Jan Cerny

Age-related changes in antibody repertoire: contribution from T cells

Summary: The immune system of aged mice produces antibodies that are characterized by low affinity, diminished protection against infections and autoreactivity. It has been shown that these antibodies may be encoded by different immunoglobulin V genes and that the mechanism of somatic hypermutation in the V genes is inefficient. Studies on scid mice reconstituted with B and T cells from donors of different ages suggested that both lymphocyte subsets may contribute to the age‐related changes in antibody repertoire. With help provided by T cells from young mice, the response to a hapten, nitrophenyl(acetyl), became gradually dominated by B‐cell clones that rearranged a particular germline VH gene (V186.2). However, help from the aged T cells resulted in A heterogeneous response of B cells expressing many different V segments. Analysis of discrete foci of primary antibody‐forming cells suggested that the aged T‐helper cells are unable to govern the normally‐occurring competition between the B‐cell clones that have different affinities for the hapten. It is proposed that a signaling disequilibrium from the aged T cells, which provide less efficient help in quantitative terms, supports the growth of low‐affinity B cells. This process may be exacerbated due to the apparent hyperactivity of aged B cells to CD40‐mediated mitogenic signal.

The repertoire diversity and magnitude of antibody responses to bacterial antigens in aged mice: I. Age-associated changes in antibody responses differ according to the mouse strain

Aging influences the host immune responses in various ways. In aging mice we have studied the antibody responses to two unrelated bacterial antigens. Streptococcus pneumoniae R36a vaccine (Pn) and TNP coupled to Brucella abortus (TNP-BA). Aged animals (20-24 months old) of the C57BL/6 strain had markedly reduced numbers of IgM antibody plaque-forming cells (PFC) to Pn as compared to young/adult mice (2-3 months old). In contrast, the anti-Pn IgM PFC responses of aged BALB/c mice were consistently higher than they were in the young/adult mice. The increased anti-Pn responses were not due to a nonspecific immunostimulation, because the responses of aged BALB/c mice to TNP-BA were lower as compared to the adults. However, the aged BALB/c mice responded relatively poorly to Pn challenge, and their IgG responses (as determined by ELISA plaque assay) demonstrated a very high individual variability. The clonotypic diversity of anti-Pn response in young BALB/c and C57BL/6 is limited, such that the majority of PFC produce antibody that express all idiotopes (Id) of the T15 immunoglobulin encoded in the VH-S107/Vk22 genes. In contrast, the PFC from aged mice are diverse, expressing incomplete T15 Id or none at all, suggesting that the antibodies are encoded by altered T15 genes and by different, non-T15 genes. Our data demonstrate that the age-related changes in the magnitude of antibody response to certain antigens are influenced by the host genetic make-up, and that the changes in magnitude and diversity of antibody response may be unrelated to each other.

Antibody feedback and somatic mutation in B cells: regulation of mutation by immune complexes with IgG antibody.

Summary: In response to an appropriate antigenic stimulus, and with help from T lymphocytes, naive B cells differentiate into plasmacytes which produce the primary (germline‐encoded) IgM and IgG antibody with low affinity for the antigen. The isotype switch from IgM to IgG coincides with the burst of germinal center reaction and the onset of “somatic hypermutation. Here we propose that formation of immune complexes between the residual antigen and the primary IgG antibody, which activate complement and localize specifically in the network of follicular dendritic cells, provides an important signal for triggering the mutation mechanism in germinal center B cells. This hypothesis has been supported by studies on immunogenicity of immune complexes in vivo. The experiments have included an immunization with pre‐formed antigen/IgG antibody complex and/or an administration of IgG and body shortly after the antigen injection. Either of these strategies, which are known to augment the germinal center formation, resulted in earlier onset of somatic mutation and increased mutation frequency in VDJ rearrangements in antigen‐reactive B cells, provided that help from T cells was also present. It is presumed that the antigen/antibody/complement complex is able to deliver this important signal by cross‐linking of antigen receptor with the CD21/CD19/CD81 molecules on B cells. As a corollary, the signaling by immune complexes may lower the threshold of cell activation determined by receptor affinity for antigen and stimulate diverse V‐gene repertoire of B‐cell clones in germinal centers.

Repertoire of Antibody Response in Bone Marrow and the Memory Response Are Differentially Affected in Aging Mice

The primary burst of Ab and germinal center (GC) formation in response to T-dependent Ag is compromised in aging mice. Here we examine the effects of aging on the post-GC phase of memory B cell differentiation and the late Ab repertoire maturation in bone marrow (BM) in mice immunized with a hapten nitrophenyl coupled to chicken γ-globulin. Specific Ab-forming cells (AFC) with mutated VH genes accumulated preferentially in the BM of aged mice, although the AFC numbers and average number of mutations per VH were lower, and the D gene usage was less restricted compared with those in the young animals. However, the repertoire of AFC after an Ag boost demonstrated the hallmarks of Ag selection, including the recurrent mutations and canonical VD rearrangements, similar to the late primary response in young animals. It is postulated that the Ab repertoire maturation in aged mice is delayed and may be notably improved by repeated immunizations.

