Natural Antibody Repertoire Research Articles

IgM antibodies to P1 cytoadhesin of mycoplasma pneumoniae are part of the natural antibody repertoire expressed early in life

IgM antibodies to P1 cytoadhesin of mycoplasma pneumoniae are part of the natural antibody repertoire expressed early in life

Natural antibody repertoire is dependent on complement receptors CD21/CD35

Natural antibody repertoire is dependent on complement receptors CD21/CD35

Genetic control of natural antibody repertoires: I. IgH, MHC and TCR beta loci.

Global analysis of natural antibody repertoires has revealed a marked conservation of reactivity patterns within inbred mouse strains, and characteristic strain-specific differences. We have now analyzed the genetic control of reactivity repertoires, aiming at identifying the respective selection mechanisms. Multiparametric statistics of a large number of serum antibody reactivities scored by quantitative Western blot analyses using extracts from homologous tissues and bacteria readily distinguish the reactivity patterns of C57BL/6 and BALB/c, revealing homogeneity among genetically identical individuals. Antibody repertoires in the prototype strains can also be segregated from those expressed by the respective IgH congenics, BC.8 and CB.20, demonstrating that IgH-linked genes contribute to determining natural antibody repertoires. Conversely, strains sharing IgH haplotype also express distinct reactivity patterns, indicating that other genes participate in the selection of serum IgM repertoires. Two such non-IgH loci were now identified. Thus, analysis of four MHC-congenic strains demonstrated that MHC-linked control of natural antibody repertoires is likely to operate through differential selection of T cell repertoires, since (1) mice that are congenic at the TCR beta locus, and (2) BALB/c nude mice grafted at birth with pure thymic epithelium from either C57BL/6 or BALB/c also differ in their natural antibody repertoires.

Genetic control of natural antibody repertoires: I. IgH, MHC and TCRβ loci

Genetic control of natural antibody repertoires: I. IgH, MHC and TCRβ loci

An innate natural antibody is reactive with a cryptic sequence of lactoferrin exposed on sperm head surface.

Lactoferrin (LF), an 80-kDa glycoprotein of ubiquitous occurrence in body fluids, is multifunctional and capable of assuming different configurations to serve those functions. The capacity of LF to undergo endocytosis and the recent demonstration of LF binding to sequence specific DNA indicate that a function or capability of LF, in addition to iron chelation, bacteriostasis, and receptor-specific lymphocyte binding, may be that of gene activation or silencing. The data of this report present a human physiological system, that of sperm entry into the oocyte in performance of fertilization in which, since LF is a component of the sperm protein coat, that capability could be expressed. However, the configuration of LF in that locus is one in which a revealed cryptic sequence provides the specific binding site for a natural antibody present in the fertilization milieu. The presence of that antibody suggests that a system of control of the potential interaction of LF with the intra-ooplasmic DNA, that of gametes or pronuclei, is operative. The configuration of LF on the sperm surface and designation of the reactive site for the natural antibody were enabled by a monoclonal antibody secreted by a hybridoma derived from a human cord blood B cell. Thus, in addition to information concerning the molecular flexibility of LF, these observations support the proposition that the repertoire of natural antibodies provides an innate homeostatic system, with each antibody serving a specific role.

The role of natural anti-galα1–3gal antibodies in hyperacute rejection of pig-to-baboon cardiac xenotransplants

Xenoreactive natural antibodies in humans and higher primates are directed predominantly at galα1–3Gal. These antibodies are thought to initiate hyperacute rejection of porcine organ xenografts. The contribution of anti-Galα1–3Gal antibodies to the xenoreactive natural antibody repertoire and to the initiation of hyperacute rejection was tested in a pig-to-baboon cardiac xenograft model. Anti-Galα1–3Gal antibodies were depleted from baboons by extracorporeal absorption of anti-Galα1–3Gal antibodies from plasma using columns with a matrix bearing Galα1–3Galb1–4GlcNAc. Specific removal of antiGalα1–3Gal antibodies was achieved prior to transplantation as demonstrated by immunoassay. Porcine hearts were then transplanted into these baboons and the outcome of the transplants was analysed. Immunofluorescence revealed little deposition of baboon antibodies in the grafts. The porcine hearts did not undergo hyperacute rejection even though complement activity was ∼90% of baeline at the time of transplantation. These findings demonstrate that anti-Galα1–3Gal antibodies constitute a major fraction of xenoreactive natural antibodies in primate blood and that these antibodies contribute significantly to the pathogenesis of hyperacute xenograft rejection.

Instability of natural antibody repertoires in systemic lupus erythematosus patients, revealed by multiparametric analysis of serum antibody reactivities.

