Cell Recognition Sites Research Articles

Protease-activated lymphoid cell and hepatocyte recognition site in the preS1 domain of the large woodchuck hepatitis virus envelope protein.

A site capable of strictly host- and cell type-specific recognition was identified in the preS1 domain of woodchuck hepatitis virus (WHV) through the use of antipeptide antisera generated against the extreme N-terminal fragment of the large virus envelope protein. The crucial determinant of this binding site was mapped to amino acids 10-13. Although a synthetic analogue of the site was highly immunogenic, natural WHV envelope did not display the site activity unless it was modified by proteolysis or acidic pH treatment, indicating an internal location of the determinant in viral envelope. Synthetic peptides encompassing the sequence of this site bound woodchuck lymphoid cells and hepatocytes in a species-restricted manner which followed characteristics of a specific ligand-receptor interaction, although their ability to interact with lymphoid cells was considerably greater than that for hepatocytes. In WHV-infected animals, a natural antibody to the identified cryptic cell-binding site arose independently of that directed against epitopes of unmodified virus envelope and its appearance constituted the earliest immunovirological indicator of virus invasion. Our results demonstrated that the preS1 domain of the large WHV envelope protein is endowed with the species- and cell type-specific recognition site which is protected against antibody surveillance by the natural tertiary structure of the protein and we suggest that proteolytic cleavage is required to induce the binding activity.

The Novel Recognition Site in the C-Terminal Heparin-Binding Domain of Fibronectin by Integrin α4β1 Receptor on HL-60 Cells

The hematopoietic cell recognition sites of human fibronectin (FN) are the Arg–Gly–Asp–Ser (RGDS) sequence recognized by widely distributed integrin receptor α5β1 and the type III connecting segment (III CS) containing two cell-binding sites, designated CS1 and CS5, that are recognized by the α4β1 receptor. The C-terminal heparin-binding domain of FN (Hep II) has recently been demonstrated to support adhesion of α4β1-dependent melanoma cells [A. P. Mould and M. J. Humphries (1991)EMBO J.10, 4089–4095]. Previously we demonstrated that this region of FN mediated binding of FN to HL-60 cells (acute promyelocytic leukemia cell line) by direct interaction independently of RGD and CS1 [H. Fujitaet al.,(1995)Exp. Cell Res.217, 484–488]. In this study we have characterized a novel site in the Hep II region for binding to HL-60 cells. α4β1 and α5β1 were expressed on HL-60 cells, while α2β1 and α3β1 were not present, as shown by flow cytometry using monoclonal antibodies specific for the different integrins. Anti-α4β1 (P4C2) and anti-β1 (JB1a) antibodies inhibited binding of a 29-kDa dispase-digestive fragment of FN to HL-60 cells. This fragment contains the C-terminal heparin-binding domain of FN but lacks CS1 and CS5. Only the peptide representing the sequence from Val1866to Arg1880, designated E1, inhibited the binding of the 29-kDa fragment to HL-60 cells. The active region of this peptide was a sequence of Thr–Asp–Ile–Asp–Ala–Pro–Ser (TAI- DAPS), which is homologous to Leu–Asp–Val–Pro–Ser (LDVPS) derived from the active site of CS1. Furthermore, labeled E1 peptide directly bound to HL-60 cells. The anti-α4β1 antibody (P4C2) inhibited this interaction. These results indicate that the site of binding to hematopoietic cells is present in the Hep II region of FN and the definition of the chemical structure of FN clarifies a fundamental mechanism of cell invasion of the extracellular matrix.

