Sherman Fong

Treatment of primary Sjögren's syndrome with hydroxychloroquine☆

Sjögrens syndrome is an autoimmune disease characterized by lymphocytic infiltration of the salivary/lacrimal glands, autoantibody production, and polyclonal hyperglobulinemia. In view of the efficacy and relative safety of hydroxychloroquine in other autoimmune disorders, the potential benefit of hydroxychloroquine (200 mg per day for 12 months) in 10 patients with Sjögrens syndrome was evaluated. Changes in levels of total immunoglobulin, antibody against Sjögrens syndrome-associated antigen B, rheumatoid factor, and in vitro production of immunoglobulin in the serum were evaluated. For comparison, 10 patients matched according to age and sex, who did not receive hydroxychloroquine were studied. In the hydroxychloroquine-treated group, the following observations were made: (1) significantly decreased total immunoglobulin G (IgG) and IgA levels with little change in IgM levels; (2) significant decrease in IgA-rheumatoid factor with a smaller decrease in IgM-rheumatoid factor; (3) decreased IgG anti-Sjögrens syndrome-associated antigen B autoantibody; and (4) decreased erythrocyte sedimentation rate and increased hemoglobin level. Further, a specific idiotype present on their rheumatoid factor (defined by monoclonal antibody 17-109) was significantly decreased, with disappearance of detectable circulating paraprotein in two hydroxychloroquine-treated patients. Finally, rheumatoid factor production in vitro by lymphocytes from hydroxychloroquine-treated patients using a T cell-dependent mitogen was significantly decreased. These results suggest that hydroxychloroquine modulates lymphoproliferation in patients with Sjögrens syndrome and may prevent progression to extraglandular sites of neoplastic transformation.

A common idiotope on human rheumatoid factors identified by a hybridoma antibody

Human monoclonal and polyclonal anti-IgG autoantibodies [rheumatoid factors (RFs)] are composed primarily of kappa light chains, and may display cross-reactive idiotypes. However, the nature of the shared idiotope(s) has remained unclear. We have prepared a murine hybridoma antibody (17-109) that recognizes an idiotope present on 30% (3/10) of human IgM-RF paraproteins, and absent on immunoglobulins without RF activity. The idiotope was measurable on isolated, intact kappa light chains, but not on light-chain tryptic peptides, nor on isolated heavy chains. A comparison of the binding to 17-109 of five IgM-RF paraproteins, with known kappa chain amino acid sequences, suggested a relationship between the idiotope recognized by the hybridoma and the complementarity-determining regions. The serum of patients with rheumatoid arthritis contained idiotope positive material that bound specifically to a 17-109 immunoadsorbent column. Moreover, the 17-109 anti-idiotope antibody partially inhibited the binding to IgG of IgM-RF and IgA-RF in serum, but did not effect the binding to antigen of IgM and IgA anti-tetanus toxoid antibodies. These results suggest that a significant proportion of IgM-RF paraproteins share an idiotope located at or near the complementarity-determining regions of the kappa light chain. Human serum RFs include a kappa light chain family that is idiotopically related to the kappa chains on IgM-RF paraproteins.

Different populations of rheumatoid factor idiotypes induced by two polyclonal B cell activators, pokeweed mitogen and Epstein-Barr virus

Abstract In vitro stimulation of peripheral blood lymphocytes from two rheumatoid arthritis patients with pokeweed mitogen (PWM) and Epstein-Barr virus (EBV) induced differing sets of IgM rheumatoid factor (IgM-RF) idiotypes. Thus, in either patient. 71–82% of the IgM-RF induced by PWM, but only 30% of the IgM-RF induced by EBV, shared individually specific idiotypes with serum IgM-RF. These results suggest that differing populations of autoreactive B lymphocytes were induced by the two mitogens. PWM, a T-cell-dependent mitogen, induced the same RF-bearing B cells that were active in vivo . In contrast, EBV, a T-cell-independent mitogen, stimulated predominantly an inactive population of RF precursor B lymphocytes.

IgM rheumatoid factor autoantibody and immunoglobulin-producing precursor cells in the bone marrow of humans

The natures of the IgM rheumatoid factor (RF)-, IgM-, and IgG-secreting cells in the human bone marrow as compared to the peripheral blood, have been investigated by (1) response to the polyclonal B-cell activator, the Epstein-Barr virus (EBV), (2) sensitivity to the S-phase specific antimetabolite hydroxyurea, (3) presence of the BA-1 and Ia antigens on the cell surface, and (4) cell size, as determined by counter flow elutriation. The EBV-inducible bone marrow IgM-RF precursors derived from medium to large B cells that were inhibited by hydroxyurea pretreatment. The marrow total IgM response derived from small to medium size cells, and was only partially inhibited by hydroxyurea. Hydroxyurea had no effect on IgM-RF or IgM synthesis by peripheral blood cells. These results indicate that the marrow EBV-induced IgM-RF response is not representative of the response by peripheral blood cells, moreover; the marrow RF secreting response arises from a dividing cell pool that may represent newly generated autoreactive B cells.

Solid-phase selection of human T lymphocyte subpopulations using monoclonal antibodies

This report describes a novel solid-phase technique for the positive selection of human T lymphocyte subsets labeled by indirect immunofluorescence with monoclonal anti-bodies. Fluorescein labeled normal human T cells or a T cell line were fractionated on plastic culture dishes precoated with affinity chromatography purified anti-fluorescein antibodies. Cell binding was specific for fluorescein, and was both time and temperature dependent. Bound cells were eluted at 37 degrees C with fluorescein-L-lysine. The eluted cells were enriched with highly viable and functional human T cell subsets. Thus Leu3 monoclonal antibody selected cells were shown to provide helper activity in the pokeweed mitogen induced IgM and IgG immunoglobulin secretory response of autologous B cells. The Leu2 antibody selected T cells suppressed both IgM and IgG secretory responses. In addition, studies with the monoclonal antibody 1G11, which binds to an antigen expressed on acute lymphocytic leukemia (T-ALL) cells and the T-ALL derived cell line RPMI-8402, demonstrated that this solid-phase technique can be used to select for cells which are present at low frequencies in a mixed population. It thus provides a simple and reproducible means for the preparative isolation of lymphocyte subsets associated with autoimmune and neoplastic disease for functional and biochemical analysis.

