Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans.

Vaccines have been used as a successful tool in medicine by way of controlling many major diseases. In spite of this, vaccines today represent only a handful of all infectious diseases. Therefore, there is a pressing demand for improvements of existing vaccines with particular reference to higher efficacy and undisputed safety profiles. To this effect, as an alternative to available vaccine technologies, there has been a drive to develop vaccine candidate polypeptides by chemical synthesis. In our laboratory, we have recently developed a technology to manufacture long synthetic peptides of up to 130 residues, which are correctly folded and biologically active. This paper discusses the advantages of the molecularly defined, long synthetic peptide approach in the context of vaccine design, development and use in human vaccination.

A potential role for CD1a molecules on human epidermal Langerhans cells in allogeneic T-cell activation.

The structural similarities of CD1a molecules to major histocompatibility complex (MHC) class I antigens, as well as their expression on epidermal antigen-presenting cells suggest that CD1a molecules might be involved in the cutaneous immune response. In the present study, we investigated the effect of different anti-CD1a monoclonal antibodies (BL6, DMC1, and Na1/34) on T cell proliferation induced by allogeneic epidermal cells in vitro. A significant inhibition of the mixed skin cell-lymphocyte reaction was obtained with BL6 and DMC1 monoclonal antibodies (MoAb), which recognize the same epitope on CD1a molecule. The observed inhibition could not be related to a steric hindrance of MHC class II molecules, because Na1/34 MoAb, which reacts with another epitope on CD1a molecule, had no significant effect. BL6 and DMC1 MoAb interfered with an early event of T-cell activation, as shown by a time-course study. In the presence of these MoAb, the addition of exogenous interleukin 2 did not restore T-cell proliferation. Furthermore, the inhibitory effect of anti-CD1a MoAb was not mediated by a suppressor factor released by Langerhans cells (LC). These present data suggest that CD1a molecule may have an important function in self peptide presentation by human Langerhans cells.

MHC-dependent and -independent activation of human nickel-specific CD8+ cytotoxic T cells from allergic donors.

T lymphocytes are critical effectors in the pathogenesis of contact hypersensitivity. Nickel is the most common contact sensitizer in humans and nickel-specific CD4+ T helper cells have been extensively characterized. Because recent observations have suggested the activation of CD8+ T cells in murine models of contact hypersensitivity, we investigated the existence of CD8+ hapten-specific T lymphocytes in patients with allergy to nickel. Nickel-specific T cell lines were generated from the peripheral blood of three allergic donors. The T cell lines were composed of a majority of CD4+ T cells, but CD8+ T cells were also present and their percentage increased with repeated in vitro stimulations. In addition to nickel-reactive helper T cell-0-type or helper T cell-2-type CD4+ T cell clones, CD8+ T cell clones could be derived from these cell lines and a total of 15 clones were further studied. Cytokine production was evaluated for 11 CD8+ T cell clones that were either cytotoxic T cell-0- or cytotoxic T cell-1-type clones. Additional effector functions were investigated on the complete panel of T cell clones. These CD8+ T cells did not only display hapten-specific proliferation, but also specific cytotoxic activities towards autologous EBV-B cells in the presence of nickel. Two different types of CD8+ T cells were characterized. Most of the clones lysed only autologous targets in the constant presence of nickel; however, one clone was able to lyse numerous targets in the presence of NiSO4, irrespective of the expression of either major histocompatibility complex class I or class II molecules. The characterization of nickel-specific cytotoxic CD8+ T cells with different requirements for nickel-specific target lysis, may have important implications in the development or in the control of human contact hypersensitivity reactions to nickel in vivo.

Functional expression and analysis of a human HLA-DQ restricted, nickel-reactive T cell receptor in mouse hybridoma cells.

