Induction of immunological tolerance to the penicilloyl antigenic determinant--IV. The effect of BPO-oligolysines and cholestanol-bearing BPO-oligolysines on murine IgE responses.

Penta-, deca- and eicosalysine carriers were synthesized in solution and conjugated with benzylpenicillin to give BPO-conjugates of high haptenic density. Each oligolysine conjugate was prepared in two forms--with a free C-terminus and with an esterified C-terminus carrying via a benzylester bridge in essence a lipophilic cholestanol moiety [p-oxymethylbenzylcholestan-3 beta-yl succinate (OSuco group)]. Decalysines that carried a single haptenic BPO group and succinyl groups on the other amino functions were also prepared. Suppression of IgE responses was studied in BALB/c mice. It was found that BPO-specific suppression could be induced by injecting OSuco-bearing deca- or eicosalysine conjugates before immunization with BPO-Asc in A1(OH)3. The pentalysine conjugate was only slightly effective as were all OSuco-deficient conjugates. Ongoing IgE responses were only slightly suppressed and OSuco-bearing conjugates were not more effective than OSuco-deficient derivatives. When the monohaptenic OSuco-bearing decalysine, which exhibited weak tolerogenic effects on primary as well as on ongoing responses, was applied under conditions that favour suppressor T-cell induction, a pronounced unresponsiveness resulted. Direct evidence for suppressor T-cell involvement in the abrogation of anti-BPO responses by OSuco-bearing BPO-conjugates was obtained from cell transfer experiments. The study shows that relatively small haptenic conjugates, the lower limit of effectiveness being approximately represented by decalysine conjugates, may be effective tolerogens depending on the immune status.

Potentiation of the tolerogenicity of benzylpenicilloylated eicosa-L-lysine by conjugation with 4-(hydroxymethyl)benzyl 3 beta-cholestanyl succinate.

It was previously found that amino acid polymers such as oligolysines bearing haptenic groups in high densities efficiently suppress anti-hapten IgE antibody formation. Such conjugates are strong elicitors of anaphylaxis and therefore may not be used for desensitization of drug allergic patients. Here we report on the synthesis and immunological evaluation of benzylpenicilloylated (BPO) eicosa-L-lysines containing none, one, or two lipophilic p-(hydroxymethyl)benzyl cholestan-3 beta-yl succinate (OSuco) groups. The lipophilic derivatives suppress primary as well as ongoing anti-BPO IgE antibody formation in mice much more efficiently than their hydrophilic counterpart. The lipophilic but not the hydrophilic derivatives form stable micelles in water and suppress the antibody formation according to different cellular mechanisms. The relationship between structure, hydrophobicity, and mode of action is discussed.

Oligomerization and ring closure of immunoglobulin E class antibodies by divalent haptens.

Cross-linking of antibodies constitutes a widespread initiation signal for their respective effector functions. Cross-linking IgE-class antibodies provide the triggering signal to mast cells for their degranulation process. To obtain a quantitative insight into these cross-linking processes, the interactions between a DNP-specific monoclonal antibody of the IgE class and a series of divalent DNP haptens with spacers of different length and flexibility have been studied by fluorescence titration experiments. These were analyzed by employing the theoretical model developed by Dembo and Goldstein [Dembo, M., & Goldstein, B. (1978) J. Immunol. 121, 345-353] in a fitting procedure. Equilibrium constants that describe the aggregation and ring-closure processes caused by divalent hapten binding have been used as free parameters. The intrinsic binding constants were determined by fluorescence titrations with corresponding monovalent haptens. The main results are the following: (1) The divalent haptens with a short and flexible spacer [i.e., N alpha, N epsilon-di-(DNP)-L-lysine,meso-bis[(DNP-beta-Ala)amino]succinate, and bis[(DNP-tri-D-Ala)amino]heptane, having a maximal DNP-DNP distance of gamma = 14, 21, and 45 A, respectively] effect aggregation of the antibodies mainly into closed dimers. (2) The divalent hapten family with long and rigid oligoproline spacers di(DNP)-Ahx-Asp-(Pro)n-Lys with n = 24, 27, and 33 (i.e., gamma = 100, 110, and 130 A) causes aggregation of the antibodies predominantly into closed dimers and trimers. The corresponding equilibrium constants of the respective ring-closure processes decrease significantly with longer spacer length. (3) Evidence was found that intramolecularly monomeric ring closure of the IgE antibodies is caused by haptens containing oligoproline spacers with n = 37 or 42 (gamma = 130-150 A). The equilibrium constant of the ring-closure process increases with spacer length. This increase in stability indicates a difference in the imposed strain. Furthermore, the latter results imply that the distance between the two binding sites of the IgE molecule lies in the range dictated by the rigid oligoproline part of the respective hapten's spacer, i.e., 115-130 A. (4) Nearly all oligomeric ring-closure processes proceed relatively slowly with an approximate lower limit of a half-life of 5-10 s. This slowing down of the aggregation and ring-closure processes most probably reflects steric factors.

The introduction of one or two 3 beta-cholestanyl residues into benzylpenicilloyl-eicosa-L-lysines greatly potentiates their tolerogenicity for anti-benzylpenicillol IgE antibody formation.

