living cells, forms misfolded, disulfide-linked homooligomers when translated alone. However, co-translation of one of its first binding partners in the course of assembly, CD3-gamma or -delta, led to the expression of mainly monomeric and correctly folded epsilon subunits, the only form we could detect as part of a properly assembled TCR complex. In the absence of these subunits, the ER-resident chaperone calnexin interacted with oligomeric, i.e. misfolded, structures of CD3-epsilon in a glycan-independent manner. A glycan-dependent interaction between CD3-epsilon and calnexin was mediated by CD3-gamma and concerned only monomeric CD3-epsilon complexed with CD3-gamma, but was dispensable for proper folding of CD3-epsilon. We suggest that in addition to its signaling function, CD3-epsilon serves as a monitor for proper subunit assembly of the TCR.

Qa-1b binds conserved class I leader peptides derived from several mammalian species.

Qa-1b binds a peptide (AMAPRTLLL), referred to as Qdm (for Qa-1 determinant modifier), derived from the signal sequence of murine class Ia molecules. This peptide binds with high affinity and accounts for almost all of the peptides associated with this molecule. Human histocompatibility leukocyte antigen (HLA)-E, a homologue of Qa-1b, binds similar peptides derived from human class Ia molecules and interacts with CD94/NKG2 receptors on natural killer cells. We used surface plasmon resonance to determine the ability of Qa-1b to bind related ligands representing peptides derived from the leaders of class I molecules from several mammalian species. All of the peptides reported to bind HLA-E bound readily to Qa-1b. In addition, peptides derived from leader segments of different mammals also bound to Qa-1b, indicating a conservation of this "Qdm-like" epitope throughout mammalian evolution. We have attempted to define a minimal peptide on a polyglycine backbone that binds Qa-1b. Our previous findings showed that P2 and P9 are important but not sufficient for binding to Qa-1b. Although a minimum peptide (GMGGGGLLL) bound Qa-1(b), its interaction was relatively weak, as were peptides sharing five or six residues with Qdm, indicating that multiple native residues are required for a strong interaction. This finding is consistent with the observation that this molecule preferentially binds this single ligand.

A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import.

We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol. NBPs are phosphorylated and phosphatase treatment abolished NLS binding. The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells. Proper import of a fluorescent protein containing the large T antigen NLS requires cytosol and ATP. In the absence of cytosol and/or ATP, NLS-containing proteins are bound to cytosolic structures and the nuclear envelope. Addition of cytosol and ATP results in movement of this bound intermediate into the nucleus. Anti-NBP antibodies specifically inhibited the binding part of this import reaction. These results indicate that a phosphoprotein common to several eukaryotes acts as a receptor that recognizes NLSs before their uptake into the nucleus.

Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins.

We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.

Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain.

Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence. These experiments demonstrate that iso-1, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain.

Signals arising from antigen-presenting cells.

It has been customary to consider that antigen-presenting cells provide, in addition to the presented antigen, a second or co-stimulatory signal that leads to T-cell growth and effector function. The recent literature indicates that this two-signal notion oversimplifies the function of antigen-presenting cells. Instead it is useful to consider four groups of events: the formation of peptide-MHC complexes, the role of soluble cytokines, the action of antigen-presenting cell-T cell molecular couples distinct from the receptor for peptide MHC, and the function of antigen-presenting cells in situ.

The proteasome inhibitor lactacystin prevents the generation of an endoplasmic reticulum leader-derived T cell epitope.

The presentation of viral antigens on MHC class I molecules requires their intracellular fragmentation into peptides of appropriate length and anchor residue positions. Evidence has accumulated that the proteasome is the endoprotease in charge of the generation of MHC class I ligands in the cytoplasm. The generation of T cell epitopes derived from the leader peptides of endoplasmic reticulum (ER) targeted proteins, however. has been reported to be independent of the proteasome. Here we show that the H-2Db restricted antigen presentation of the immunodominant T cell epitope derived from the ER leader of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) is completely abolished by administration of the proteasome inhibitor lactacystin. Thus our data support the role of the proteasome in class I restricted antigen processing and extend it to an ER leader derived epitope from a viral glycoprotein.

A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies.