Hybrid hepatitis B virus core antigen as a vaccine carrier moiety: I. Presentation of foreign epitopes

Hepatitis B virus (HBV) core antigen (HBcAg) is a highly immunogenic subviral particle. Here, we review recent progress in the use of HBcAg as a carrier moiety for heterologous epitopes. To define surface exposed and immunogenic insertion sites for foreign epitopes in HBcAg, peptidic epitopes representing binding sites for virus neutralizing antibodies on the HBV surface antigens were inserted at different positions within HBcAg using genetic engineering in an Escherichia coli expression system ( Schödel et al. (1992) J. Virol. 66, 106–114). While fusion to the N-terminus required a linker to become surface accessible, both fusion to the N-terminus and to the C-terminus was compatible with particle assembly and preserved the native antigenicity and immunogenicity of HBcAg. Fusion to an immunodominant internal site of HBcAg reduced the HBcAg immunogenicity and antigenicity and most drastically enhanced the immunogenicity of the inserted foreign epitope. This internal site of HBcAg was used to express circumsporozoite antigen (CS) repeat epitopes of two rodent malaria parasites and of Plasmodium falciparum (Schödel et al. (1994b) J. Exp. Med. 180, 1037-1046 and Schödel et al. (1995a) 95th ASM General Meeting, Washington DC, Abstr. E61). When purified from recombinant Salmonella typhimurium, the hybrid HBcAg-CS proteins were particulate and displayed CS antigenicity as well as reduced HBc antigenicity, as compared to native HBcAg. Immunization of several mouse strains with HBcAg-CS hybrid particles resulted in high titered serum anti-CS antibodies representing all murine IgG isotypes. Immunization of mice with HBcAg or HBcAg-CS particles formulated on alum, complete Freunds or incomplete Freunds adjuvant resulted in equivalent anti-CS and anti-HBc serum antibody titres. The possible influence of carrier-specific immunosuppression was examined and pre-existing immunity to HBcAg did not significantly alter the immunogenicity of hybrid HBcAg particles suggesting that they would be useful carrier moieties for repeated immunizations against multiple haptens or in immune subjects after HBV infection. Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site. This indicates that the internal amino acid position in HBcAg is permissive for the inclusion of heterologous functional T helper as well as B cell epitopes. BALB/c mice immunized with HBcAg-CSl were protected against P. berghei challenge to 90% and 100%, respectively, in two independent experiments.

Synthetic Peptides Coupled to the Lipotripeptide P 3CSS Induce in vivo B and T helper Cell Responses to HIV-1 Reverse Transcriptase

To evaluate the ability of the lipotripeptide P 3CSS to increase peptide-specific immune responses in vivo, we immunized mice from different inbred strains (BALB/c, C3H/HeJ, C57BL/6) with the 22-mer lipopeptide conjugates P 3CSS-[RT-(522-543)] and P 3CSS[RT-(528-549)] of HIV-1 reverse transcriptase (RT) which included an immunodominant T h epitope [ i.e. RT-(528-543)] characterized previously. Analysis of T and B cell responses to these lipopeptide conjugates indicated that specific T h responses could be readily induced in vivo. The peptide segments could also efficiently prime mice for secondary recognition of native RT. The use of shorter peptides permitted a delineation of the minimal T cell recognition site of this RT C-terminal region [ i.e. RT-(528-540)]. Close to this T cell epitope we identified a B cell determinant containing the motif EQVD [RT-(546-549)] which was recognized in three different strains of mice (H-2 b, H2 d and H-2 k). A comparison with X-ray analysis of the C-terminal region of HIV-1 reverse transcriptase indicated exposed positions of these T h and B cell epitopes. Both the presence of T and B cell sites and its limited polymorphism make the region RT-(528549) a promising candidate for vaccine design. The use of the P 3CSS adjuvant/carrier principle as a nontoxic adjuvant may be of major importance in the development of vaccines applicable to humans.

The use of collagen-based model peptides to investigate platelet-reactive sequences in collagen.

