Independent association of T cell receptor beta and gamma chains with CD3 in the same cell.

We have demonstrated that the PEER cell line, which expresses a CD3-associated TCR gamma chain on the cell surface, synthesizes TCR beta chain intracellularly. A percentage of this TCR beta chain associates with the CD3 complex intracellularly. These results indicate that TCR beta and gamma chains can be synthesized by one cell line, and that these chains can independently associate with the CD3 complex. However, the results argue against the formation of TCR beta gamma chain complexes in this cell line.

Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex alpha/beta chains.

Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.

Mutations in the alpha 2 helix of HLA-A2 affect presentation but do not inhibit binding of influenza virus matrix peptide.

Previous studies have suggested that MHC class I molecules bind and present peptides to CTL in a manner that is analogous to the presentation of peptides by class II molecules to Th. Crystallographic studies of HLA-A2 have led to the assignment of a putative peptide binding site that is bordered by two alpha helices consisting of residues 50-84 and 138-180. In this study, we have investigated whether residues in the alpha 2 helix are involved in the binding and/or presentation of a peptide to CTL. We have generated CTL to type A influenza virus by stimulation of human PBL with a synthetic peptide from the influenza A virus matrix protein (M1 residues 57-68) in the presence of rIL-2. Such HLA-A2.1-restricted influenza virus-immune CTL do not recognize infected HLA-A2.3+ targets. A2.1 and A2.3 differ by three amino acids in the alpha 2 domain: Ala vs. Thr at position 149, Val vs. Glu at position 152, and Leu vs. Trp at position 156. Site-directed mutants of the A2.1 gene that encode A2 molecules that resemble A2.3 at positions 149, 152, and 156 have been constructed, transfected into human cells, and assayed for their ability to present the M1 peptide. The results demonstrate that most, but not all, A2.1-restricted M1-peptide-specific CTL fail to recognize M1 peptide-exposed transfectants with certain single amino acid substitutions at positions 152 and 156. In contrast, M1 peptide-exposed transfectants that express A2 molecules with an Ala----Thr substitution at position 149 were recognized by all CTL tested, but they exhibited an apparent difference in the kinetics of peptide binding. These results indicate that amino acid substitutions at positions 152 and 156 of the putative peptide binding site of the A2 molecule can affect presentation without eliminating binding, and indicate that the failure to recognize complexes between the peptide and the mutant A2 molecules is due to different TCR specificities and not to the failure to bind the peptide.

Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.

Genetic control of the immune response in mice to a Plasmodium falciparum sporozoite vaccine. Widespread nonresponsiveness to single malaria T epitope in highly repetitive vaccine.

Different H-2 congenic strains of mice were immunized with a P. falciparum sporozoite vaccine currently being tested in humans, or with different segments of the vaccine molecule. Specific IgG production or lymph node cell proliferation in response to different antigens was then determined. Only four of seven strains (representing three of eight possible different class II restriction molecules) responded to the vaccine. Of those restriction molecules, only one, I-Ab, was associated with a response to a malaria-encoded T epitope [contained within NP(NANP)3NA], while the other two molecules (E alpha dE beta d and E alpha kE beta s) were associated with a T cell response to a nonmalarial epitope(s) carboxyterminal to the malaria sequence and encoded by a tetracycline resistance gene, read out of frame. If an analogous situation applies in humans, natural boosting by sporozoites will be very restricted. This has serious implications for the effectiveness of the vaccine, since constant high levels of antisporozoite antibodies and possibly antibody-independent T cell effector functions are required for immunity.

T cell receptor gamma/delta chain diversity.

The TCR-gamma and -delta chains of six murine hybridomas were compared by one-dimensional SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analysis. This allowed the identification of three distinct gamma chains (gamma a, gamma b, and gamma c) and three distinct delta chains (delta a, delta b, and delta c). Four gamma/delta chain combinations (gamma a delta a, gamma b delta b, gamma b delta c, and gamma c delta a) were observed. These results indicate that multiple forms of the delta chain are expressed and suggest that the delta chains are encoded for by an Ig-like rearranging gene. This delta chain polymorphism significantly enhances the potential diversity of TCR-gamma/delta, which may be of importance for a better understanding of the putative ligand(s) recognized by this receptor.

Identification of a T3-associated gamma delta T cell receptor on Thy-1+ dendritic epidermal Cell lines.