Nickel-induced contact dermatitis represents a T cell mediated delayed type hyperreactivity. The elucidation of the molecular basis of T cell activation by Ni2+ ions may serve as a model for the understanding of other metal allergies. We describe here the expression of hybrid T cell antigen receptor (TCR) alpha- and beta-genes, containing rearranged human Ni-reactive variable and mouse constant regions, together with human CD4 in a mouse T cell hybridoma. The resulting hybridoma specifically responds to IL-2 secretion to Ni, but not to other metal ions in the presence of HLA-matched antigen-presenting cells. Loss of CD4 decreases, but does not completely abrogate this reactivity. The restricting HLA-DQ element is identified as consisting of DQA1*0101 and DQB1*0501; however, only some of the B cell lines homozygous for these molecules effectively present Ni to the hybridoma. We interpret these data to show that (i) Ni-reactivity is definitely mediated by alpha beta TCR variable regions; (ii) as for peptide-specific TCR, the CD4 co-receptor enhances Ni-reactivity, but is not absolutely essential; (iii) Ni2+ ions like nominal peptide antigens require HLA (here class II) molecules of the APC for presentation; (iv) the restricting molecule may require a special conformation or the association with a particular type of peptide or an as yet unidentified other surface structure on the antigen-presenting cell for effective Ni-presentation.

T cell immune responses to haptens. Structural models for allergic and autoimmune reactions.

Protein-reactive chemicals, metal salts and drugs, commonly classified as immunological haptens, are major environmental noxes targeted at the immune system of vertebrates. They may not only interfere with this defense system by toxicity alone, but more often by evoking hapten-specific immune responses resulting in allergic and eventually autoimmune responses. Here, we review recent developments in the analysis of the structural basis of hapten recognition, particularly by T lymphocytes, which represent central elements in cell-mediated, as well as in IgE dependent, allergies. A break-through in this field was the finding that T cells detect haptens as structural entities, attached covalently or by complexation to self-peptides anchored in binding grooves of major histocompatibility antigens (MHC-proteins). Synthetic hapten-peptide conjugates were shown to induce hapten-specific contact sensitivity in mice, opening new routes for studying hapten-induced immune disorders.

Isolation and short-term culture of human epidermal langerhans' cells.

The human epidermis consists of a heterogeneous population of cells including keratinocytes in various stages of differentiation, Merkel cells, melanocytes, and Langerhans' cells (LC). The latter account for only around l-3% of the epidermal population, but they represent the major antigen-presenting cells of the epidermis (1).

Characterization of processing requirements and metal cross-reactivities in T cell clones from patients with allergic contact dermatitis to nickel.

Metal ions such as nickel, cobalt, copper and palladium are known to be potent sensitizers in humans, but the antigenic determinants created by these metals as well as the mechanisms of recognition by specific T cell clones are still not elucidated. In this paper, nickel-specific T lymphocyte clones were isolated from four patients exhibiting contact dermatitis to this metal. A panel of 42 independent T cell clones was studied. They were shown to recognize nickel in the context of major histocompatibility complex (MHC) class II molecules and to belong to the CD4 subset. Using fixed autologous Epstein-Barr virus-transformed B cells as antigen-presenting cells (APC), we could distinguish two distinct groups of T cell clones on the basis of processing requirements: 40% of the T cell clones were strictly processing dependent, whereas the remaining 60% could proliferate in response to nickel even in the presence of glutaraldehyde-fixed APC. Furthermore, we present arguments indicating that individual Ni-specific T cell clones cross-react with some transition metals (e.g. Cu or Pd), but not with others (e.g. Co, Cr and Pt), presented by identical MHC class II molecules. These results thus provide an explanation for the multiple metal-reactivities observed in vivo in human patients: they indicate that for Cu and Pd, these co-reactivities in vivo might be due to cross-reactivity at the clonal level. Our findings also suggest that this is not the case for cobalt allergy, which might result from cosensitization of the patient to cobalt in addition to nickel.

The antigen presenting capacity of human epidermal Langerhans cells.

Dendritic cells (DC) represent a distinct system of antigen-presenting cells (APC) that function to initiate primary T cell responses and are found as a trace population in both lymphoid and non-lymphoid organs. This paper is focused on some properties of epidermal Langerhans cells (LC), the best characterized non-lymphoid DC. The phenotypic markers of LC are reviewed and their immunological function is presented in view of some recent findings partly obtained in our laboratory.

TCR reactivity in human nickel allergy indicates contacts with complementarity-determining region 3 but excludes superantigen-like recognition.

