Evidence of antigen receptor-influenced oligoclonal B lymphocyte expansion in the synovium of a patient with longstanding rheumatoid arthritis.

Plasma cell infiltration of synovium is common in longstanding rheumatoid arthritis (RA). The mechanism(s) underlying synovial B cell proliferation remains unclear. One theory invokes nonspecific polyclonal stimuli; another implicates antigen as the driving force. Antigen-driven repertoires are characteristically enriched for related sets of V gene segments containing similar sequence in the antigen binding site (complementarity-determining regions; CDRs). To study the forces shaping B cell proliferation, we analyzed V kappa transcripts expressed in the synovium of an RA patient. We found Humkv325, a developmentally regulated V kappa III gene segment associated with autoantibody reactivity, in > 10% of randomly-chosen synovial C kappa cDNAs. Two sets of sequences contained identical charged amino acid residues at the V kappa-J kappa join, apparently due to N region addition. We generated "signature" oligonucleotides from these CDR3s and probed PCR amplified V kappa products from the synovium and PBLs of the same patient, and from PBLs and spleen of individuals without rheumatic disease. Significant expression of transcripts containing these unique CDR3 sequences occurred only in the patient's synovium. Thus, in this synovium there is expansion of a limited set of B cell clones expressing antigen receptors that bear evidence of antigen selection.

Towards the design of an antibody that recognises a given protein epitope.

We have explored the possibility of designing repertoires of antibodies complementary to a given protein epitope, specifically the face of the ribonuclease inhibitor barstar that binds to the enzyme barnase. An antibody repertoire was created by mutation of ten residues in the hypervariable loops of a synthetic antibody fragment and displayed on filamentous bacteriophage. The positions of three of the ten residues of the antibody (VL 32, 50 and 94) were chosen to match a triangle of three negative charges on the face of barstar and mutated to favour residues of opposite charge or those with hydrogen-bonding potential. The other seven residues, chosen to allow for variation in the surface of interaction, were mutated at random. One of the antibody fragments isolated after selection of the repertoire (10(8) clones per library) was shown to bind to barstar with an affinity of 1.0x10(-7) M and the binding was competed by barnase. Furthermore, the binding of the antibody to barstar was highly sensitive to mutation of any of five residues of barstar known to contact barnase. This indicates that it may be possible, by a combination of design and selection, to build antibodies to a given epitope.

Biophysical methods for the determination of antibody-antigen affinities.

Binding specificities against virtually any antigen can be isolated from antibody libraries displayed on filamentous phage. The determination of antibody-antigen affinity constants and binding kinetics is an important part of antibody characterization, and may be predictive of antibody performance in biotechnological applications. This article, intended as a guideline for the scientist who isolates a novel antibody and wishes to characterize its binding properties, presents the authors' view on widely used methodologies for the quantitative determination of antibody-antigen interactions.

Developmental regulation of the human antibody repertoire.

The ability to respond to antigen develops in a programmed fashion during ontogeny. In human, "fetal" immunoglobulin gene segment utilization appears biased towards a small set of evolutionarily conserved V gene segments. Many of these gene segments are also used in antibodies with antigen specificities that do not arise until after infancy. The human fetus primarily regulates the diversity of the antibody repertoire through control of the H (heavy) chain CDR 3, which is generated by VDJ joining and forms the center of the antigen-binding site. Molecular modeling suggests that limitations in the length and composition of fetal CDR 3 intervals result in antibodies that contain a relatively "flat" antigen-binding surface that could serve to maximize the number of different interactions possible between the antibody and potential antigens. We propose that these limitations in the sequence and structure of H chain CDR 3 contribute to the low affinity and multireactivity of fetal antibody repertoires. The specific mechanisms used to generate a restricted fetal repertoire appear to differ between human and mouse. Nevertheless, included in the final products of both human and mouse fetal B cells will be antibodies that are quite homologous in composition and structure. The precise role that these antibodies play in the development of immunocompetence remains to be elucidated.

Antibody structure and the evolution of immunoglobulin V gene segments.

Immunoglobulin V domains can be divided into eight unique segments, each of which plays a separate structural role in the creation of an antigen binding site. Three of these segments encode the VH/VL core and are preserved in all V domains. V family identity depends on sequence similarity in two segments which provide support for the antigen binding site. Within a family, gene segments primarily diverge in CDR1 and CDR2. H chain CDR3, flanked by H chain CDR1 and L chain CDRs 2 and 3, builds specificity at the center of the antigen binding site. These findings provide a structural context for interpreting the preferential use of V gene segments in specific immune responses.

Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding.

Mammalian immunoglobulin VH families can be grouped into three distinct clans based upon sequence conservation in two of the three framework (FR) intervals. Through replacement/silent site substitution analysis, molecular modeling and mathematical evaluation of known immunoglobulin crystal structures, we demonstrate that this conservation reflects preservation of protein sequence and structure. Each clan contains a characteristic FR 1 interval that is solvent-exposed and structurally separated from the antigen binding site. Families within a clan contain their own unique FR 3 interval that is capable of either influencing the conformation of the antigen binding site or interacting directly with antigen. Our results provide a structural context for theories that address differential use of VH families in the immune response.

The human immunoglobulin VH7 gene family consists of a small, polymorphic group of six to eight gene segments dispersed throughout the VH locus.

In this report we describe the analysis and mapping of members of the human immunoglobulin VH7 gene family. VH7 and VH1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from VH1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity-determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162-base pair probe spanning the family-specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The VH7 gene family was found to be a small but discrete VH gene family consisting of five to eight germ-line elements, of which at least three are polymorphic. Four different VH7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence-specific hybridization. Two of the four VH7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel VH families. A combination of one and two-dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these VH7 elements. Individual VH7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other VH gene families. The polymorphism of the VH7 gene segments and their scattered location throughout the VH locus makes them potentially useful markers for mapping and linkage studies.

Immunoglobulin gene expression in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by inflammation of synovium, in which immunoglobulin-secreting plasma cells are generally present. The forces driving immunoglobulin expression in RA synovium are unknown. Sequences of VH and VK transcripts from an RA synovial cDNA library demonstrate patterns of somatic mutation typical of an antigen-driven response. Moreover, 5% of the kappa repertoire appears to derive from the same B cell progenitor, suggesting an oligoclonal response. Immunoglobulin expression in this synovium thus appears to result from antigen stimulation. In addition, this patient's synovium is enriched for unusually long VK-JK joins (CDR3s), suggesting abnormal selection or regulation of the B cell response in RA.

IgM heavy chain complementarity-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth.

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an "inappropriate" length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.