Peter J. Delves

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3‐dioxygenase. An undefined factor capable of down‐regulating T‐cell activity has recently been reported [1] as being produced by short‐term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL‐2‐dependent proliferation of mouse CTLL‐2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)‐2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX‐1/COX–2 inhibitors indomethacin and diclofenac, or with the COX‐2‐selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac‐inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL‐2 cells is exerted via the EP4 receptor. Thus, addition of an EP4‐selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL‐2 cells. This may have implications for attempts to selectively manipulate T‐cell responses.

Does hCG or hCGβ play a role in cancer cell biology

The role that hCG might play in the oncogenic process in cancer is certainly complex. We know that the expression of hCG and its beta subunit is a widespread phenomenon which has been described in many cancer subtypes. However, hCGs involvement in breast cancer has been antithetical: the detection of ectopically expressed hCG(β) by breast tumors has been employed as a biomarker of malignancy, and hCG has been proposed as a ligand vehicle for toxic drugs, with the aim of targeting the LH/hCG receptor which is reported to be expressed by malignant breast tissue. However, it has also been proposed that hCG is a protective agent against the development of breast cancer, leading some to advocate hCG administration to non-pregnant women as a prophylactic measure against cancer. Nevertheless, suggestions that hCG is involved in the angiogenesis, metastasis and immune escape that are central to cancer progression - are phenomena which clearly apply to breast cancer. Indeed, a tumor vaccine based upon hCG has very recently been shown to protect against mammary tumors in mice. We propose that this apparent paradox is resolved if the free beta subunit of hCG produced by tumors acts as an autocrine anti-apoptotic and angiogenic growth factor, whilst intact heterodimeric hCG, as in pregnancy, is part of developmental signaling that initiates tissue differentiation (including breast ductal tissue development), and hence reduces the population of stem-like cells which are susceptible to oncogenic factors.

Identification and selective destruction of shared epitopes in human chorionic gonadotropin beta subunit.

The feasibility of producing epitope-specific antigens by mutation of the gene is demonstrated, the aim being to eliminate unwanted surface epitopes yet allowing the natural folding of the protein to maintain the desired epitope(s). The model protein is the beta subunit of human chorionic gonadotropin (hCG beta) which previously has been used as an immunological contraceptive vaccine but has extensive cross-reaction with human luteinizing hormone. Of a series of mutants made, the mutant with substitutions of Glu for Arg 68, Ser for Arg 74, His for Gly 75 and His for Val 79, lost the ability to react with a panel of cross-reacting monoclonal antibodies while retaining the discontinuous and linear epitopes specific to the holo-hormone. In addition, allocation of amino acid residues to established epitope clusters could be made: residues 24, 25, 68 and 71 probably contribute to the cluster termed beta 3, residues 20, 21, 22, 75 and 77 to cluster beta 6 and residue 68 to clusters beta 2, beta 4 and beta 5.

The Role of Glycosylation in Autoimmune Disease

Oligosaccharide structures play a key role in the antigenicity of a number of clinically important antigens such as blood group determinants. Interest in glycobiology has increased dramatically amongst immunologists during the last few years due to the fact that oligosaccharides also play a central role in adhesion and homing events during inflammatory processes (1), comprise powerful xenotransplantation antigens (2), and may provide targets for tumor immunotherapy (3). Additionally, alterations in glycosylation are now known to occur in a number of autoimmune diseases. This review will first discuss some general aspects of protein glycosylation and then explore some of the autoimmune diseases in which the role of glycosylation has been examined.

Comparison of nucleic acid and protein immunization for induction of antibodies specific for HIV-1 gp120

We have compared the antibody response to HIV‐1 gp120 type LAI in mice immunized with either a gp120 expression plasmid or with baculovirus‐derived recombinant gp120 (rgp120) formulated with Freund’s complete adjuvant, TiterMax, Alum, Ribi R‐700, AF‐A or QuilA. DNA immunization resulted in variable levels of antibody, with endpoint titres ranging from 104 to 105, whereas mice immunized with rgp120 mixed with Ribi R‐700, AF‐A or QuilA produced antibody levels with endpoint titres > 105. Both types of immunization failed to elicit antibodies able to recognize denatured rgp120. The V3 region was immunogenic in animals immunized with nucleic acid, whereas only a few animals immunized with recombinant protein produced antibodies specific for V3 or other linear epitopes, irrespective of the adjuvant used. These data suggest that the immunogenicity of gp120 is dependent upon the mode of antigen delivery, and that in vivo expressed gp120 following nucleic acid immunization elicits, at least with respect to V3, an antibody response which more closely reflects that seen following natural infection in man.

