The response in old mice: positive and negative immune memory after priming in early age.

To analyze the effect of age in both B and T cell compartments of the immune system, we have studied the anti-dextran (Dx) B512 humoral immune response in aged C57BL/6 mice. We have used Dx in its native form, which induces a thymus-independent (TI) response, or conjugated to chicken serum albumin (CSA), which induces a thymus-dependent (TD) response. We have also analyzed the adjuvant effect of cholera toxin (CT) in both types of responses. Our results show that the B cell compartment is not greatly affected by age as demonstrated in the TI responses and that CT is a powerful adjuvant despite the age of the animals. However, we found a severe age-associated impairment of TD responses. We conclude that the first antigenic challenge deeply influences further antigenic responses in a positive or negative manner. Priming in early life with native Dx (TI) inhibited late TD responses in aged mice, even when the primary immunization had occurred a long time ago. This negative memory affects posterior TD responses both in the quantity and in the affinity of the response. However, immunization at an early age with TD priming (CSA-Dx) provoked a long-lasting immune memory that abolished in part the age-associated impairment of the response. Our results suggest that protocols of vaccination with TI antigens may not be a convenient strategy, because the development of further optimal immune responses to the same antigen can be impaired.

The ADP-ribosylating CTA1-DD adjuvant enhances T cell-dependent and independent responses by direct action on B cells involving anti-apoptotic Bcl-2- and germinal center-promoting effects.

We recently developed a novel immunomodulating gene fusion protein, CTA1-DD, that combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant was found to be nontoxic and greatly augmented T cell-dependent responses to soluble protein Ags after systemic as well as mucosal immunizations. Here we show that CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injections, but, rather, it binds directly to B cells of all isotypes, including naive IgD+ cells. No binding was observed to macrophages or dendritic cells. Immunizations in FcepsilonR (common FcRgamma-chain)- and FcgammaRII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that FcgammaR-expressing cells are not required for the adjuvant function. Whereas CT failed to augment Ab responses to high m.w. dextran B512 in athymic mice, CTA1-DD was highly efficient, demonstrating that T cell-independent responses were also enhanced by this adjuvant. In normal mice both CT and CTA1-DD, but not the enzymatically inactive CTA1-R7K-DD mutant, were efficient enhancers of T cell-dependent as well as T cell-independent responses, and both promoted germinal center formation following immunizations. Although CT augmented apoptosis in Ag receptor-activated B cells, CTA1-DD strongly counteracted apoptosis by inducing Bcl-2 in a dose-dependent manner, a mechanism that was independent of the CD19 coreceptor. However, in the presence of CD40 stimulation, apoptosis was low and unaffected by CT, suggesting that the adjuvant effect of CT is dependent on the presence of activated CD40 ligand-expressing T cells.

Kappa-deficient mice are non-responders to dextran B512: is this unresponsiveness due to specialization of the kappa and lambda Ig repertoires?

In the dextran B512 high-responder strain C57BL, the response to dextran is restricted to the preferential expression of the V(H)B512 and the V(kappa)OX1 gene combination. The importance of the heavy chain is suggested by the fact that mice with the Ig C(H) allotype, different from C57BL, are low or non-responders to dextran, but the light chain could also play a role. All anti-dextran B512 mAb described to date (>200) use kappa light chains. No anti-dextran antibody using lambda has ever been observed. To ascertain if the restriction of the use of V(kappa) genes in response to dextran B512 was more stochastic or due to other factors, we have studied the response to dextran B512 in C57BL/6 mice where the C(kappa) domain has been disrupted (C57BL.C(kappa)T). These mice are unable to express kappa light chains and their humoral antibodies bear light chains of the lambda type. We found that C(kappa) knockout mice are unable to respond to dextran given in a thymus-independent or -dependent form. The lack of responsiveness is specifically directed to the dextran epitopes since these mice are fully competent to respond to other antigenic structures present in the same immunogenic molecule. These mice are also apparently normal regarding the expression of V(H) genes. Finally, we tested the response to dextran in C57BL.C(kappa)T mice carrying the lpr mutation that was introduced to favor an increase in the life span and make the response to dextran more easily detectable. The introduction of the lpr mutation was not sufficient to change the pattern of unresponsiveness in the C57BL.C(kappa)T mice. We concluded that there are deficiencies in the light chain repertoire because the V lambda light chain could not reconstitute the response to dextran. We discuss the possible mechanisms for this new type of unresponsiveness to dextran B512.