Nickel is the most common inducer of contact sensitivity in humans. We previously found that overrepresentation of the TCRBV17 element in Ni-induced CD4+ T cell lines of Ni-allergic patients relates to the severity of the disease. Amino acid sequences of these beta-chains suggested hypothetical contact points for Ni2+ ions in complementarity-determining region (CDR) 1 and CDR3. To specifically address the molecular requirements for Ni recognition by TCR, human TCR alpha- and beta-chains of VB17+ Ni-reactive T cell clones were functionally expressed together with the human CD4 coreceptor in a mouse T cell hybridoma. Loss of CD4 revealed complete CD4 independence for one of the TCR studied. Putative TCR/Ni contact points were tested by pairing of TCR chains from different clones, also with different specificity. TCRBV17 chains with different J regions, but similar CDR3 regions, could be functionally exchanged. Larger differences in the CDR3 region were not tolerated. Specific combinations of alpha- and beta-chains were required, excluding a superantigen-like activation by Ni. Mutation of amino acids in CDR1 of TCRBV17 did not affect Ag recognition, superantigen activation, or HLA restriction. In contrast, mutation of Arg95 or Asp96, conserved in many CDR3B sequences of Ni-specific, VB17+ TCR, abrogated Ni recognition. These results define specific amino acids in the CDR3B region of a VB17+ TCR to be crucial for human nickel recognition. CD4 independence implies a high affinity of such receptor types for the Ni/MHC complex. This may point to a dominant role of T cells bearing such receptors in the pathology of contact dermatitis.

Antigen contacts by Ni-reactive TCR: typical alphass chain cooperation versus alpha chain-dominated specificity.

VB17(+) TCR dominate in Ni-driven T cell cultures from highly Ni-sensitized patients. Using transfection of TCR from three CD4(+), VB17(+), Ni-specific human T cell clones, we studied their Ni-MHC contacts by site-directed TCR mutation and combination of alpha and ss chains between different TCR. All three TCR exhibited N-nucleotide-determined Arg-Asp motifs in their CDR3-ss sequences. Two of them were specifically restricted to HLA-DR13, while the third one accepted a variety of HLA-DR alleles. The highly similar alpha or ss chains of the DR13-restricted TCR were interchangable without loss of specificity, but alpha or ss chains of other TCR were not tolerated. Mutations of their Arg-Asp motif revealed loss of reactivity upon exchanging Asp for Glu or Ala and of Arg for Ala but not of Arg for Lys or the Ni binding His. Reactivity was also destroyed by mutation of alpha chain position 51, proposed as a general contact site for MHC. Hence, in these two TCR the Arg-Asp motif is clearly involved in contacting Ni-MHC complexes, and close cooperation between alpha and ss chain is required. In contrast, the third TCR retained Ni reactivity upon mutation of alpha chain position 51 or of its ss chain Arg-Asp motif, which rather affected the pattern of DR cross-restriction. Moreover, its alpha chain paired with various ss chains from other, even mouse TCR, irrespective of their specificity, retaining Ni reactivity as well as promiscuous HLA-DR restriction. This preponderance of an alpha chain in defining specificity indicates fundamental differences in Ni interactions of individual TCR and implies that ss chain similarities may not necessarily result from antigen selection.

Interleukin-10 inhibits the primary allogeneic T cell response to human epidermal Langerhans cells.

In this study, we analyzed the effect of interleukin-10 (IL-10) on the primary allogeneic T cell response induced by human Langerhans cells (LC), the dendritic cells from epidermis. We showed that IL-10 strongly inhibited the T cell response, provided it was added at the beginning of the mixed epidermal cell lymphocyte reaction (MELR). Proliferation of both CD4+ and CD8+ T cell subsets was affected by the cytokine. An inhibitory effect of IL-10 on human LC allostimulatory function was evidenced by the fact that IL-10-preincubated LC, but not IL-10-preincubated T cells, can display inhibitory effect. LC treatment with IL-10 partially inhibited the increase of HLA-DR expression on cultured LC as the percentage of highly positive HLA-DR cells was lower than that observed in the absence of the cytokine. IL-10 inhibited T cell alloreaction induced by 2-day-cultured human LC which constitutively display high levels of HLA class II, as well as ICAM-1 and LFA-3 antigens. This suggests that the suppressive effect of the cytokine was not merely related to an impaired up-regulation of these molecules. Addition of IL-1 during the MELR potentiated the allogeneic T cell proliferation and could reverse, at least partly, the inhibitory effect of IL-10. Collectively, these data indicate that IL-10 can prevent the alloreaction induced by human dendritic cells, providing new insights into the potential clinical use of this cytokine.

Dominance of the BV17 element in nickel-specific human T cell receptors relates to severity of contact sensitivity.