Simple collagen-like peptides comprising a repeat Gly-Pro-Hyp sequence are highly platelet-reactive when presented to platelets in triple-helical and polymeric form. This activity is not mediated by the platelet collagen receptor integrin alpha 2 beta 1. This may imply the existence of an intrinsic platelet reactivity associated with the collagen triple helix as such or perhaps that the Gly-Pro-Hyp sequence in collagen serves as a specific cell-recognition site. In our view this basic alpha 2 beta 1-independent reactivity is modulated by the presence in collagen of sequences that may either enhance or diminish the interaction with platelets. Inhibition studies with short linear peptides have allowed the tentative identification of sequences in collagen such as XPGEP(Q)GPX and D(N)GE(Q)X that may promote the activation of platelets and so enhance collagen-platelet interaction. Sequences serving as integrin alpha 2 beta 1-binding sites may also promote platelet reactivity by permitting interaction with the collagen receptor. Using triple-helical peptides based on the sequence of the platelet-reactive collagen type III fragment alpha 1(III)CB4, we have been able to locate an alpha 2 beta 1-binding site in collagen type III within a 30-mer sequence representing residues 508-537 of the alpha 1(III) constituent alpha-chain. Despite their alpha 2 beta 1-independent platelet reactivity, signalling by the (Gly-Pro-Hyp)n-based peptides shows many features in common with signalling by collagen fibers, including activation of p72SYK and p125FAK the latter of which has until now been considered a specific consequence of ligand binding to alpha 2 beta 1.

The hepatitis nucleocapsid as a vaccine carrier moiety.

The "carrier effect," defined as the provision of T cell recognition sites physically linked to B cell epitopes in order to provide Th cell function for antibody synthesis, is well known. Peptides, proteins, and more recently particulate protein antigens have been used for this purpose. The hepatitis B core antigen represents a highly immunogenic antigen in humans as well as in experimental animal models. Studies in mice have provided insight into this enhanced immunogenicity. For example, HBcAg directly activates B cells (i.e., T cell independence), HBcAg elicits strong T cell responses, and HBcAg is efficiently processed and presented by antigen presenting cells (APCs). These characteristics suggested that HBcAg may be an ideal carrier moiety for B cell epitopes requiring additional Th cell function. Therefore, a number of HBV and non-HBV B cell epitopes have been chemically linked or fused by recombinant methods to HBcAg as a method to increase immunogenicity with significant success. We have designed bacterial expression vectors that allow insertion of heterologous B cell epitopes at various positions within HBcAg particles and permit efficient purification of hybrid HBcAg particles. Studies of positional effects have demonstrated that an internal insertion into a dominant HBcAg-specific B cell site represents a superior location for enhanced antibody production. Immunogenicity studies have been extended to protection against experimental challenge in several systems. For example, a malaria CS repeat sequence derived from P. berghei was inserted into HBcAg at the internal site, and purified hybrid HBcAg/CS particles were highly immunogenic and protected 100% of experimentally challenged BALB/c mice. This system has also been exploited for purposes of oral vaccination by expressing genes coding for hybrid HBcAg particles in live, avirulent vaccine strains of Salmonella species.

Function and receptor specificity of a minimal 20 kilodalton cell adhesive fragment of fibronectin.

Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti-fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin.

T lymphocyte recognition sites on peripheral nerve myelin P 0 protein

Synthetic peptides corresponding to the extracellular and cytoplasmic domain of bovine (b) or rat (r) peripheral myelin P 0 protein were used to establish a total of 50 short-term T cell lines (TCL) from blood of eight healthy subjects. Despite expressing different HLA-DR and HLA-DQ specificities, one or more TCL (range 1–16) specific for peptide bovine P 0 19–38 could be isolated from the blood of each donor. Therefore, this peptide covers an immunodominant T cell recognition site in humans. However, when testing seven bP 0-19-38-specific TCL derived from blood of two healthy subjects for recognition of the corresponding human P 0 sequence, no TCL showed any proliferative response. Bovine P 0-19-38 differs in only two amino acid residues from the human peptide. This observation stresses the necessity for using homologous antigens when screening for T cell-mediated autoreactivity to myelin antigens in humans. Unexpectedly, we failed to establish a single P 0 peptide-specific TCL from blood of four patients with acute Guillain-Barré syndrome (GBS), in which P 0 is considered a putative target autoantigen. As already suggested by others, this could indicate that T cell responses to P 0 do not play a pathogenic role in all GBS cases. Alternatively, in these four patients neuritogenic P 0-specific T lymphocytes may have been sequestrated to peripheral nerves.

