Eckehart Kölsch

Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL‐4 and IL‐10 but reduced levels of IL‐12. The release of IFN‐γ is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4+ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose‐dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.

Antigen dose‐dependent predominance of either direct or sequential switch‘qc in IgE antibody responses

Priming of CBA/J mice with minute doses of protein antigens (Ag) leads to high IgE antibody (Ab) titres in the immune sera of these animals. In contrast priming with large doses elicits only a marginal production of IgE Ab. In vitro restimulation of spleen cells from animals primed with large doses and lacking in vivo IgE Ab leads to a burst of IgE Ab‐forming cells. This in vitro anamnestic response is lacking in mice primed with minute doses of Ag. In order to trace the cellular basis of the in vitro IgE memory response we have extended the analysis of the distribution of Ab isotypes to Ag‐primed IgG1‐deficient Δ5′Sγ1 mice. The data presented here must be interpreted as followed. Priming of mice with minute doses of Ag leads to a direct switch from IgM to IgE Ab expression in both strains. These animals have high IgE Ab titres without establishing an IgE memory. The direct switch was verified by polymerase chain reaction and Southern blot analysis of switch circle DNA isolated from Ag‐specific B cells of CBA/J mice primed with minute doses of Ag. In contrast to immunization with minute doses, priming with large doses of Ag fails to induce in vivo IgE Ab production in CBA/J and Δ5′Sγ1 mice but establishes a Bɛ memory in CBA/J mice which involves IgG1‐bearing intermediate B cells. In vivo these Bɛ memory cells do not enter the status of IgE Ab‐producing cells. In vitro they can be released from this anergy and presumed suppression and develop in an anamnestic response into a large population of IgE Ab‐forming B cells. This increase in the number of IgE Ab‐producing cells after restimulation in vitro is lacking in Δ5′Sγ1 mice, apparently because of their inability to generate IgG1‐expressing precursor cells. The notion of a sequential switch and an IgG1 intermediate Bɛ memory status is also supported by depletion and inhibition experiments. Elimination of IgG1‐expressing B cells in CBA/J mice primed with high doses of Ag prevents the IgE Ab burst after in vitro challenge with Ag. The data further suggest that the two switch pathways are not mutually exclusive and that the Ag dose can decide which pathway is preferentially used.

Antigen Dose-Dependent Regulation of Bε-Memory Cell Expression

The data presented in this study document that the phospholipase A2 (PLA2)-specific IgE antibody response in high responder CBA/J mice is solely dependent on the antigen dose used for immunization. Repeated injections of minute doses (MD) of antigen (0.1 μg/mouse) induce a persisting high level of PLA2-specific IgE antibody titer, whereas large doses (LD) (10 μg/mouse) induce a persisting low level of IgE. The IgG antibody titers are the same under both conditions. The low level IgE immune status induced by repeated LD is irreversible and cannot be boosted by MD. In contrast a single LD primes for a secondary IgE response which can be recalled by MD. A high level of PLA2-specific IgE antibodies induced by MD can be downregulated by a single intervening LD of antigen. A low level IgE immune status can be transferred with spleen cells of mice immunized with LD into naive syngeneic recipients, which then fail to mount a high level IgE response upon injection of MD of antigen. The experiments reveal two countercurrent processes, induction of Be-memory cells after a single LD and additional activation of a persisting IgE-specific cellular suppression mechanism after repeated LD of antigen. These properties make the system suitable for the analysis of cellular interactions and of potential desensitization protocols.

Prostaglandins, but not tumor-derived IL-10, shut down concomitant tumor-specific CTL responses during murine plasmacytoma progression

