Factors affecting production of antibodies to prostate antigens by in vitro-primed human splenocytes.

Efficiency of immunization of splenocytes in vitro by three kinds of prostate antigen has been studied. Intact cells of the human prostate cell line LNCaP, a membrane preparation (PMA) of LNCaP cells, and prostate specific antigen (PSA) were used as antigens. Three different schemes of in vitro immunization were tested: male spleen cells vs female; single donor spleen cells vs. mixed lymphocyte culture (MLC); addition of exogenous IL-2 vs. no lymphokine addition. The supernatant antibodies were tested for reactivity to the immunizing antigen by ELISA. Subsequently hybridomas were generated by fusing the primed lymphocytes to a heteromyeloma cell line, K6H6/B5. Only antigen specific IgM responses could be detected. Intact LNCaP cells induced the highest responses from mixed lymphocyte cultures. PMA also induced the highest responses from mixed lymphocyte cultures of male origin, whereas both single donor and mixed donor spleen cell cultures of female origin responded to PMA. However, anti-PMA responses by mixed lymphocyte cultures of female cells were significantly augmented by addition of IL-2. PSA only induced specific responses from mixed female splenocyte cultures supported with IL-2. Several anti-PMA and -PSA antibodies were generated after somatic fusion of the in vitro primed cells. Two monoclonal IgG antibodies from LNCaP primed spleen cells could be competitively inhibited with tumor membrane antigen.

Generation and molecular analyses of two rheumatoid synovial fluid-derived IgG rheumatoid factors.

OBJECTIVE: To study the Ig genes that encode IgG rheumatoid factor (IgG-RF) from rheumatoid synovial fluid. METHODS: We used rheumatoid synovial fluid B cells to generate IgG-RF-secreting hybridomas. We then characterized their binding properties and determined their nucleotide sequences. RESULTS: Two monospecific IgG-RFs were obtained. Sequence analysis of the RFs revealed a new V lambda gene family (designated V lambda 9) and extensive somatic diversification, including a duplication-insertion of 18 nucleotides (6 amino acid residues) into a hypervariable region. CONCLUSION: The data provide further support for an antigen-driven response in the sustained production of potentially pathogenic IgG-RFs in rheumatoid synovium.

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.

Generation and characterization of two monoclonal self-associating IgG rheumatoid factors from a rheumatoid synovium.

OBJECTIVE: To study IgG rheumatoid factor (RF) from rheumatoid synovium. METHODS: We fused the K6H6/B5 human-mouse heterohybridoma with unstimulated rheumatoid synovial B cells to generate IgG-RF-secreting hybridomas. RESULTS: The RFs from 2 such hybridomas bound specifically to the Fc fragment of human IgG and self-associated to form immune complexes. Such immune complexes are a major characteristic of the pathogenic IgG-RFs in rheumatoid synovium. CONCLUSION: IgG-RF-secreting hybridomas have been obtained. Analyses may reveal the underlying mechanisms of the induction of IgG-RF.