Immunity to malaria elicited by hybrid hepatitis B virus core particles carrying circumsporozoite protein epitopes.

The hepatitis B virus (HBV) nucleocapsid antigen (HBcAg) was investigated as a carrier moiety for the immunodominant circumsporozoite (CS) protein repeat epitopes of Plasmodium falciparum and the rodent malaria agent P. berghei. For this purpose hybrid genes coding for [NANP]4 (C75CS2) or [DP4NPN]2 (C75CS1) as internal inserts in HBcAg (between amino acids 75 and 81) were constructed and expressed in recombinant Salmonella typhimurium. The resulting hybrid HBcAg-CS polypeptides purified from S. typhimurium were particulate and displayed CS and HBc antigenicity, however, the HBc antigenicity was reduced compared to native recombinant HBcAg. Immunization of several mouse strains with HBcAg-CS1 and HBcAg-CS2 particles resulted in high titer, P.berghei- or P.falciparum-specific anti-CS antibodies representing all murine immunoglobulin G isotypes. The possible influence of carrier-specific immunosuppression was examined, and preexisting immunity to HBcAg did not significantly affect the immunogenicity of the CS epitopes within HBcAg-CS1 particles. Similarly, the choice of adjuvant did not significantly alter the immunogenicity of HBcAg-CS hybrid particles. Immunization in complete or incomplete Freund's adjuvant or alum resulted in equivalent anti-HBc and anti-CS humoral responses. Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site. This indicates that the internal site in HBcAg is permissive for the inclusion of heterologous pathogen-specific T as well as B cell epitopes. Most importantly, 90 and 100% of BALB/c mice immunized with HBcAg-CS1 particles were protected against a P. berghei challenge infection in two independent experiments. Therefore, hybrid HBcAg-CS particles may represent a useful approach for future malaria vaccine development.

Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B.

Four monoclonal antibodies (mAbs) were produced binding to four nonoverlapping epitopes on the superantigen staphylococcal enterotoxin B (SEB). The mAbs were tested for their ability to detect SEB bound to major histocompatibility complex (MHC) class II, to inhibit SEB binding to MHC class II, to inhibit SEB stimulation of T cell hybridomas, to bind to various nonfunctional mutants of SEB, and to capture and present SEB and its mutants to T cells in the absence of MHC class II. We concluded that two mAbs, B344 and B327, bound to epitopes not required for superantigen function, one mAb, 2B33, blocked an MHC interaction site on SEB, and the fourth mAb, B87, blocked the T cell recognition site on SEB. Moreover, two mAbs (B344 and 2B33) were capable of presenting SEB, although much less efficiently than APC, to CD4- but not CD4+ T cell hybridomas. The results confirm the functional domains on SEB originally defined by mutation and show that MHC class II is not always an essential component of the superantigen ligand.

Soluble CTLA-4 can suppress autoantibody production and elicit long term unresponsiveness in a novel transgenic model.