CD5 mRNA Expression and Auto‐Antibody Production in Early Human B Cells Immortalized by EBV

A number of studies have suggested that lymphocytes producing polyreactive antibodies belong to the CD5+ B‐cell subset. In this study we have examined CD5 at the cell surface and mRNA levels in EBV‐driven cord blood and fetal liver clones previously characterized in terms of their antibody specificities. We show that EBV‐immortalized cells can express surface CD5, and that some of the clones not expressing surface CD5 express it at the mRNA level. The complete absence of CD5 mRNA in some polyreactive clones is consistent with the proposition that the production of auto‐antibodies and multispecific antibodies is not restricted to the CD5+ B‐cell subset.

Refocusing of B-cell responses following a single amino acid substitution in an antigen

Intranasal immunization of BALB/c strain mice was carried out using baculovirus‐derived human chorionic gonadotrophin (hCG) β‐chain, together with Escherichia coli heat‐labile enterotoxin. Gonadotrophin‐reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross‐reactive antibodies when holo‐hCG is used as an immunogen. In contrast to wild‐type hCGβ, a mutated hCGβ‐chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross‐react with LH. Furthermore, the epitopes utilized in the B‐cell response to the mutated hCGβ shifted away from the immunodominant region of the parent wild‐type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross‐reactive epitope region and towards a normally weakly immunogenic but antigen‐unique area. Similar mutational strategies for epitope‐refocusing may be applicable to other vaccine candidate molecules.

Can epitope-focused vaccines select advantageous immune response?

One of the great success stories of preventive medicine is the achievement of protection against many pathogens by the simple procedure of vaccination. The rationale behind vaccination is to generate a protective immune response and an expanded population of memory cells ready to encounter the infectious agent, which will then elicit a potent secondary immune response. However, the development of effective vaccines against many pathogens has, so far, been unsuccessful. Many vaccines in current use fail to direct the immune response towards the epitopes that will ensure optimal protection. In these circumstances, can vaccines be produced that focus the immune system in a calculated, epitope-specific manner?

Elimination of luteinizing hormone cross-reactive epitopes from human chorionic gonadotropin

The beta-chain of human chorionic gonadotropin (hCG) has been shown to have efficacy in clinical trials when used as a contraceptive vaccine. This hormone is a heterodimer, the alpha-chain being shared with the other members of the glycoprotein hormone family but the beta-chain being unique to hCG. Nevertheless, there is sequence homology between the hCG beta-chain and the beta-chain of human luteinizing hormone (hLH) which results in cross-reactive antibodies being produced following immunization with wild-type hCGbeta. To reduce or eliminate such cross-reactions we generated a number of mutants of the hCGbeta-chain. One mutant (hCGbeta(R68E)), containing an arginine to glutamic acid replacement at position 68, has been expressed as a recombinant protein in High Five insect cells. The recombinant BAChCGbeta(R68E) form of this molecule was used to immunize rabbits and the antibody response compared to the response following immunization with the recombinant wild-type protein BAChCGbeta and with the native hCGalphabeta heterodimer isolated from pregnancy urine. The mutant elicited the production of antibodies which avidly recognize native hCG. Compared to immunization with wild-type hCG, the response showed very little cross reactivity with hLH. This is demonstrated to be due to a radically altered epitope usage in the response to the mutant, which now focuses mainly upon the C-terminal region of the beta-chain.

IGG GLYCOSYLATION IN AUTOIMMUNE-PRONE STRAINS OF MICE

The relationship between increased levels of IgG oligosaccharide chains lacking galactose (GO) and the development of rheumatoid arthritis is unclear. In order to further our understanding of the observed correlation between raised serum GO and arthritis, we have studied GO levels in arthritis prone and non‐susceptible (i.e., non‐arthritis‐prone) mice and the effects on GO of mycobacterial antigens, which have been postulated to play a role in the early events leading to the development of arthritis. We have shown that different age‐matched mouse strains have characteristic “resting” levels of GO which (in six out of seven strains of mice) increase with age. We have also shown that these increases can be enhanced by immunization of arthritis‐prone strains of mice with an adjuvant containing mycobacteria (Freunds complete adjuvant (FCA)), suggesting that deflects in the ability to regulate these GO changes may be related to susceptibility to arthritis.