Objective.A qualitative and quantitative analysis of the functional, antigen-specific B cell receptor repertoire of patients with rheumatoid arthritis (RA) in synovial and peripheral compartments.Methods. B cells were activated to grow and differentiate at high efficiency in vitro under limitingdilution conditions. Isotype and specificity of the secreted Ig were tested by enzyme-linked immunosorbent assay.Results. In contrast to peripheral B cells, most synovial B cells had already switched to IgG/IgA in vivo. The frequencies of B cells specifically recognizing foreign antigens were decreased within the synovial population, whereas the frequencies of B cells specific for type I1 collagen, mycobacterial heat-shock protein 60 (hsp60), or IgG Fc fragments were significantly increased, revealing a negative correlation in terms of frequencies.Conclusion. B cells specific for human type I1 collagen, hsp60, and IgG Fc fragments are produced and/or expanded locally within the affected joints of RA patients. Thus, the specific immune system is definitely involved in the local inflammatory and destructive processes.

SUMMARY Using a potent in vitro limiting dilution culture system, we have activated human peripheral blood B cells to proliferate and to differentiate into antibody‐secreting cells (ASC). Under these conditions 25–100 % of B cells are clonally expanded and produce IgM, IgG or IgA. Culture supernatants were tested for antibodies binding to human IgG‐Fc fragments (RF), the 65‐kD heat shock protein of Mycobacterium bavis (hsp60), human collagens type I, II, IV, V, transferrin, lactoferrin, albumins, and gelatine. All blood samples contained precursors of ASC (p‐ASC) able to produce IgM binding to these antigens in frequencies above 003% of B cells. Most interestingly, a significant difference exists between rheumatoid arthritis (RA) patients and controls, concerning the relative frequencies of p‐ASC able to produce monospecific or multircactive RF. Whereas most p‐ASC(RF) in RA patients are monospecific (mean ratio 3.7), most p‐ASC(RF) in healthy control persons are cross‐reactive with at least one of five other antigens tested (mean ratio 0.2). The data suggest a disease‐specific expansion of p‐ASC committed to the production of monospecific rheumatoid factors.

Four type II collagen-specific T cell lines established from the peripheral blood of a healthy donor were studied in detail. These CD4+ T cell lines with an alpha/beta T cell receptor proliferated in response to native and denatured human and chicken type II collagen and human type I collagen, but not to human type IV collagen or other potentially arthritogenic antigens. The T cell response showed typical dose response characteristics, peaked between 30 and 48 h, was major histocompatibility complex class II restricted and not due to a mitogenic effect. Type II collagen-reactive T cells could hardly be detected in freshly isolated peripheral blood mononuclear cells from healthy donors, as revealed by limiting dilution analysis (frequency less than 1/100,000). By prestimulation in bulk cultures for 10 days, type II collagen-reactive T cells could be approximately 1000-fold enriched. However, in these limiting dilution cultures, collagen-reactive T cells could only be observed in a narrow window of cell concentrations, suggesting that type II collagen-reactive T cells may be controlled by suppressive mechanisms in healthy persons.

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Ligation of interleukin 2 (IL2) is known to regulate both protein tyrosine and serine/threonine phosphorylation. A family of leukocyte transmembrane proteins whose cytoplasmic domain exhibits intrinsic protein tyrosine phosphatase activity is collectively called CD45 and is identified by a set of common cell surface epitopes. Although CD45 is known to be a phosphoprotein, it is not known how phosphorylation specifically regulates its function. We therefore identified a cell line, the IL4-dependent line CTLL-2.4, in which CD45 could be phosphorylated in response to addition of IL2. These cells are a variant of an IL2-dependent murine cell line which were selected for long-term growth on IL4 but which retain the ability to proliferate on exposure to IL2. Incubation of CTLL-2.4 in low serum concentrations followed by stimulation with IL2 caused a three- to fivefold increase in the phosphorylation of CD45 in a time- and concentration-dependent manner. CD45 in non-stimulated cells contained one major tryptic phosphopeptide, whereas, after exposure of the cells to IL2, two new phosphopeptides were present in CD45. The pattern of IL2-induced phosphorylation was different from that found following addition of phorbol 12-myristate 13-acetate (PMA) to the cells. Although IL2 induced rapid and potent tyrosine phosphorylation in CTLL-2.4 cells, all of the basal and cytokine-activated phosphorylation of CD45 occurred on serine residues. The IL2-stimulated phosphorylation caused no change in the amount of cell surface CD45 and no alteration of its catalytic activity using an artificial tyrosine phosphorylated substrate-RCM-lysozyme. We speculate that the increase in phosphorylation of CD45 may modify its association with potential substrates. The differences in the phosphorylation patterns induced by IL2 and PMA further suggest that more than one kinase can use CD45 as substrate and that IL2 activates a protein serine/threonine kinase different from protein kinase C.