Origin and Age-Associated Changes in the Expression of a Physiologic Autoantibody

Aging is accompanied by increased prevalence of serum autoantibodies. One commonly detected autoantibody, IgM rheumatoid factor, is also found associated with rheumatoid arthritis and other autoimmune disorders. Much evidence indicates that this autoantibody plays a physiologic role in the immune response. The potential of human subjects to secrete this autoantibody and the age-related changes in its expression by human peripheral blood and bone marrow lymphocytes have been investigated. The size of this self-reactive B cell pool increases with advancing age. The lymphocytes expressing this potential are found predominantly in an early B cell subset in elderly individuals as compared to a more mature B cell subset in individuals with rheumatoid arthritis. Preliminary data show that IgM rheumatoid factors share idiotypes implying a common origin, possible from a single light chain gene or a closely related family of light chain genes.

Molecular interactions in human T-cell-mediated cytotoxicity to Epstein-Barr virus. I. Blocking of effector cell function by monoclonal antibody OKT3.

Abstract The ability of different anti-human T-cell lymphocyte monoclonal antibodies to inhibit the effector function of the cytotoxic T-cell response against autologous Epstein-Barr virus (EBV)-infected B-cell targets has been tested. It was found that monoclonal antibody, OKT3, which reacts with most human T cells, blocks the effector cell function in the absence of complement, an effect that was dose dependent. When monoclonal antibody OKT3 was tested at a concentration of 1 μg/ml, inhibition of cytotoxicity ranged between 50 and 80%. The F(ab′) 2 fragment of OKT3 inhibited as well as the intact IgG molecule, indicating that the Fc portion of the antibody is not necessary for the cytotoxicity blocking. The Fab fragment of OKT3 had lower blocking activity per microgram of protein tested. Antibodies SC1, OKT11 (anti-pan T cell), OKT8 (anti-cytotoxic/suppressor subset), and L368 (anti-HLA) did not have any discernible blocking effects. However, antibodies SC1, OKT8, and L368 could abrogate the cytotoxic activity in the presence of complement. Blocking by OKT3 was not due to its being present on the cell surface in higher concentrations than the other monoclonal antibodies since cytofluorographic analysis demonstrated that the amount of OKT8 or L368 antibodies bound on the cells was greater than OKT3. In addition, blocking was not due to antigenic modulation since incubation with antibody OKT3-F(ab′) 2 was not associated with a significant decrease in the amount of its reactive antigen. Under the conditions tested OKT3 did not affect cell viability or cause agglutination.

Lysis of autologous Epstein-Barr virus-infected B cells by cytotoxic T lymphocytes of rheumatoid arthritis patients

Abstract Since B lymphocytes of patients with rheumatoid arthritis (RA) have a higher rate of Epstein-Barr virus (EBV)-induced transformation than normal B cells, we have investigated whether this phenomenon is related to defective cytotoxic T-cell control of emerging B-cell clones. We have studied the in vitro generation of cytotoxic T lymphocytes specific for antigens appearing on autologous B cells after EBV infection using lymphocytes from six patients with RA. Cytotoxic cells were generated by two cycles of stimulation with mitomycin C-treated autologous B cells, and their lytic activity was assessed against 51Cr-labeled targets. As previously reported for lymphocytes of normal individuals, RA cytolytic T lymphocytes were induced only by EBV-infected B cells, not by noninfected B cells, and expression of the cytotoxicity required EBV-transformed targets. After primary in vitro stimulation with autologous EBV-infected B cells, the RA cytotoxic cells expressed higher lytic activity than the normal counterparts, but the difference was not statistically significant (P > 0.1). However, after secondary in vitro stimulation both groups displayed similar cytotoxic activities. In the presence of complement, monoclonal antibodies OKT8 (characterizing cytotoxic/suppressor cells), SC1, and OKT3 (characterizing most T cells) reduced cytotoxicity by 80–90%; antibody OKT3 also reduced the lytic activity by 68% in the absence of complement. We conclude that the higher EBV-induced transformation rate seen with RA lymphocytes is not explained by a deficiency in this EBV-specific cytotoxic T cell.

Idiotypes and the structural diversity of human rheumatoid factors.

ConclusionIt has been shown that IgM-RF paraproteins can be subdivided into two major kappa light chain CRI families, namely the 17.109 CRI and the 6136.6 CRI. The two CRIs have been shown to be distinct and together account for nearly two-thirds of all the kappa light chains of IgM-RF paraproteins. The 17.109 CRI is associated with the VkIIlb sub-subgroup. Both CRIs may also be detected on a non-RF immunoglobulins. The 17.109 CRI is encoded by the conserved Humkv325 (or VkRF) gene. The 6B6.6 CRI is likely encoded by the recently isolated Humkv328 gene. Both CRIB are associated with RFs from patients with rheumatic diseases. The widespread appearance of RFs that are encoded by germline genes or their somatic variants suggest that the molecules are necessary for the survival of the human species. The conservation of genes for RF autoantibodies may be of physiological importance for antigen presentation, for the clearance of immune complexes, and for immune regulation.

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus☆

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.