Recent views on autoimmune diseases invoke generalized but specific perturbations in antibody repertoires, rather than the clonally restricted or non-specific polyclonal alterations proposed thus far. The present experiments analyse serum antibody reactivities in 24 systemic lupus erythematosus (SLE) patients and 17 healthy controls, using a method that quantitatively scores a large number of antibody reactivities and allows for multiparametric statistical analyses. The results show global but relatively specific perturbations in SLE antibody repertoires, and identify novel disease-associated reactivity patterns. Furthermore, a time series analysis of serum antibodies over 3 months demonstrates instability of natural antibody repertoires in individual SLE patients, contrasting with their remarkable conservation in healthy donors. Moreover, the method used clusters controls and patients independently, and might prove of diagnostic value, once large data bases are established.

Self Assertion Modeled as a Network Repertoire of Multi-Determinant Antibodies

We study repertoire selection in a network of natural antibodies that is maintained by simulatory idiotypic interactions. The natural antibody repertoire develops in an environment of self epitopes to which the self-reactive B cell clones are completely tolerant. For the modeling formalism, we extend the shape space framework so that each antibody is represented by several randomly chosen shapes. B cell clones are generated stochastically and are removed whenever their density falls below an extinction threshold. The idiotypic interactions are governed by a log bell-shaped interaction function. The natural antibody repertoire in the model is formed by percolation: the network is autonomously activated following a point stimulation. Our main conclusion is that the natural antibody repertoire organizes itself in such a way that most self epitopes are included in the repertoire. We find an over-representation of antibody determinants that are similar to self epitopes. We speculate the the network forms a “smoke screen” covering the somatic self.

How adaptive antibodies facilitate the evolution of natural antibodies

It is shown how Ig specificities, randomly generated in conventional B cells, come to be expressed in the genetically-determinate B1 population. Thus the adaptive antibody population facilitates the evolution of the natural antibody repertoire, in accordance with the Baldwin effect in the evolution of instinct. The evolution of these two populations is discussed under both the 'proximal usage' and 'preferential expression' hypotheses of biased Ig gene segment usage. This process is independent of theories of B1 function.

Analysis of the natural human IgG antibody repertoire: life‐long stability of reactivities towards self antigens contrasts with age‐dependent diversification of reactivities against bacterial antigens

We used a quantitative immunoblotting technique to analyze the repertoires of IgG antibody reactivities in the serum of healthy young children, young adult males and aged males with self and non-self antigens. Densitometric patterns of reactivity of purified IgG with self antigens were highly conserved between individuals within a given age group and across age groups. Inter-individual differences were observed, however, upon analysis of self reactivities of IgG in whole serum. A striking heterogeneity between individuals within a given age group and across age groups characterized the reactivity of purified IgG and of IgG in whole serum with bacterial antigens. Inter-individual differences were more marked among aged individuals than among individuals of other age groups. Analysis of variances of reactivities of IgG with bacterial antigens further demonstrated an increased diversity of repertoires of aged donors compared with those of young adults and children. Our results document the stability of the self-reactive repertoires of IgG throughout life, which contrasts with the diversification of the repertoire of IgG antibody reactivities directed toward foreign antigens with aging. These findings support the concept that self-reactive antibody repertoires are positively selected throughout life by a restricted set of self antigens shared by all individuals.

Studies on the T cell dependence of natural IgM and IgG antibody repertoires in adult mice

The present experiments address the thymic dependence of IgM and IgG natural antibody repertoires in adult euthymic and athymic BALB/c mice, as well as in athymic animals reconstituted with a fixed number of syngeneic T cells. Within 3 weeks of the transfer of 10(7) syngeneic splenic T lymphocytes to athymic mice, the T cell compartment is essentially reconstituted in the peritoneal cavity (up to 80% of the numbers in euthymic animals), but is only 10-20% of controls in the spleen and lymph nodes. Early after transfer, there is an increase in the numbers of activated B cells and of immunoglobulin-secreting cells in the spleen, and within 1-2 weeks, the serum concentrations of IgG1 and IgG2a are fully reconstituted to control levels (30-40-fold increased). Multiparametric analyses of serum IgM and IgG repertoires revealed that euthymic and athymic mice share essentially all natural antibody reactivities towards syngeneic extracts of liver and muscle. When tested at the same immunoglobulin concentrations, however, nude sera consistently show higher values of reactivity in all detectable bands. The transfer of 10(7) splenic T cells into athymic mice results in a general decrease of serum IgM reactivities, some of which become undetectable, and in alterations of the serum IgG repertoire as early as 1 week, and for at least 4 weeks after transfer. T cell transfer, however, fails to restore the euthymic IgM and IgG repertoires within 4 weeks. The present observations demonstrate that, after limited T cell reconstitution of nude mice, there is a rapid and quantitatively important increase of serum IgG1 and IgG2a production; the serum IgM reactivity repertoire is qualitatively similar in euthymic and athymic animals, but is generally decreased by T cell activity; and the serum IgG repertoire, which is qualitatively similar in euthymic and athymic animals, is amplified by T cell activity and partially altered by T cell transfer into athymic animals. These results raise questions on the mechanisms of B cell activation and natural antibody repertoire selection in T cell-deficient adult individuals.