Unresponsiveness following immunization with the T-cell-independent antigen dextran B512. Can it be abrogated?

The bacterial carbohydrate dextran B512 is a thymus-independent (TI) antigen and a poor immunogen. Humoral responses consist primarily of IgM and no memory response is observed; rather, secondary responses to native dextran are similar to or suppressed compared with primary responses. However, immune responses to dextran can be enhanced. In this study we have used a protein-dextran conjugate that elicits a thymus-dependent (TD) immune response against dextran. Furthermore, we used the potent adjuvant cholera toxin (CT) for the dextran immunizations. This enables us to re-evaluate the phenomenon of poor secondary response to dextran and whether it can be abrogated. We show that native dextran-primed mice were not able to mount IgG anti-dextran antibody responses after repeated immunizations with the TD, protein-dextran conjugate. This was also apparent in the spleen, where almost no dextran-specific germinal centres were detected. However, the anti-protein antibody response was normal in these mice, demonstrating that it is only the anti-dextran-responding cells that are affected. The effect of CT adjuvant on these events was also evaluated. CT enhanced the humoral IgM anti-dextran responses as well as the splenic responses to dextran. But, the isotype profile was not altered, still no IgG was produced. In contrast, mice primed with the TD conjugate and repeatedly re-immunized with native, TI, dextran generated IgG anti-dextran responses. Our results indicate that it is probable that the lack of proper costimulation in the initiation of the response to dextran causes the suppressed secondary dextran responses. Furthermore, these results suggest that TI and TD forms of dextran activate the same type of B cells, since TI dextran-priming abrogated TD dextran IgG responses. The importance of the priming event for the induction of a classical memory response to carbohydrate antigens and the implications for vaccination strategies, are discussed.

Role of T cells and germinal center formation in the generation of immune responses to the thymus-independent carbohydrate dextran B512.

Immunization with the thymus-independent (TI) Ag native dextran (DX) B512 induces germinal center (GC) formation in the spleen. However, despite this GC formation, the anti-DX response is poor, and no affinity maturation can be observed. Using cholera toxin (CT) as an adjuvant, splenic as well as humoral responses to DX are improved. In this study, we investigated immune responses against DX in mice lacking TNF receptor I and in athymic mice. The adjuvant effect of CT on these responses was also evaluated. Mice lacking the TNF receptor I allowed us to investigate the role of follicular dendritic cell networks and GC formation in the spleen for the generation of Ab responses to DX, whereas we could investigate the role of T cells in GC development to TI Ags using athymic mice. We found that the humoral immune response to TI DX B512 was not dependent upon T cells or the presence of GCs, although GC development occurred after DX immunization. However, T cells were required for this GC formation, since athymic mice could not develop GCs after immunization with DX. We also show that even if CT is able to directly activate B cells when administered as an adjuvant, the major effect may require T cell participation; this is also the case for TI Ags. In contrast, CT adjuvancy is independent of GC formation.

Germinal center formation following immunization with the polysaccharide dextran B512 is substantially increased by cholera toxin.

We have compared the splenic responses following immunization with the T cell-independent (TI)-2 antigen native dextran B512 and with a thymus-dependent (TD) protein-dextran conjugate. Interestingly, primary immunization with native dextran induced germinal center (GC) formation in the spleen to the same extent as the protein-dextran conjugate. The GC were antigen-specific as characterized by the presence of peanut agglutinin (PNA)-positive areas that were also binding FITC-conjugated dextran. Dextran-binding B cells were also detected outside the GC as sites of antibody-producing cells. The secondary splenic response to native dextran was suppressed compared to the primary response, with almost no dextran-specific GC or extra-follicular sites with dextran-specific B cells present in the sections. This suppression could be reverted by using cholera toxin (CT) as an adjuvant for the dextran immunizations. Following native dextran immunization with CT adjuvant a secondary splenic GC response similar to a TD secondary splenic GC response was generated, with almost all the dextran-specific B cells located in the GC. Collectively, this indicates that the difference between TI and TD antigen responses is not due to different abilities in inducing GC development, rather the GC reaction is less productive for a TI antigen than for a TD antigen. CT can both increase secondary GC formation in particular for the TI form of dextran and ameliorate the GC reaction, as reflected by increased anti-dextran antibody levels.