BALB/c mice were repeatedly immunized with microgram doses of benzylpenicilloylated Ascaris protein(s) (BPO9Asc) in alum. At different stages of the immune response, BPO21 eicosa-L-lysine or two analogs containing one or two hydrophobic p-oxymethylbenzyl-3 beta-cholestanyl succinate (OSuco) groups were injected. When injected early in the immune response, the anti-BPO IgE antibody formation was much more strongly and permanently suppressed by the lipophilic conjugates than by the hydrophilic BPO21 eicosa-L-lysine. A similar, but less marked, suppressive effect was observed on the anti-BPO IgG1 response. By adoptive cell transfer experiments, it was found that the OSuco-containing derivatives induce and act via suppressor T lymphocytes, since this cell-mediated suppression was sensitive to cyclophosphamide or to treatment with anti-Lyt-2.2 antibody plus complement. When these compounds were injected into repeatedly immunized mice producing late ongoing antibody responses no differences in suppression between hydrophilic and hydrophobic derivatives were observed. In this case, the IgE response was suppressed by about 50%, while the IgG1 response was not affected. These results are compatible with the suggestion that early IgE responses are most sensitive to T cell-mediated suppression and that T suppressor cells are better induced by lipophilic than by hydrophilic antigens. The late ongoing IgE response, on the other hand, is less amenable to T cell-induced suppression and tolerogenic effects brought about by plurivalent BPO antigens operate directly on hapten-specific IgE-bearing B cells, regardless of their lipophilic character.

A 3 beta-cholestanyl-containing mono-benzylpenicilloyl oligoamide and peptide suppress anti-benzylpenicilloyl antibody formation in mice.

A long-chain linear mono-benzylpenicilloyl (BPO) oligoamide and a succinoylated mono-BPO decalysine were tested in BALB/c mice for suppression of IgE and IgG1 antibody formation. Both compounds were available with either a free C-terminal end or were C-terminally linked to a hydrophobic 3 beta-cholestanyl residue. Only the sterol-containing derivatives suppressed hapten-specific IgE and IgG1 responses. Substantial suppression was obtained when the compounds were administered before primary or secondary, but not later immunizations. In an adoptive cell transfer experiment, spleen cells from tolerized animals actively suppressed anti-BPO IgE antibody formation of immune spleen cells. This effect was reversed by pretreatment of the tolerized spleen cells with anti-Lyt-2.2 antibody plus complement. The requirement for macrophages in the induction of T suppressor cells was demonstrated by injecting antigen-pulsed macrophages into naive recipients; upon immunization, only mice treated with tolerogen-pulsed macrophages showed suppressed anti-BPO IgE responses. It is suggested that lipid modification of antigens alters their processing and presentation by macrophages in a manner that leads to the induction of T suppressor cells. Injection of the cholestanyl derivatives into passively sensitized guinea pigs elicited anaphylactic reactions. By immune precipitation analysis and molecular weight estimation, these derivatives were shown to form micelles in aqueous solution. Therefore, the anaphylactic response appeared to be due to their behavior as multivalent antigens.

Chain-length dependence for secondary structure formation of homo-oligopeptides from epsilon-tert.-butyloxycarbonyl-L-lysine with a lipophilic C-terminal group.

A solid-state and solution analysis of the homo-oligopeptides from epsilon-tert.-butyloxycarbonyl-L-lysine with p-oxymethylbenzylcholestan-3 beta-yl succinate as C-terminal group, using infrared absorption and circular dichroism, is described. The occurrence of intermolecular beta-structure is seen in the solid state and in solvents of low polarity, e.g. methylene chloride, for peptides of intermediate size (from pentamer to decamer). Conversely, the eicosapeptide exhibits a high percentage of alpha-helical structure both in the solid state and in 2,2,2-trifluoroethanol. The influence of the C-terminal group on the conformational preferences of the epsilon-blocked homo-oligolysines in the solid state and in organic solvents appears negligible.

Human CD8(+) T cells expressing HLA-DR and CD28 show telomerase activity and are distinct from cytolytic effector T cells.

Cycling lymphocytes may express the enzyme telomerase which is involved in maintenance of telomere length and cell proliferation potential. In CD8(+) T cells freshly isolated from peripheral blood, we found that in vivo cycling cells expressed HLA-DR. Furthermore, CD28-positive cells are known to have longer telomeres than CD28-negative T cells. Therefore we used HLA-DR- and CD28-specific antibodies to sort CD8(+) T cells and measure telomerase activity ex vivo. Relatively high levels of telomerase activity were found in HLA-DR/CD28 double-positive cells. In contrast, HLA-DR-negative and CD28-negative cells had almost no telomerase activity. In summary, HLA-DR expression correlates with proliferation, and CD28 expression with proliferative potential. We have previously identified that ex vivo cytolytic CD8(+) T cells are CD56 (NCAM) positive. Here we show that HLA-DR(+) cells were rarely CD56(+) and vice versa. This demonstrates that telomerase-expressing and cytolytic CD8(+) T cells can be separated on the basis of the cell surface markers HLA-DR and CD56. Thus, activated CD8(+) T cells specialize and exert distinct functions correlating with surface molecule expression.