Activation of Ag-specific T cells often requires costimulatory signals in addition to the primary signal mediated through the TCR. The CD28-B7 interaction provides one important costimulatory signal. Previous studies have shown that a soluble CD28 homologue, CTLA4lg, binds B7 with high affinity and can inhibit CD28-B7-mediated costimulation in vitro and in vivo. In this study we examined the ability of soluble human CTLA4lg to inhibit autoantibody production in vivo. For this purpose we used a novel transgenic (Tg) model of autoantibody production. Hepatitis B eAg-expressing Tg mice can be induced to produce autoantibody to the circulating autoantigen (HBeAg) by the injection of a T cell recognition site that fails to elicit T cell tolerance in these mice. Autoimmunity in this model is quantitative because serum autoantibody and autoantigen concentration are inversely correlated and easily measurable by ELISA. In this system a single regimen of CTLA4lg treatment significantly suppressed primary autoantibody production and variably led to long term unresponsiveness. Furthermore, in vivo treatment with CTLA4lg inhibited both T cell activation and T cell-B cell interactions.

Human immunodeficiency virus-1 reverse transcriptase immunodominant CD4+ T cell epitopes: a peptide-based multiparametric assessment in the mouse.

We previously identified an immunodominant CD4+ T cell determinant in the carboxy-terminal region of HIV-1 reverse transcriptase (RT528-543). The present study aimed at enumerating all the potential sites of HIV-1 RT recognized by Th cells in the BALB/c (H-2d) mouse model. To achieve this we used a panel of 62 overlapping 15-mer synthetic peptides covering the whole RT sequence to assay the following parameters: (i) immunogenicity in naive BALB/c mice injected either with peptides pools or individual peptides; (ii) antigenicity, as detected by their ability to restimulate in vitro T cells from BALB/c mice primed with native RT; (iii) MHC class II (Ad)-binding capacity as measured by the inhibition of the antigen-specific, Ad-restricted presentation of unfolded apamin (4-Acm) by fixed antigen-presenting cells to Ad/4-Acm-specific, interleukin-2-producing T hybridoma cells; and (iv) the presence of typical or degenerate consensus Ad-binding motifs. The results in this study permitted identification of three novel immunodominant RT mouse CD4+ T cell sites (RT276-290, RT375-389 and RT411-425) located in regions of limited polymorphism among RT from several HIV isolates. Some of these RT segments were found to be in the vicinity of B cell or H-2Kk- or HLA-A2-restricted cytotoxic T lymphocyte epitopes. Finally, the approach used in this study was found to be very efficient for enumerating most T cell recognition sites in a complex protein, a result that would have not been achieved by a single parameter-based analysis.

The role of the ninth and tenth type III domains of human fibronectin in cell adhesion

Fibronectins (FN) contain sites, in addition to the cell recognition site RGD in the tenth type III domain (FIII10), that are required for adhesive activity. The role of FIII10 and the adjacent FIII9 was analysed in functional cell adhesion assays recombinant FIII domains in which the domain boundaries were strictly conserved. FIII9 had no adhesive activity. FIII10, and FIII9 plus FIII10 had less activity than FN, whereas the activity of FIII9-10 was similar to FN. We conclude that FIII9 acts synergistically with FIII10 in cell adhesion, and that this synergy is dependent upon the structural integrity of the FIII9-10 pair of domains.

In vitro T cell immune responses to the preS2 antigen of the hepatitis B virus envelope protein in preS2 + S vaccine recipients. Absence of cross-reactivity of subtypes at a major T cell recognition site.

The immune responses to hepatitis B virus envelope antigen were investigated in 16 vaccine recipients after immunization with a recombinant yeast-derived preS2 + S (adw) vaccine for hepatitis B virus. After the completion of the three-slot immunization series, all vaccine recipients developed antibody to the S domain and anti-preS2 antibody. In vitro proliferative responses to preS2 (120-174) peptide were demonstrated in 10 of 16 vaccine recipients. Although reactivity could be demonstrated through the length of the preS2 peptide, the principal site of proliferative activity was contained within the preS2 (146-165) region of the peptide. The principal T cell reactive site coincides with a region of significant amino acid variability of the different hepatitis B virus serotypes. Cross-reactivity with a serotype (ayw) not present in the preS2 + S vaccine could not be demonstrated at this widely recognized T cell epitope. The low level of cross-reactivity demonstrated in a limited subset of the vaccine recipients was mediated through nondominant T cell reactive sites contained in the relatively conserved preS2 (120-146) region of the molecule. The identification of widely recognized but serotype-specific T cell epitopes in the preS2 region of the hepatitis B virus envelope antigen may be an important consideration in future vaccine development.