Immunogenicity of bacterial carbohydrates: cholera toxin modulates the immune response against dextran B512.

Native dextran B512 is a T-cell-independent (TI) antigen. By conjugating low molecular weight (MW) dextran to protein, a T-cell-dependent (TD) response against dextran can be obtained. We have previously reported the effects of native dextran and two different protein-dextran conjugates on the immune system. While one type of conjugate induced an optimal TD response, the other conjugate ('suboptimal') evoked a response more similar to that induced by native dextran, i.e. with little immunoglobulin class switch and with a secondary response of similar magnitude to the primary response. In order to investigate if it was possible to augment the anti-dextran response we examined the effects of cholera toxin (CT) in our dextran model system. CT is a potent mucosal, as well as systemic, adjuvant with effects on T cells, B cells and antigen-presenting cells. We show that CT is a very efficient adjuvant for both the TD and TI forms of dextran. A major increase in IgM and IgG anti-dextran antibody production was detected after administration of CT together with the conjugates compared with a conventional alum adjuvant. The effect was most pronounced for the suboptimal TD conjugate. CT was also able partially to abrogate the unresponsiveness to dextran in the TI type 2 (TI-2) non-responder strain CBA/N. CT was also found to be a very potent adjuvant for native dextran, secondary IgM levels were enhanced eightfold by the co-administration of CT. Furthermore CTB-Dx, which is a conjugate of the non-toxic part of CT and low MW, non-immunogenic dextran, elicited an anti-dextran response in nude mice. Collectively, our data show that it is possible to improve the immunogenicity of both TD and TI forms of a carbohydrate by co-administration of CT. This is indicative of two components of the adjuvant effect, one could enhance antigen presentation and costimulation of T cells and the other could have a direct stimulatory effect on B cells.

Polyreactive binding of antibodies generated by polyclonal B cell activation. I. Polyreactivity could be caused by differential glycosylation of immunoglobulins.

The aim of this study was to gain knowledge of the pre-immune repertoire with reactivity directed to the carbohydrate antigen dextran B512 (Dx). Polyclonal activation of spleen cells in mice has been estimated to be a method of revealing the available repertoire. Hybridoma cell lines derived from lipopolysaccharide (LPS)-stimulated C57BL/6 spleen cells were screened for reactivity with Dx and with the non-related protein bovine serum albumin (BSA). Despite the lack of structural similarity between Dx and BSA we observed that nearly all of the Dx positive monoclonal antibodies (MoAbs) cross-reacted with BSA. The Dx and BSA crossreactive MoAbs were also found to bind to several foreign-and auto-antigens, and therefore we concluded that these MoAbs fulfilled the criteria of polyreactivity. The Dx and BSA cross-reactive antibodies were produced in an apparently random fashion as judged by the use of kappa and lambda light chains and the use of V(H) J558 subfamily genes. There are two possible explanations for this type of polyreactivity: (1) LPS induces a randomly generated polyreactive and a randomly generated specific immunoglobulin (Ig) repertoire; (2) polyreactivity can be the result of post-translational modifications. Since immunoglobulins are glycoproteins we considered the possibility that post-translational modifications such as glycosylation could be responsible for the generation of the polyreactive pool. Dextran-specific and Dx/BSA cross-reactive MoAbs showed different degrees of sensitivity to inhibition of glycosylation performed by treatment with tunicamycin (Tm), an inhibitor of the formation of N-glycosidic linkages. Other polyreactive, connective and specific MoAbs were also tested. The authors found that Tm treatment had a more profound effect in reducing the binding capacity of the polyreactive antibodies (Abs), suggesting that the polyreactive Abs may be more dependent than the specific Abs on the carbohydrate content of the molecule for binding to the antigens. The authors propose that lymphocytes may use differential glycosylation as a means to generate polyreactive or monospecific Abs.