The murine epidermis contains a subpopulation of bone marrow-derived lymphocytes that have a dendritic morphology and that express Thy-1 and T3 cell-surface antigens but not other markers (L3T4 or Lyt-2) characteristic of mature peripheral T lymphocytes. An alternative type of T cell receptor was earlier identified on a subpopulation of murine thymocytes with a similar phenotype (T3+, L3T4-, Lyt-2-), but not on peripheral murine T lymphocytes. Two independently derived Thy-1+, L3T4-, and Lyt-2- dendritic cell lines of epidermal origin that express a T3-associated disulfide-linked heterodimer composed of a 34-kilodalton gamma-chain and 46-kilodalton partner (the delta chain) have now been identified. Analysis of N-linked glycosylation revealed that this receptor is similar to that detected on thymocytes. These results demonstrate that Thy-1+ dendritic epidermal cell lines can express gamma delta T cell receptors in vitro and suggest that Thy-1+ dendritic epidermal cells express such receptors in vivo. The localization of these gamma delta T cell receptor-expressing cells in the epidermis may be of importance for understanding the function of these receptors.

Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development.

As part of a study of potential vaccines against malaria, the protective efficacy of sporozoite subunit vaccines was determined by using the Plasmodium berghei murine malaria model. Mice were immunized with recombinant DNA-produced or synthetic peptide-carrier subunit vaccines derived from the repetitive epitopes of the Plasmodium berghei circumsporozoite gene, or with radiation-attenuated sporozoites. Immunization with subunit vaccines elicited humoral responses that were equivalent to or greater than those elicited by irradiated sporozoites, yet the protection against sporozoite challenge induced by either of the subunit vaccines was far less than that achieved by immunization with attenuated sporozoites. Passive and adoptive transfer studies demonstrated that subunit vaccines elicited predominantly antibody-mediated protection that was easily overcome whereas irradiated sporozoites induced potent cell-mediated immunity that protected against high challenge doses of sporozoites. These studies indicate that new strategies designed to induce cellular immunity will be required for efficacious sporozoite vaccines.

Characterization of T cell receptor gamma chain expression in a subset of murine thymocytes.

While much information exists about the structure and function of the clonally distributed T cell receptor (TCR) alpha beta heterodimer, little is known about the gamma protein, the product of a third rearranging TCR gene. An antiserum to a carboxyl-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex were used to identify products of this TCR gene family in a subpopulation of Lyt2-, L3T4- thymocytes. This subpopulation does not express TCR alpha or full-length TCR beta messenger RNA. The gamma chain is a 35-kilodalton (kD) protein that is disulfide-bonded to a 45-kD partner and is associated with the T3 complex. Analysis of the glycosylation pattern of this thymic gamma chain revealed that the major variable region gamma (V gamma) gene transcribed in activated peripheral T cells is absent from this subpopulation. The cells that bear this second T cell receptor may therefore represent a distinct lineage differentiating within the thymus.

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein.

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum.

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum.

The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.

Anticancer efficacy of Magainin2 and analogue peptides.

Linear helical channel-forming peptides structurally similar to the Xenopus-derived antibiotic, Magainin2-amide, were synthesized. Because activity resides in the physicochemical properties of the peptides, an all-D-amino acid as well as an all-L-amino acid sequence were tested for anticancer activity. In vitro activity against carcinoma cells and in vivo efficacy against four murine ascites tumors were determined. The novel peptides proved to have enhanced potency in vitro and in vivo as compared to the parent compound. The 50% inhibitory concentrations against A549 cells for the all-D, the all-L, and Magainin2 were 6, 10, and 110 micrograms/ml, respectively. All three peptides had activity against P388 leukemia, S180 ascites, and a spontaneous ovarian tumor when injected i.p. Increase in life span of over 100% was produced for the analogues in the latter two models. The maximally effective concentrations for the analogues were 20 to 25 mg/kg while Magainin2 required 50-60 mg/kg for in vivo efficacy. The all-D-amino acid peptide, MSI-238, proved as effective as doxorubicin at a more advanced stage of the ovarian tumor and this activity may be attributed to its resistance to proteolytic degradation. Therefore, this class of amphiphilic alpha-helical cationic peptides has potential in the peritoneal treatment of ovarian cancer.

Production of heterologous antibodies specific for murine B-cell leukemia (BCL1) immunoglobulin by immunization with synthetic peptides homologous to heavy chain hypervariable regions.

Three peptides homologous to each heavy chain hypervariable region expressed by murine B-cell leukemia, BCL1, were synthesized in vitro by solid phase peptide synthesis. All three synthetic peptides elicited responses in rabbits which were immunized with synthetic peptide or synthetic peptide conjugated to the carrier keyhole limpet hemocyanin. Six individual rabbits were immunized, five of which responded by producing antisera which react specifically in radioimmunoassay with the synthetic peptide used as immunogen. One antiserum has specificity for the peptide homologous to the first hypervariable region, three antisera have specificity for the peptide homologous to the second hypervariable region, and one has specificity for the peptide homologous to the third hypervariable region. The five antisera with high titers of antibody recognizing synthetic peptide also specifically recognize native immunoglobulin M secreted by BCL1 tumor cells as demonstrated by immunoprecipitation followed by sodium dodecyl sulfate: polyacrylamide gel electrophoresis and autoradiography. These five antisera do not show reactivity with immunoglobulin secreted by spleen cells from normal BALB/cAn mice or by B-cells from unrelated tumors and cell lines. However, as determined by absorption experiments, the majority of antibodies in each antiserum are directed against the respective synthetic peptide, and only a small portion are reactive with native immunoglobulin M. Nonetheless, these results indicate use of synthetic peptides as a potential alternative source of immunogen for production of antitumor antibody.