The Cell Attachment and Spreading Activity of Vitronectin Is Dependent on the Arg-Gly-Asp Sequence: Analysis by Construction of RGD and Domain Deletion Mutants

The cell attachment activity of vitronectin has been ascnbed to an Arg-Gly-Asp (RGD) sequence near the amino terminus. To verify the importance of the RGD sequence for cell binding, we created RAD and RGE vitronectin mutants and also deleted either the somatomedin B (ΔS-rVN) or heparin (ΔH-rVN) binding domains. These mutants were expressed as fusion proteins, purified using Ni +2 affinity chromatography, and assayed for cell attachment. EAhy.926 cells bound equally well to wild-type, ΔS-rVN, and to ΔH-rVN, but binding to RAD-rVN and RGE-rVN was inhibited by more than 90%. We therefore conclude that the RGD sequence of vitronectin is the most important cell recognition site and that neither the somatomedin B nor heparin domains contribute signficantly to the cell adhesive activity of vitronectin.

Immune recognition of a molecule naturally presented as a monomeric or an oligomeric structure: The model of the human chorionic gonadotropin α subunit

The immune recognition of a molecule naturally presented as a monomeric or an oligomeric structure is analyzed using the human chorionic gonadotropin a subunit (hCG-α) as a model. Indeed, hCG-a circulates as either a free subunit or combined to the β subunit (hCG-β) to form the dimeric hCG hormone. A T cell study was performed in BALB/c (H-2 d) mice which were found to be high responders to hCG-α. Mice were immunized with the free hCG-α or the dimeric hCG α/β, and their lymph node cells were challenged in vitro with either α subunits from different species, hCG or peptides spanning the entire primary structure of hCG-α. Proliferation and IL-2 assays demonstrated that hCG-α-primed lymph node cells responded equally well to hCG-α and hCG α/β, suggesting that both the free and combined hCG-α subunits are processed in a similar way. Among the various synthetic peptides used, only those mimicking the hCG-α (59–92) C-terminus portion were able to stimulate hCG-α-primed lymph node cells, demonstrating that this region contains immunodominant T cell recognition site(s). The hCG-α(23-–43) and (32–59) peptides, although incapable of stimulating T cells primed with hCG-α, elicited a T cell response when used as immunogens. These regions encompassed cryptic epitopes which were not generated during hCG-α processing in H-2 d mice. The T cell epitopes of hCG-α above described as immunodominant or cryptic on the free α subunit, had similar characteristics when the α/β dimer was used as the immunogen. In contrast, T cells primed with peptides mimicking immunodominant sites recognized differently the hCG-α and the hCG α/β antigens. Moreover, the analysis of the B cell response to all the immunogenic hCG-α peptides indicated that they bear B and T cell epitopes as well. Antibodies elicited against the hCG-α (59–92) or (32–59) peptide were capable of recognizing the α subunit in its free form but not in the α/β hCG dimer. Such study deserves attention for the comprehensive mechanisms of the immune response to hCG as well as for the design of anti-hCG vaccines.

T cells in allergic responses to haptens and proteins.