Polyreactive binding of antibodies generated by polyclonal B cell activation. II. Crossreactive and monospecific antibodies can be generated from an identical Ig rearrangement by differential glycosylation.

In this work the authors tested the hypothesis that differential glycosylation may be one of the mechanisms by which B cells could be able to produce monospecific or polyreactive antibodies flexibly. The same genetic information will be used in both situations. To prove this hypothesis the authors used mice transgenic for the SP6 (mu + kappa) hybridoma cell line producing monoclonal antibodies (MoAbs) with specificity for the hapten 2,4,6-trinitrophenyl (TNP). In these animals most cells in the periphery express exclusively the transgene SP6. To obtain polyreactive antibodies (Abs) spleen cells from transgenic animals were stimulated in vitro with lipopolysaccharide (LPS). The authors used LPS derived B cell blasts to produce a hybridoma cell collection. A high proportion of the monoclonal antibodies were found to bind to TNP and to crossreact with unrelated antigens. Endogenous immunoglobulins (Igs) were not responsible for crossreactivity since the crossreactive clones only expressed the transgene products. This was demonstrated by polymerase chain reaction (PCR) amplification of cDNA and by sequencing analysis of the PCR products. The nucleotide sequences of the expressed mono- and crossreactive genes were identical to the sequences of the rearranged V(H) and V(K) SP6 which undoubtedly demonstrates that crossreactive Igs are derived from the same rearrangement and also that no mutations in the V(H) or V(K) or in the CDR3 could account for the observed crossreactivity. It is also shown here that the crossreactive antibodies bear the idiotype Id 20-5 characteristic of SP6 binding Abs. Crossreactive monoclonal antibodies were found to be slightly more glycosylated than the TNP-monospecific Abs. Furthermore, binding to TNP was not influenced by treatment with tunicamycin, an inhibitor of glycosylation, while, in the same molecule, other types of binding were considerably reduced. This supports the hypothesis of the importance of glycosylation in polyreactivity.

Anti-IgM-Ficoll conjugates activate B cells from CBA but not CBA/N mice.

We have compared the stimulating effects of an anti-IgM-Ficoll conjugate on B cells from the two mouse strains CBA and CBA/N. CBA/N mice have a recessive defect on their X chromosome which make them unable to respond to T1-2 antigens and B cells from these mice are supposed to be in an immature state. We found that the anti-IgM-Ficoll conjugate stimulated B cells of the CBA strain to proliferate even without the addition of interleukins, but was not able to stimulate B cells from the CBA/N strain. To rule out that the unresponsiveness of CBA/N mice to anti-IgM-Ficoll was due to the immaturity of their B cells, we cultured B cells from both strains in the presence of an anti-IgM-Sepharose conjugate. This conjugate stimulated B cells from both mouse strains to proliferate in the presence of IL-4. These results agree with the hypothesis that the T1-2 antigen Ficoll is able to deliver activating signals to B cells and therefore cannot be considered as an inert carrier.

Immune responses to bacterial polysaccharides: terminal epitopes are more immunogenic than internal structures.

Two types of dextran-protein conjugates can be produced depending on the size of dextran and the method chosen for coupling. Dextran of 4 x 10(4) molecular weight, randomly coupled to keyhole limpet hemocyanin evoked "incomplete" T cell-dependent (TD) immune responses. This atypical response only affected the dextran epitope since the response to the protein carrier was as expected for TD secondary immune responses. A second type of TD conjugates can be derived by coupling dextran (Dx) of 10(3) Da to the protein chicken serum albumin (CSA) via the reducing end (CSA-Dx-1). Immunization with CSA-Dx-1 induced the classical pattern of TD immune responses. Interestingly, immunization with CSA-Dx-1 favored the production of antibodies directed against terminal structures of the dextran molecule. These results were also confirmed at the hybridoma cell level. In contrast to other protein-dextran conjugates, CSA-Dx-1 was able to induce anti-dextran antibodies in CBA/N mice and in neonatal animals. We have interpreted these results to mean that conjugates exposing carbohydrate terminal nonreducing end structures could be more "physiological" and able to be recognized by helper T cells. This opens a new possibility for the production of vaccines against bacterial polysaccharides.