We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

There is evidence that certain alleles at the HLA-DQ locus are correlated with susceptibility to insulin-dependent diabetes mellitus (IDDM) and in particular that DQ beta-chain alleles containing aspartic acid at position 57 are protective. The availability of a large group of patients with IDDM enabled us to assess the role of HLA-DQ alleles in susceptibility to the disease in order to confirm and extend recent observations derived from studies of smaller numbers of patients. Using allele-specific oligonucleotide probes and the polymerase chain reaction, we studied 266 unrelated patients with IDDM and 203 unrelated normal subjects for eight HLA-DQ beta-chain alleles. Two major findings emerged from these studies. First, the presence of an HLA-DQw1.2 allele was protective. Only 6 of the 266 patients with IDDM (2.3 percent) were positive for HLA-DQw1.2, as compared with 74 of the 203 normal subjects (36.4 percent; P less than 0.001). Thus, persons with the HLA-DQw1.2 allele, which is one of the polymorphic forms of the beta chain of the HLA-DQ molecule, rarely had IDDM, no matter which other HLA-DQ beta-chain allele they inherited ("dominant protection"). Second, the presence of the HLA-DQw8 allele increased the risk of IDDM. The relative risk of IDDM was 5.6 in persons homozygous for HLA-DQw8, and it was similar in persons with the HLA-DQw1.1/DQw8 or HLA-DQw2/DQw8 haplotype ("dominant susceptibility"). However, the relative risk of IDDM in persons who had the HLA-DQw1.2/DQw8 haplotype was 0.37, demonstrating that the protective effect of HLA-DQw1.2 predominated over the effect of HLA-DQw8. We conclude that the presence of the HLA Class II antigen DQw1.2 is strongly protective against the development of IDDM, and that complete HLA-DQ typing is necessary for accurate assessment of susceptibility to IDDM.

During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (∼110,000 nucleotides) from CD38+IgD− germinal center, and CD38−IgD− memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38− follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

Both the B cell-surface trigger(s) and the intracellular molecular mechanism(s) of somatic hypermutation in immunoglobulin (Ig) variable region genes remain unknown, partly because of the lack of a simple and reproducible in vitro model. Here, we show that upon surface immunoglobulin cross-linking followed by co-culture with activated cloned T cells, the Burkitt's lymphoma cell line BL2 is induced to mutate its IgV(H) gene. Repeated activation of BL2 cells increased the frequency of mutation. The in vitro-induced mutations, which do not affect the IgM constant region, are point mutations distributed over the entire V(H)DJ(H) gene segment and do not show evidence of antigen-driven selection.

Structural studies of human monoclonal rheumatoid factors (RF) derived from patients with monoclonal gammapathies have revealed a restriction in the usage of heavy and light chain variable regions. These studies have suggested that germline genes with little if any somatic mutation can generate RF specificity. However, there is no information presently available regarding the primary structure and genetic origin of RF in rheumatoid arthritis. In this study, we have isolated and sequenced the VH regions of six monoclonal RF derived from the synovial membranes of two patients with rheumatoid arthritis and one with the juvenile polyarticular form of the disease. We found the same VH families as previously reported among monoclonal paraproteins with RF activity. However, our sample was diverse regarding the VH, DH, and JH gene segments used. Among VHI RF there was conservation in the length of CDRIII as well as restriction in the amino acid generated at the V-D junction, as opposed to VHIII RF and non-RF VHI molecules that are highly heterogeneous in these two aspects. We also found that different JH gene segments may contribute to RF specificity. The VH, DH, and JH elements of one RF in our study all had clearly identifiable germline counterparts. This RF displays a nearly germline configuration throughout its entire heavy chain and represents another example of an autoantibody encoded by one of the VH gene segments from the preimmune fetal repertoire. (J. Clin. Invest. 1990Invest. . 86:1320Invest. -1328

Certain antiserums prepared against isolated cold agglutinins demonstrate specificity for gamma M globulins with this activity and not for similar gamma M globulins lacking such activity. The cold agglutinin specific antigens fall into several major and minor groups. Their exact relation to the presumed antibody-combining sites remains to be determined.

To better understand the structural basis for rheumatoid factor activity, the nucleotide sequence of the light chain variable regions of nine human monospecific IgM rheumatoid factors were analyzed. Rheumatoid factors were isolated from three patients with rheumatoid arthritis, a patient with systemic lupus erythematosus, and a normal individual. The VL gene segments used by these rheumatoid factors are not as restricted as previous work on mixed cryoglobulin rheumatoid factors had suggested. Each of the different VK families is represented and there are two examples where a V, gene segment is used. Molecules with structures similar to those of the Wa and Po CRI, characteristic of mixed cryoglobulin rheumatoid factors, are not common among these rheumatoid factors isolated from patients with rheumatoid arthritis. While there are clear examples of rheumatoid factors that are direct copies of germline genes, most of the sequence data suggest that the processes of antigenic selection and somatic mutation contribute significantly to the generation of monospecific rheumatoid factors in patients with autoimmune disease. (J. Clin. Invest. 1991.

Recent studies have indicated the presence of cross-idiotypic determinants among monoclonal IgM proteins possessing anti-T-globulin activity that were not found among IgM proteins lacking this activity (1). The pattern obtained was very similar to that previously described for the IgM cold agglutinins (2) with antigenic specificities in proteins from different individuals which appeared to relate to the combining specificity of these proteins toward their respective antigens. Evidence was obtained both for the cold agglutinins and the anti-T-globulins that the antigen-combining site was involved in these specificities. Through the use of the idiotypic antisera which showed crossspecificity, it was possible to delineate two entirely separate groups of anti-Tglobulins (1). One, the major Wa group, made up approximately 60 % of the anti-'y-globulins; the other, the minor Po group, included approximately 20 % of the total; a few anti-T-globulins could not be classified in either of these two groups.The relative role of the H and L chains of these proteins in the cross-specificities was not readily determined because the antigenic determinants involved required the combination of the chains for expression. However, amino acid sequence studies on the minor Po group of anti-3,-globulins demonstrated an extraordinary similarity in H chain sequence through two hypervariable regions (3) ; the L chains were quite distinct. This suggested that heavy chain similarities were primarily involved in the cross-specificity determinants of the minor Po group. The present studies were carried out to define these relationships further by both antigenic and sequence analyses with special emphasis on the major Wa group of anti-~-globulins. A marked similarity in the L chains of this group was found. Materials and MethodsThe IgM anti-T-globulins utilized in this study were the same as those described in a previous report (1). They were initially purified in most instances as cryoglobulins from sera where they appeared as monoclonal bands; a combined procedure using Pevikon electrophoresis and Sephadex columns as described previously (1) was then utilized for final isolation. All of the proteins precipitated with aggregated Fr II 3'-globulin and this was the primary method of