Regulation of VH gene repertoire and somatic mutation in germinal centre B cells by passively administered antibody

Immunization with T‐dependent antigens induces a rapid differentiation of B cells to plasmacytes that produce the primary immunoglobulin M (IgM) and IgG antibodies with low affinities for the immunogen. It is proposed that the IgG antibody forms immune complexes with the residual antigen which provide an important stimulus for the formation of germinal centres (GC) and the activation of somatic mutation. This hypothesis was tested by passive administration of hapten‐specific antibody into mice shortly after the immunization with nitrophenyl (NP) coupled to chicken gamma globulin (NP‐CGG) in an environment of limited T‐cell help. Athymic mice that received normal T helper cells at 72 hr after the administration of antigen produced low levels of anti‐NP antibody and the splenic GC formation was delayed until day 12 after the antigen administration. The analysis of VDJ segments from NP‐reactive GC B cells showed very few mutations in the VH genes. Passive injection of anti‐NP IgG1 monoclonal antibody – but, not IgM – stimulated the GC formation up to normal levels and the somatic mutation activity in the GC B cells was fully restored. In addition, GC B cells in the recipients of IgG1 antibody demonstrated a change in the usage of germline‐encoded VH genes which was not apparent among the primary antibody‐forming cells. These results suggest the existence of a specific feedback mechanism whereby the IgG antibody regulates the GC formation, clonotypic repertoire and somatic mutation in GC B cells.

Insertion of the Dibasic Motif in the Flanking Region of a Cryptic Self-Determinant Leads to Activation of the Epitope-Specific T Cells

Efficient induction of self tolerance is critical for avoiding autoimmunity. The T cells specific for the well-processed and -presented (dominant) determinants of a native self protein are generally tolerized in the thymus, whereas those potentially directed against the inefficiently processed and presented (cryptic) self epitopes escape tolerance induction. We examined whether the crypticity of certain determinants of mouse lysozyme-M (ML-M) could be attributed to the nonavailability of a proteolytic site, and whether it could be reversed to immunodominance by engraftment of a novel cleavage site in the flanking region of the epitope. Using site-directed mutagenesis, we created the dibasic motif (RR or RK; R = arginine, K = lysine), a target of intracellular proteases, in the region adjoining one of the three cryptic epitopes (46–61, 66–79, or 105–119) of ML-M. Interestingly, the mutated lysozyme proteins, but not unmutated ML-M, were immunogenic in mice. The T cell response to the altered lysozyme was attributable to the efficient processing and presentation of the previously cryptic epitope, and this response was both epitope and MHC haplotype specific. In addition, the anti-self T cell response was associated with the generation of autoantibodies against self lysozyme. However, the results using one of three mutated lysozymes suggested that the naturally processed, dibasic motif-marked epitope may not always correspond precisely to the cryptic determinant within a synthetic peptide. This is the first report describing the circumvention of self tolerance owing to the targeted reversal of crypticity to dominance in vivo of a specific epitope within a native self Ag.

A study of autologous anti-idiotypic antibody-forming cells in mice of different ages and genetic backgrounds.

Antibody response to phosphorylcholine, an immunodominant epitope of Streptococcus pneumoniae R36a (Pn), is characterized by a public idiotype, T15, that is expressed on a large proportion of antibody molecules produced by all mouse inbred strains. The ability of the immune system to produce an autologous antibody to T15 upon immunization with Pn vaccine was investigated using a modified ELISA plaque assay for detection of single antibody-forming cells (AFC). The limit of ELISA assay for detection of specific anti-T15 AFC is approximately 300 cells/spleen. However, our studies failed to detect any autologous anti-T15 AFC in the course of the primary antibody response to Pn vaccine in young/adult (2-4 months) BALB/c and C57BL/6 mice. Aged mice (20-22 months) also failed to develop any specific auto-anti-T15 AFC upon the primary Pn immunization, despite the fact that the anti-Pn response in these animals changes both quantitatively and qualitatively. In order to generate specific anti-T15 AFC, BALB/c mice had to be immunized repeatedly with Pn vaccine (four weekly injections) or immunized directly with T15 protein in CFA. Different results were obtained with D1.LP mice that are low responders to Pn and express lower levels of T15 Id as compared to BALB/c. Young D1.LP mice produced high numbers of auto-anti-T15 AFC of both IgM and IgG isotypes following a single immunization with Pn vaccine. The kinetics of auto-anti-T15 response in D1.LP mice was similar to that of the antigen-specific response. These results demonstrate that the ability of the immune network to produce autologous antibody to a shared Id depends on the genetic makeup of the host, and that this response may be regulated by the level of Id expression.