Global Analysis of Antibody Repertoires. 1. An Immunoblot Method for the Quantitative Screening of a Large Number of Reactivities

This paper describes a procedure for analysing multiple antibody reactivities that explores a commercially available immunoblot system, and is based on a double staining of nitrocellulose membranes, revealing both antibody reactivities and the migration position of the blotted proteins in the membrane. Quantification of both stainings by densitometry allowed the accurate superposition of the immunoreactivity and total protein profiles of each lane. Moreover, the protein stainings of the different lanes could be adjusted with a simple-scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison of the immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 10(4) activated B cells, and to define strain-specific natural antibody repertoires. The utility of this immunoblot method as an assay for simultaneously scoring multiple reactivities to hundreds of antigens in complex mixtures of antibodies, and thus defining antibody repertoires in a global manner, is discussed.

Analysis of immunoglobulin variable region genes from human IgG anti‐DNA hybridomas

The molecular mechanisms leading to anti-double-stranded (ds) DNA antibody production in systemic lupus erythematosus (SLE) are poorly understood. We describe here the immunoglobulin variable region genes of six human hybridomas secreting IgG anti-dsDNA antibodies derived from three SLE patients. The monoclonal IgG anti-dsDNA antibodies have been shown to be of high affinity and no multireactivity was observed (Winkler et al., Clin. Exp. Immunol., 1991. 85: 379). The comparison of the variable region genes expressed in the hybridomas with known germ-line genes as well as with the germ-line counterparts from one patient shows that the VH and VL sequences are somatically mutated. The pattern and extent of the observed somatic mutations are suggestive for an antigen-driven selection of at least four of these B cell clones. Several VH and VL genes used by the hybridomas were found to be expressed in the natural antibody repertoire, in the restricted fetal repertoire and in B cell malignancies expressing the CD5 antigen.

Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response.

Although much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (RFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG RFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG RF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this RF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM RF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed RF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with L1's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG RF must have been driven by the Fc antigen.

Population dynamics of natural antibodies in normal and autoimmune individuals.

We have measured the quantities of naturally occurring autoantibodies in the serum of normal, unmanipulated individuals. These changes over time following broad-band complex dynamical patterns that are similar in mouse and man. The patterns more likely reflect the network architecture of the natural antibody repertoire, regulating the activation and decay of individual clones. The temporal changes of both disease-specific and nonspecific autoantibodies are consistently modified in autoimmune individuals.

Establishment and functional implications of B-cell connectivity.

We have discussed some aspects of the structure of the normal immune system, particularly the B-cell compartment. We have argued: that a basic property of the natural antibody repertoire is constituted by high degrees of connectivity within the immune system as well as between the system and other components of the organism; that the complementarities constituting this connectivity are based on self-self interactions, high degrees of degeneracy or somatically selected interactions and that these properties are conserved through evolution, to ensure self-reference; that by evolutionary selection, antibody V-genes encoding such structural properties are ensured to be expressed early in ontogeny. The set of highly connected cells will be kept through ontogeny and form the basis for a compartment of naturally-activated lymphocytes making up 10-15% of the total lymphocyte population. As suggested before, this pool of connected cells may be responsible for maintenance of normal network dynamics and prevention of autoaggression.

Idiotypic determinants of natural IgM antibodies that resemble self Ia antigens.

A collection of immunoglobulin-secreting B-cell hybridomas was derived from normal neonatal BALB/c spleen and searched for reactivity against a panel of monoclonal anti-H-2 antibodies. We describe here one IgM antibody which was found to react with the monoclonal anti-Ia.7 antibody 14-4-4S. The characterization of this clone (BA.N 4:4.57) revealed its anti-trinitrophenyl specificity and demonstrated specific binding to five different monoclonal anti-Ia.7 antibodies but not to other anti-H-2 antibodies. The variable region specificity of these interactions was shown by the use of pepsin Fab fragments of the IgM antibody. Anti-Ia.7 antibodies were shown to specifically inhibit plaque formation by the hybridoma cells, and dinitrophenylglycine was shown to inhibit the reaction between the IgM antibody and anti-Ia.7 molecules. We interpret these results as to indicate that BA.N 4:4.57 expresses an idiotope or idiotopes which mimic Ia.7 determinants. This idiotypic family is naturally expressed in both newborn and adult BALB/c mice, as shown by the presence in normal serum of IgM molecules that specifically react with the F(ab')2 fragment of the 14-4-4S antibody. We speculate on the importance of idiotypic mimicry with major histocompatibility complex determinants, for both the selection of natural antibody repertoires and the evolution of antibody genes.