IL‐10 is assumed to be a major immunosuppressive factor produced by most B‐cell tumors. The immunosuppressive role of tumor‐derived IL‐10 was analyzed using the MHC class II‐negative BALB/c plasmacytoma ADJ‐PC‐5 as a model tumor. Immune monitoring of tumor‐bearing mice was based on the measurement of tumor burden, tumor‐specific CTL cytotoxicity and intracellular cytokine staining using FACS. ADJ‐PC‐5 tumor progression in syngeneic recipients is associated with strong, concomitant, tumor‐specific CTL responses during early stages of tumor progression which are sufficient to cause rejection of small s.c. autologous test tumors. These initial CTL responses gradually decline during later tumor stages. Blocking of IL‐10 in vivo did not abolish CTL suppression or retard tumor growth. More strikingly, application of anti‐IL‐10 antibodies during early tumor stages abrogated CTL induction and markedly accelerated tumor growth. In contrast to anti‐IL‐10 treatment, application of cyclo‐oxygenase inhibitors to ADJ‐PC‐5 tumor‐bearing mice led to enhanced tumor‐specific CTL responses throughout all stages of tumor progression, paralleled by retarded tumor growth and a significantly delayed onset of suppression. Both findings contradict a dominant immunosuppressive role of IL‐10 during B‐cell tumor progression. Tumor‐derived IL‐10 must therefore be considered an immunostimulating factor, which accounts for the high immunogenicity of B‐cell tumors, whereas prostaglandins, which are not produced by the tumor cells themselves, are the dominant immunosuppressors in this system.

Structural comparison of I-A antigens produced by a cloned murine T suppressor cell line with B-Cell-Derived I-A

A cloned, antigen-specific T suppressor cell line derived from a CBA mouse expresses large amounts of I-A and I-E antigens. Comparative two-dimensional polyacrylamid gel electrophoresis of biosynthetically labeled I-A antigens immunoprecipitated with a variety of monoclonal I-Ak-specific antibodies suggested that α,β and Ii polypeptide chains are identical with B-cell-derived I-A. Dimeric complexes formed by I-A chains derived from B or T suppressor cells were also similar with two major exceptions. Pulse-labeled T-cell-derived la antigen was complexed with two additional unknown components of about 31K. These components were not visible in pulse-chased (processed) materials. In addition, T suppressor-cell-derived I-A antigens did not contain S-S linked dimers consisting of processed α andβ chains, which are usually formed during solubilization of B cells. We consider the possibility that in T cells these chains are associated with other structures, thus preventing S-S linkage between α andβ chains.

Epitope-dependent nonreciprocal regulation of IgE and IgG2a antibody formation.

The antigen dose-dependent production of IgE versus IgG2a antibodies has been investigated using keyhole limpet hemocyanin (KLH) and bee venom phospholipase A2 (PLA2) as antigens. Repeated injections of minute doses (0.1 microgram/mouse) of KLH into CBA/J mice lead to relatively high titers of IgE antibodies which are gradually reduced in animals that had received higher doses of antigen. In contrast, the IgG2a antibody titers are inversely correlated, resulting in low titers after repeated injection of 0.1 microgram KLH and high titers after repeated doses of 10 micrograms/mouse. With PLA2 as antigen, one finds a comparable dose-dependent downregulation of IgE antibody production. In contrast to the response to KLH, there is, however, only a marginal IgG2a response at all doses tested, and no dose-dependent increase can be found. The data show that an inverse regulation of IgE and IgG2a responses is not mandatory. Instead there seems to be an additional regulation dictated by antigen epitopes.

T‐Cell Enforced Invariance of the Antibody Repertoire in the Immune Response Against a Bacterial Carbohydrate Antigen

The humoral response against the bacterial polysaccharide antigen α(1→3) dextran (Dex) is controlled by J558 idiotype‐(Id) specific T cells. These T cells of which the cell clone 178–4 Ts is a representative by all relevant criteria, recognize J558 Id‐bearing B cells in an I‐Ed‐restricted manner. Costimulation via CD28/B7‐1 but not via CD40/CD40L leads to T‐cell activation. These T cells do not only suppress B cells producing the immunoglobulin (Ig)G3 isotype but also support the survival and clonal expansion of J558 Id positive B cells both in vivo and in vitro. This T‐cell mediated dominance of the J558 idiotype limits the appearance of antibodies carrying other more diverse idiotypes which appear in immunized BALB/c nu/nu mice where no regulatory T cells occur. This T‐cell mediated antibody invariance could be a strategy of the immune system responding to highly conserved antigens like polysaccharides, different from those against protein antigens, where diversity is assumed to be the basis for a successful response.