Hypersensitivity to nickel (Ni) represents the most common manifestation of contact allergy in humans. The role of metal-specific T cells in this disease is well established, but the molecular interactions involved in their activation are poorly understood. We examined the T cell receptor (TCR) repertoire in T cells activated with either NiSO4 or NiSO4-treated human serum albumin from six allergic patients. For the three most hyperreactive donors, we found a strong over-representation of the TCR BV17 element. TCR sequencing for one of these donors revealed an additional skewing for AV1 as well as a selection for an N region encoded argine at position 95 of the BV17 complementarity determining region (CDR)3. Since Arg is not known to participate in Ni complexing, we suppose that this selection is driven by contacts with peptide rather than nickel. However, the CDR1 of BV17 contains a unique combination of amino acids (HDA) that bears similarities to known motifs in Ni-binding proteins or peptides. We therefore propose that the severe hypersensitivity reactions found in BV17 over-expressors may be the result of Ni2+ ions bridging the germ-line-encoded BV17 CDR1 loop to corresponding sites in the major histocompatibility complex/peptide complex and thereby creating a superantigen-like enhancement of weak TCR-peptide contacts.

In vitro primary sensitization of hapten-specific T cells by cultured human epidermal Langerhans cells--a screening predictive assay for contact sensitizers.

BACKGROUND: The need to develop predictive tests which could identify potential allergens has been recognized for many years. There is as yet no accepted in vitro method for the assessment of contact sensitizers. OBJECTIVE: We have tested the ability of a range of contact allergens to induce in vitro primary sensitization of autologous T cells. METHOD: T-cell proliferation induced by haptens using 2-day cultured human Langerhans cells as antigen-presenting cell was assessed by 3H thymidine incorporation. Antigen specific stimulation was calculated as stimulation indexes. RESULTS: Strong allergens induced in vitro a primary T-cell response in all (trinitrophenyl, TNP: 13/13) or in the majority (fluorescein isothiocyanate, FITC: 7/10) of experiments. An irritant, sodium dodecyl sulfate (SDS), failed to generate a significant T-cell proliferation in any of the experiments (0/10). We obtained a significant lymphoproliferative response to weak sensitizers only in a limited number of experiments: (coumarin: 1/12, citronellal: 0/10, hydroxycitronellal: 2/8). p-Phenylenediamine (PPDA), a prohapten and highly sensitizing chemical in vivo, generated primary sensitization in vitro in only one of six experiments, while Bandrowski's base (BB), a metabolization product of PPDA induced a significant T-cell response in all six experiments. CONCLUSION: The present in vitro model allows discrimination between two groups of substances: strong contact sensitizers (TNP, FITC, BB) on the one hand and weak sensitizers (coumarin, citronellal and hydroxycitronellal) and irritants (SDS) on the other hand. It could be used as a screening in vitro assay to eliminate strong contact allergens before further predictive animal tests have to be performed.

Kinetics of CXCR4 and CCR5 up-regulation and human immunodeficiency virus expansion after antigenic stimulation of primary CD4(+) T lymphocytes.

The chemokine receptors CCR5 and CXCR4 are coreceptors for the human immunodeficiency virus (HIV) and determine the cell tropism of different HIV strains. Previous studies on their regulation were performed under conditions of unspecific T-lymphocyte stimulation and provided conflicting results. To mimic physiologic conditions, highly purified primary Staphylococcus enterotoxin B (SEB)-reactive CD4 T lymphocytes were stimulated in the presence of autologous antigen-presenting cells and the kinetics of CCR5 and CXCR4 surface expression and HIV replication were studied. Both chemokine receptors were transiently up-regulated with maximal expression at day 3 after stimulation. The stimulated T cells were equally susceptible to productive infection with R5-and X4-tropic virus strains. Thus, antigenic stimulation of T cells promotes efficient replication of both, T cell-tropic and macrophage-tropic HIV. (Blood. 2000;96:1853-1856)

In vitro primary sensitization and restimulation of hapten-specific T cells by fresh and cultured human epidermal Langerhans' cells.