Evidence for the association of I-A and I-E molecules in d-haplotype mice.

Previous studies using sequential immunoprecipitation indicated the existence of two I-E molecules in d-haplotype mice. Using N-terminal amino acid radiosequence analysis, we have shown that the sequence of the alpha- and beta-chains from the I-E molecule, which is immunoprecipitated by a monoclonal antibody (14.4.4S), is consistent with that determined for the gene of the I-Ed molecule. The material, which is immunoprecipitated by an anti-I-Ed alloantiserum [(B10 X D2.GD)F1 anti-B10.D2] after preclearing with 14.4.4S, has a N-terminal amino acid sequence consistent with it being a mixture of the I-Ed and I-Ad alpha- and beta-chains. The amount of I-Ad varied with each preparation, ranging from 15 to 40% of the total. Nonequilibrium pH gradient gel electrophoresis fractionation of the beta-chain pool precipitated by the alloantiserum yielded two molecules. One molecule had an Ad beta N-terminal amino acid sequence and the other had an Ed beta N-terminal sequence. The existence of antibodies cross-reactive with I-A in the anti-I-E alloantiserum were ruled out because this antiserum could not immunoprecipitate any I-Ad molecules from a D2.GD spleen cell lysate. Therefore, we conclude that the alloantiserum is recognizing determinants that are formed by an interaction between I-Ad and I-Ed molecules or their subunits.

Influenza basic polymerase 2 peptides are recognized by influenza nucleoprotein-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) play an important role in limiting viral infections and in eradicating virus from host tissues. Recent progress in understanding the processing and presentation of viral antigens to CTL indicates that the CTL antigen receptor recognizes peptides derived from viral proteins that are bound to an antigen binding groove present in class I major histocompatibility complex (MHC) molecules. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccinia recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that while screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.

H-2K molecules have two different C-termini, one of which is K-region specific.

Amino acid sequences encoded by exon 8 of the H-2K and H-2D/L genes appear to be locus specific. The majority of H-2Kb molecules contain 10 amino acids that are derived from exon 8. In contrast, the H-2Db, -Dd and -Ld molecules contain only one amino acid which is encoded by exon 8, even though the genetic information exists to encode 10 amino acids analogous to those encoded by the majority of H-2Kb transcripts. We have produced a rabbit anti-peptide serum reactive with the exon 8 encoded sequence of H-2Kb (alpha K-C) that specifically immunoprecipitates a molecule of 45 K mol. wt from spleen cell lysates of b, d, p, q and k haplotype mice. Further analysis by Western blots indicated that virtually all mouse strains express a 45 K protein reactive with alpha K-C. In sequential immunoprecipitations of spleen cell lysates from b, q, p and d haplotype mice using alpha K-C followed by H-2K or H-2D private specificity alloantisera, the anti-peptide serum removed nearly all of the molecules reactive with the anti-H-2K alloantisera (except in the k haplotype) and none of the molecules reactive with the anti-H-2D serum. In addition, no D-region molecules possessing a long C-terminal sequence were detected with an antiserum directed against a representative D-region long C-terminal peptide. We conclude that even though the genetic information for an extended exon 8 exists in K, D and L locus genes, apparently only K-region molecules are expressed with such a C-terminus. Furthermore, in most haplotypes the great majority of H-2K molecules are produced using long exon 8; however, H-2Kk is produced mostly from short exon 8. The absence or presence of key adenosine residues is predicted to be responsible for the variability in class I exon 8 splicing.

Modulation of membrane activity of amphipathic, antibacterial peptides by slight modifications of the hydrophobic moment.

Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (mu) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an alpha-helical conformation when bound to phospholipid vesicles. Analogs with increased mu were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.

Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides.

The hydrophobicity (H), hydrophobic moment (mu) and the angle subtended by the positively charged helix face (phi) of a set of model and magainin 2 amide peptides with conserved charge and helix propensity have been shown to be effective modulators of antibacterial and haemolytic activity. Except peptides of low hydrophobicity which are inactive, changing the parameters has little influence on the activity against Gram-negative bacteria, thus revealing the dominance of electrostatic interactions for the effect. However, the increase of H, mu and phi substantially enhances haemolytic and Gram-positive antibacterial activity and is related to a reduction of peptide specificity for Gram-negative bacteria.