We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (truncated at amino acid residue 289 with a 6-histidine tail) to investigate properties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A and Ni-chelate chromatography extracted milligram quantities of TSHR-289 (approximately 20-40% purity) from the culture medium. Nanogram quantities of this material neutralized the TSH binding inhibitory activity in all 15 Graves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, to partially purified TSHR-289. Screening of a TSHR complementary DNA fragment expression library localized the 3BD10 epitope to 27 amino acids at the N-terminus of the TSHR, a cysteine-rich segment predicted to be highly conformational. 3BD10 preferentially recognized native, as opposed to reduced and denatured, TSHR-289, but did not interact with the TSH holoreceptor on the cell surface. Moreover, mAb 3BD10 could extract from culture medium TSHR-289 nonreactive with autoantibodies, but not the lesser amount (approximately 25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Although the active form of TSHR-289 in culture medium was stable at ambient temperature, stability was reduced at 37 C, explaining the mixture of active and inactive molecules in medium harvested from cell cultures. In conclusion, studies involving a TSHR ectodomain variant indicate the exquisite conformational requirements of TSHR autoantibodies. Even under "native" conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients' autoantibodies. Therefore, Graves' disease is likely to be caused by even lower concentrations of autoantibodies than previously thought. Finally, reciprocally exclusive binding to TSHR-289 by human autoantibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition.

The role of dendritic cells in the innate immune system.

Dendritic cells (DCs) are bone-marrow-derived leucocytes that are specialised antigen-presenting cells capable of stimulating a primary T-lymphocyte response to specific antigen. In this chapter we discuss the role DCs play in the innate response acting as a critical link with the adaptive response and the influence of the innate response on dendritic cells.

Differential induction of immunoglobulin G subclasses by immunization with DNA vectors containing or lacking a signal sequence.

The route and method used to immunize mice with antigen-expressing DNA plasmids have an impact on the resulting T-helper cell response and IgG subclass distribution. Previous findings further indicate that the intracellular targeting of expressed antigens influences the differentiation of naive T-cells into either a Th1 or a Th2 type of response. In the present study, we analyzed the levels of IgG1 and IgG2a antibodies, as correlates of Th2 and Th1 responses, respectively, after intramuscular injection of mice with plasmids encoding a chimeric protein containing a Plasmodium falciparum blood stage antigen expressed in two different forms. One plasmid expresses the antigen in a secreted form as it is preceded by a signal sequence while expression from the other plasmid, lacking this sequence, results in cytoplasmic localization of the antigen. Mice immunized with the plasmid encoding secreted antigen responded with predominantly IgG1 antibodies. In contrast, sera from mice immunized with the plasmid expressing cytosolic protein displayed a mixed IgG1/IgG2a profile. In line with previous findings, our results suggest that the intracellular targeting of proteins expressed by DNA plasmids is an important factor for the differentiation of Th cells and the resulting subclass pattern of IgG responses.

Overlapping epitopes of friend murine leukemia virus gag-encoded leader sequence recognized by single cytotoxic T-lymphocyte clones.

The leader signal sequence of the non-structural gag-encoded glycoprotein precursor, Pr75gag, of Friend murine leukemia virus (F-MuLV) contains overlapping epitopes, SIVLCCLCL (p71-79) and CCLCLTVFL (p75 83) that activate Friend virus (FV)-induced tumor (FBL-3)-specific cytotoxic T-lymphocytes (CTL) (Kondo et al., J. Virol., 69, 1995, 6735-6741; Chen et al., J. Virol., 70, 1996, 7773-7782). It was investigated whether these two peptides are recognized by a single CTL clone or by individual clones with different specificities. The results show that both hydrophobic and cysteine-containing peptides are bound to H-2Db class I major histocompatibility complex (MHC) molecules and cross-recognized by a single CTL clone as well as bulk-cultured CTL from the spleens of mice immunized with FBL-3. The peptide p71-79 was effective for sensitizing target cells to lysis by CTL in the concentration of common antigenic peptides. Moreover, peptide p75-83 was 1000-fold more potent than the peptide p71-79. Specific cytotoxicity assays with variant peptides with alanine- and serine-substitutions suggested a highly complex function of the disulfide bond-forming peptides potentially sensitive to small sequence differences. The dominance of CTL responses to the transmembrane region is discussed in light of the high affinity of a novel hydrophobic peptide to compete with other peptides for binding to MHC molecules.