We examined the capacity of human Langerhans's cells (LC) to sensitize autologous T cells to the trinitrophenyl hapten (TNP) in vitro. Two-day cultured Langerhans' cells, but not freshly prepared Langerhans' cells, can induce in vitro primary proliferative reactions to the TNP hapten. Using a CD45RA+ naive T-cell subset, similar results were found, therefore making the possibility of a previous in vivo T-cell contact with the hapten unlikely. The primary in vitro response was strongly inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I and II, CD4 antigens and ICAM-1 and LFA-3 adhesion molecules. Furthermore, we found that fresh LC can prime T cells to TNP, as revealed by a significant secondary T-cell proliferation after restimulation of the recovered T lymphocytes by fresh hapten-modified autologous LC. Nevertheless, the ability of these fresh LC to stimulate in vitro secondary hapten-specific T-cell proliferation was very limited in comparison with that of 2-day incubated Langerhans' cells. After secondary stimulation with TNP-cultured LC, sensitized T cells could be non-specifically expanded without losing hapten specificity. The TNP-specific T-cell lines were mostly of the CD4+ phenotype. The present findings extend previous studies in the mouse, showing that culture LC are potent antigen-presenting cells (APC) in primary hapten-dependent proliferation assays. Furthermore, this in vitro priming assay, using cultured human Langerhans' cells as APC, might be useful to analyse the early steps of T-cell sensitization and subsequently to develop in vitro predictive tests allowing detection of sensitizing compounds.

Long synthetic peptides as biologically active proteins: the example of the chemokines.

Chemokines constitute an expanding protein family of over 40 members which exhibit a wide variety of biological activities and are involved in many normal physiological processes, such as cellular migration, differentiation and activation, but also in pathological situations, such as inflammation and metastasis. Over the last few years, we have developed methods to manufacture long synthetic peptides of up to 130 residues, and to achieve the formation of native-like cysteine pairings. This ability prompted us to undertake the total chemical synthesis of chemokines. So far, we have successfully produced over 30 chemokine species, which exhibit biological activities similar to, or greater than, those reported by others. Chemical synthesis offers a clear advantage over recombinant technologies for the introduction of fluorochromes and haptens at molecularly defined positions. In addition, approval of chemically synthesized products for use in humans is straightforward compared with material produced by biological methods.

CD40 ligation of human keratinocytes inhibits their proliferation and induces their differentiation.

While CD40-CD40 ligand interactions are known to regulate B cell proliferation and differentiation, much less is known about the role this receptor plays on other cell types, especially those of nonhemopoietic origin. We report here that CD40 is expressed in normal human epidermis in situ, especially on the basal cell layer, and that it is maintained on cultured epidermal basal cells. Immunoprecipitation and SDS-PAGE analysis confirms that CD40 expressed by epidermal basal cells is immunologically related to the B cell CD40. IFN-gamma up-regulates CD40 expression on cultured keratinocytes, whereas other proinflammatory cytokines, such as IL-1 or TNF-alpha, have little effects. Using CD40-ligand-transfected L cells (CD40Lc), we demonstrated that CD40 triggering results in an enhanced secretion of both IL-8 and TNF-alpha by cultured epidermal basal cells, suggesting that CD40-CD40L interactions may play a role in amplifying the cutaneous inflammatory reactions. More importantly, we found that keratinocyte proliferation was significantly inhibited when the cells were grown on CD40Lc, as compared with CD32-transfected, or nontransfected, L cells. This inhibitory effect can be reversed substantially by pretreatment of keratinocytes with anti-CD40 mAb. In addition, inhibition of proliferation could be obtained by adding a soluble form of CD40 ligand to the keratinocyte cultures. Interestingly, inhibition of keratinocyte proliferation on CD40Lc correlates with differentiation of the cells, as assessed by morphologic analysis and increased profilaggrin content. Collectively, these results demonstrate that CD40 is expressed and functional on human epidermal basal cells and that, on these cells, CD40 ligation may be a signal for limitation of cell growth and induction of differentiation.

A recombinant rubella virus E1 glycoprotein as a rubella vaccine candidate.

A recombinant rubella virus E1 (rE1) glycoprotein was produced and some of its chemical and immunological features were characterized. Two animal models were then used to establish that the rE1 glycoprotein and rubella virus particles shared antigenic and immunogenic properties. In the first one, sera from rE1 glycoprotein-immunized BALB/c mice neutralized in vitro rubella virus infection. In the second model, severe combined immune deficient (SCID) mice implanted with tonsil fragments from rubella immune donors and immunized with rE1 glycoprotein produced human anti-rubella virus antibodies. Altogether, these results showed that immunization with rE1 glycoprotein elicited neutralizing anti-rubella virus antibodies. This study thus indicated that the rE1 glycoprotein could constitute a non-replicating rubella vaccine.