Haptens are defined as compounds that bind to proteins. They may induce irritant reactions that are often accompanied by DTH. This DTH is mediated by CD4+ and CD8+ T cells. Binding of haptens to cell surface proteins of epidermal cells induces irritant reactions. Trapping by APC may induce subsequent activation of hapten-specific T cells. Various data suggest that haptens are highly immunogenic as they bind directly to preformed MHC-peptide complexes on the cell surface of APC, thereby creating new T cell recognition sites. Environmental protein antigens may induce elevated levels of specific IgE in atopic individuals. There is accumulating evidence that aeroallergen-specific IgE is induced by an aberrant subset of allergen-specific TH cells showing a high IL-4/IFN-γ production ratio. There are no indications so far that allergens are aberrantly presented to allergen-specific T cells. Since nonatopic individuals have normal allergen-specific CD4+ T cells, it seems that the atopic state results from a tendency to selective maturation of TH2 cells in response to aeroallergens. The search for the factors that regulate this biased maturation pathway in atopic disease may give new clues for immunotherapy.

Computational simulations of the conformational behaviour of the adhesive proteins RGDS fragment.

Many adhesive proteins present in extracellular matrices and in blood contain the tetrapeptide sequence -Arg-Gly-Asp-Ser- (or RGDS) at their cell recognition site. Since this sequence, or similar ones, was found in many proteins involved in major biological mechanisms, conformational investigations were performed on the RGDS fragment. A preliminary review of available crystal structures indicates that the RxDy sequences exhibit 3 well-defined structural patterns: one corresponding to a strong interaction between the Arg and Asp ionic side chains which are only about 4 A apart, one with the ions separated by about 8 A, and another in which the side chains are further apart (about 11 A). The conformational behaviour of the isolated RGDS fragment was next tackled using sequential building, Monte Carlo and molecular dynamics computational techniques. Analysis of the RGDS sequence conformational possibilities, as simulated in vacuum and in water solution, indicates that they can be classified into several conformational classes, which correspond roughly to the behaviour of the RGDS fragment as observed in protein matrices. This suggests the possibility of understanding the biological role of the RGDS or parent sequences in recognition processes.

Fine specificity of the human T cell response to hepatitis B virus core antigen.

The fine specificity of the human T cell response to the hepatitis B virus core antigen (HBcAg) was investigated in 23 patients with acute hepatitis B virus (HBV) infection using a panel of short synthetic peptides covering the entire core region. An immunodominant T cell epitope which was recognized by all except one patient, was identified within the core sequence 50-69. Two further important T cell recognition sites were represented by the amino acid sequences 1-20 and 117-131, which were stimulatory for the T cells of 69% and 73% of the patients, respectively. T cell recognition of the synthetic peptides was HLA class II restricted because the peptide-induced T cell proliferation was inhibited by anti-HLA class II but not by anti-HLA class I monoclonal antibodies. These findings may be relevant to the development of future preventive and therapeutic strategies against HBV infection.

In vitro unresponsiveness to autologous sequences of the immunopathogenic autoantigen, S-antigen.

Previous analyses of T cell recognition sites on immunopathogenic neural autoantigens have demonstrated, using LEW rats, the functional dissociation of in vitro proliferative responses and the ability to actively induce autoimmune diseases. In experimental autoimmune uveoretinitis, immunization of LEW rats with bovine retinal S-Ag reveals the presence of three immunodominant T cell recognition sites located in regions containing sequence differences between bovine and rat S-Ag. Immune responses of LEW rats to self (rat) and nonself (bovine and human) peptide homologues representing these three sites were compared. The immunodominant sequences of heterologous S-Ag were found to predict new pathogenic T cell recognition sites in the corresponding autologous rat sequence. Furthermore, in vitro proliferative responses to the pathogenic autologous sequences are dramatically diminished relative to the responses of lymphocytes raised to the non-self homologues. A pathogenic T cell line, R858, efficiently transferred disease, but was unresponsive to the autologous S-Ag peptide in proliferation assays. However, responses to autologous peptides were readily detected using nonirradiated splenic APC. Detection of responses to non-self peptides was independent of this radiosensitive Ag-presenting activity. The lack of in vitro proliferative responses to pathogenic autologous sequences by T cells bearing self-specific receptors, contrasted with the strong proliferation induced by non-self peptide homologues, suggests a mechanism of unresponsiveness to self.