Peter Steinberger

B7-H1 (Programmed Death-1 Ligand) on Dendritic Cells Is Involved in the Induction and Maintenance of T Cell Anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-α, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.

Methods for the generation of chicken monoclonal antibody fragments by phage display

Phage display has become an important approach for the preparation of monoclonal antibodies from both immune and nonimmune sources. This approach allows for the rapid selection of monoclonal antibodies without the restraints of the conventional hybridoma approach. Although antibodies to a wide variety of antigens have been selected using phage display, some highly conserved mammalian antigens have proven to be less immunogenic in mammalian animals commonly used for immunization. In order to optimize methods for constructing chicken immunoglobulin phage display libraries in the pComb3 system, we have immunized chickens with the hapten fluorescein, and generated combinatorial antibody libraries from spleen and bone marrow RNA. Herein we present methods for the isolation of scFv, diabody and Fab fragment libraries from chickens. Chicken Fab fragment libraries are constructed using human constant regions, facilitating detection with readily available reagents as well as humanization. Analysis of the selected V-genes revealed that gene conversion events were more extensive in light-chain variable region genes as compared to heavy-chain variable region genes. In addition, we present a new variant of the pComb3 phage display vector system.

Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-γ production.

B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction

B7‐H3 belongs to the B7 superfamily, a group of molecules that costimulate or down‐modulate T‐cell responses. Although it was shown that B7‐H3 could inhibit T‐cell responses, several studies – most of them performed in murine systems – found B7‐H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7‐H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T‐cell subsets. We show that B7‐H3 does not costimulate human T cells. In the presence of strong activating signals, B7‐H3 potently and consistently down‐modulated human T‐cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre‐activated T cells. Furthermore, we demonstrate that B7‐H3–T‐cell interaction is characterised by an early suppression of IL‐2 and that T‐cell inhibition can be reverted by exogenous IL‐2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT‐2) has been recently described as costimulatory receptor of murine B7‐H3 we have extensively analysed interaction of human B7‐H3 with TREML2/TLT‐2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7‐H3.

Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen

Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C‐terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE‐binding capacity as the natural allergen.

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.

The capacity of the TNF family members 4-1BBL, OX40L, CD70, GITRL, CD30L and LIGHT to costimulate human T cells

Activating signals generated by members of the tumour necrosis factor receptor superfamily upon interaction with their cognate ligands play important roles in T‐cell responses. Members of the tumour necrosis factor family namely 4‐1BBL, OX40L, CD70, GITRL, LIGHT and CD30L have been described to function as costimulatory molecules by binding such receptors on T cells. Using our recently described system of T‐cell stimulator cells we have performed the first study where all these molecules have been assessed and compared regarding their capacity to costimulate proliferation and cytokine production of human T cells. 4‐1BBL, which we found to be the most potent molecule in this group, was able to mediate sustained activation and proliferation of human T cells. OX40L and CD70 were also strong inducers of T‐cell proliferation, whereas the costimulatory capacity of human GITRL was significantly lower. Importantly CD30L and LIGHT consistently failed to act costimulatory on human T cells, and we therefore suggest that these molecules might be functionally distinct from the costimulatory members of this family.

Generation and Characterization of a Recombinant Human CCR5-specific Antibody: A Phage Display Approach for Rabbit Antibody Humanization.*

We describe the isolation of a CCR5-specific antibody, ST6, from an antibody phage display library generated from an immune rabbit. ST6 was previously shown to efficiently prevent the surface expression of CCR5 when expressed intracellularly (Steinberger, P., Andris-Widhopf, J., Buhler, B., Torbett, B. E., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A.97, 805–810). Because ST6 has therapeutic potential in human immunodeficiency virus, type 1 disease, its humanization was desired to minimize the potential for immunogenicity. ST6 was humanized using a phage display-based approach. Like the parental rabbit clone, the humanized version ST6/34 efficiently prevented the surface expression of CCR5. The conserved linear peptide epitope bound by these antibodies was mapped using phage display. Both ST6 as well as the humanized anti-CCR5 antibody ST6/34 were produced as complete IgG antibodies and shown to bind to cell surface CCR5.

Immunological and structural similarities among allergens: Prerequisite for a specific and component-based therapy of allergy

It is known that allergic patients are frequently co‐sensitized against different allergen sources. Progress made in the field of allergen characterization by molecular biological techniques has now revealed that sensitization against different allergen sources can be explained as cross‐reactivity of IgE antibodies with structurally and immunologically related components present in these allergen sources. This review defines groups of cross‐reactive plant allergens with significant sequence homology. The similarities among allergens may facilitate allergy diagnosis by using a few representative cross‐reactive allergens to determine the patients IgE reactivity profile (allergogram). According to that typing, a few cross‐reactive allergens, carrying most of the relevant IgE epitopes, may then be selected for patient‐tailored specific therapy.

A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an α helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.