Recombinant human monoclonal antibody IgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates.

The CD4-binding domain of human immunodeficiency virus type 1 (HIV-1) gp120 elicits antibodies that are present in infected human sera. Monoclonal antibodies that recognize the HIV-1 gp120 CD4-binding domain have been isolated. Some of these antibodies can neutralize laboratory-adapted strains of HIV-1 and probably mediate neutralization by interfering with virus binding to its cellular CD4 receptor. However, most anti-CD4 binding domain antibodies do not neutralize primary HIV-1 isolates. We used primary HIV-1 isolates in an infectivity reduction assay to test the uniquely derived anti-CD4 binding domain recombinant human monoclonal antibody, IgG1b12. All of the tested HIV-1 isolates were neutralized by this antibody. Additional studies indicated that neutralization of a primary isolate with MAb IgG1b12 did not require continuous exposure of human peripheral blood mononuclear cell cultures to the antibody. Finally, a complete IgG1 molecule of an in vitro-selected b12 FAb mutant with a > 400-fold increase in affinity was assembled, expressed in mammalian cells, and evaluated in the infectivity reduction assay in comparative studies with the parent IgG1b12 antibody. The mutant did not retain the level of primary isolate neutralization potency that was a property of the parent molecule. Thus, we confirm that recombinant IgG1b12 has a unique specificity, and that it can neutralize all primary isolates tested in human PBMC cultures in vitro.

Comparative study of DNA-based immunization vectors: effect of secretion signals on the antibody responses in mice.

The presence of a signal sequence preceding the gene encoding a target antigen in a DNA vaccine should facilitate secretion of the in vivo translated antigen. The immune responses elicited upon injection with such a vector could differ from those induced by the same vector lacking a signal sequence. In the present study, the humoral responses elicited in mice immunized with two plasmids, either containing or lacking the human tissue plasminogen activator signal sequence, were compared. Both plasmids encode the chimeric antigen ZZN4, containing a malaria antigen Pf332-derived sequence (N4) linked to a bacterial fusion partner (ZZ). In vitro transfection of COS cells with each plasmid and treatment of the transfectants with brefeldin A confirmed that secretion of ZZN4 via the endoplasmic reticulum and Golgi pathway only occurred in cells transfected with the signal peptide-encoding plasmid. Repeated intramuscular injections of mice with either of the plasmids elicited comparable antibody responses to ZZN4 with regard to kinetics, specific IgG levels and persistence. These results indicate that in vivo transfection of muscle cells by either of these two plasmids generated comparable levels of antigen available for B-cell recognition and for uptake by antigen-presenting cells, despite the differential intracellular targeting of the encoded antigen. The relevance of these findings for the design of DNA vaccine vectors is discussed.

Dendritic cells in cattle: phenotype and function.

Dendritic cells are professional antigen presenting cells derived from the bone marrow and distributed throughout body tissues where they are located in sites that are suitable for antigen uptake. They are central to the induction of immune responses in naive animals and thus have become targets in strategies that are aimed at modulating resistance to infection. Studies in cattle have shown that the dendritic cells are phenotypically heterogeneous and that the different phenotypes have different biological properties. The molecular basis for this variation has begun to be investigated and has led to the identification of a member of the SIRPalpha family of signal regulatory proteins (MyD1) on a subset of dendritic cells in afferent lymph. Uptake of antigen by cattle dendritic cells is by a number of mechanisms that can involve endocytosis via clathrin coated pits or via caveolae as well as macropinocytosis.

Negative signalling via the P58/NKR for HLA.C alleles.

Recent studies have demonstrated that the interaction between HLA class I alleles and specific NK receptors results in negative signals which inhibit NK-mediated cytotoxicity. Such NK receptors have been identified by GL183 and EB6 mAbs which recognize distinct members of a molecular family involved in the recognition of two groups of HLA.C alleles. Now we describe a new allospecific NK group (group 0) which recognize all HLA.C alleles and we demonstrate that, on these clones, the p58 molecules EB6 and GL183 act independently to recognize Cw4 (and related alleles) and Cw3 (and related alleles) respectively. Finally we investigate whether the inhibitory signal mediated by the NK-receptor for HLA.C induce a temporary turn off on the cytolytic activity.