The murine (H-2k) T-cell epitopes of bee venom phospholipase A2 lie outside the active site of the enzyme : Implications with respect to a paracrine activation of Th2 cells for an IgE antibody response

Recombinant, enzymatic active phospholipase A2 from bee venom (PLA2) is a potent inducer of IgE antibody formation in CBA/J (H-2k) mice. In contrast, a recombinant mutant protein lacking enzymatic activity due to an amino acid exchange in the active site of the enzyme fails to induce IgE antibodies under identical immunization conditions. Peptide mapping and T-cell stimulation experiments with 18-mer overlapping peptides locate the T-helper cell-activating epitopes in the C-terminal region of the PLA2 protein. No T-cell epitopes are found in the area around position 34, the center of the enzymatically active site. The data support a model in which initially an enzymatic activation of mast cells or basophiles leads to IL-4 production which in a paracrine way drives T-helper cells, concomitantly activated by antigen, into Th2 differentiation. This ultimately favors B-cell activation for an IgE response.

The Role of Interleukin-10 in the Generation of CD4+ and CD8+ Memory T Cells (Expressing a CD44+, CD62L– Phenotype) and Their Contribution to the Regulation of Immunoglobulin E Antibody Formation

Background: Immunization of mice with low doses of protein antigens like keyhole limpet hemocyanin (KLH) results in high immunoglobulin (Ig) E Ab titers in the sera of those mice while the application of high doses leads to the production of only marginal amounts of IgE but high levels of IgG2a and IgG1 antibodies. The aim of these studies is to elucidate the role of interleukin-10 (IL-10) in the generation of memory T cells and their contribution to the production of IgE Ab. Methods: Both IL-10-deficient mice and control mice were immunized repeatedly with KLH. Serum levels of KLH-specific Ab were measured. The frequencies of memory T cells were determined by flow cytometry and the role of CD4+ and CD8+ T cells was evaluated. Results: IL-10-deficient mice show an augmented production of IgE in vivo. They exhibit enhanced ratios of CD4+:CD8+ memory T cells with a CD44+, CD62L– phenotype with a significantly raised generation of CD4+ memory T cells. On the other hand, the development of CD8+ memory T cells is reduced moderately in IL-10-deficient mice, which is an interesting fact since it has been shown that primed CD8+ T cells suppress IgE Ab production at least in vitro. The ratios of total CD4+:CD8+ T cells are augmented in IL-10-deficient mice compared to wild-type mice and in K01 mice compared to K100 mice in vivo. Conclusions: The elevated ratios of CD4+:CD8+ T cells indicate a higher capacity to provide B cell help, which results in a strongly elevated IgE response in IL-10-deficient mice. These altered ratios are furthermore interesting in view of the regulatory role of CD8+ T cells which provide a suppressive potential regarding IgE Ab production as shown in vitro. The capacity of IL-10 to suppress IgE Ab production by reduction of the CD4+:CD8+ memory T cell ratio opens new possibilities in the interference with allergic disorders.

Suppression of Tumour-Specific Cytotoxic T-Cell Responses Against the Syngeneic BALB/c Plasmacytoma ADJ-PC-5 by Tumour-Induced CD8+ Regulatory T Cells Via IFN-γ

The mechanisms of tolerance induction by tumour cells during early stages of tumourigenesis were analysed in a murine model system using the highly immunogenic BALB/c plasmacytoma ADJ‐PC‐5. Early stages of tumourigenesis were simulated in syngeneic BALB/c mice by repeated intraperitoneal injections with subimmunogenic doses of X‐irradiated ADJ‐PC‐5 tumour cells. This treatment causes a state of tumour‐specific tolerance in a high percentage of mice, involving a population of CD8+ peritoneal T cells which are able to suppress a protective tumour‐specific Tc response against this tumour. Using a primary mixed lymphocyte tumour cell culture (MLTC) as an in vitro system to study suppressive mechanisms of such regulatory T cells, the role of production or consumption of a number of cytokines was analysed. The data presented here demonstrate that inhibition of a protective Tc response against ADJ‐PC‐5 tumour cells is due to IFN‐γ production by suppressive T cells from tolerized mice, but not to IL‐2 consumption. In contrast to typical CD8+ Tc cells, ADJ‐PC‐5‐specific CD8+ Tc cells do not produce IFN‐γ and are furthermore suppressed by IFN‐γ. Thus, tumour‐induced suppressive T cells and tumour‐specific Tc cells seem to represent functionally and phenotypically different subsets of CD8+ T cells, possibly pointing towards a differential activation of type‐1 and type‐2 CD8+ T cells depending on the dose of tumour cells.