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Objective: To determine the specificity of anti-CD45 autoantibodies in systemic lupus erythematosus (SLE) for native CD45 and for CD45 expressed by T cells and B cells, and at different stages of cellular activation., Methods: CD45 purified from different types of lymphocytes was examined by immunoblotting with sera from patients with SLE. Indirect immunofluorescence experiments were performed with purified anti-CD45 autoantibodies., Results: IgM anti-CD45 autoantibodies in SLE recognize native CD45 expressed on the surface membrane of viable lymphocytes and CD45 purified from activated peripheral T cells and certain T cell lines, but not CD45 purified from B cells or resting peripheral T cells. The presence or absence of reactivity is independent of the individual isoforms expressed., Conclusion: Recognition of CD45 by IgM antilymphocyte autoantibodies in SLE varies with the lineage and state of activation of the lymphocyte target. This pattern of reactivity is consistent with autoantibody specificities involving CD45 glycoforms, rather than CD45 isoforms.

Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinant E. coli fusion proteins encoded by exons 3-7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.

Sera from patients with systemic lupus erythematosus frequently contain IgM antibodies to glycoproteins of Mr 46,000 and approximately 200,000 isolated from nonionic detergent lysates of mature T cells by affinity chromatography with solid-phase wheat germ agglutinin. Autoantibodies of this specificity correlate strongly with the presence of IgM anti-T cell autoantibodies, as determined by independent indirect immunofluorescence and complement-dependent microcytotoxicity assays, and are specifically absorbed by incubation of patient serum with viable T cells. Collectively, the data suggest that gp46 and, to a lesser extent, gp approximately 200 represent major targets of IgM antilymphocyte autoantibodies in systemic lupus erythematosus.

Cold-reactive lymphocytotoxic autoantibodies are present in the serum of most patients with active systemic lupus erythematosus (SLE) and may be important for the development of the lymphopenia and T cell dysfunction characteristic of this disorder. Neither the mechanisms of autoantibody action in this regard, nor the nature of the relevant T cell membrane target molecules have been defined, however. In the present investigation, preincubation of T cells with SLE serum at 37 degrees C reduced their reactivity with SLE IgM anti-lymphocyte autoantibodies, as demonstrated by indirect immunofluorescence and complement-dependent cytotoxicity. Modulation was restricted to SLE IgM autoantibody-reactive antigen; monoclonal antibody staining of various T cell differentiation and activation antigens remained unchanged. Loss of antigen from the surface membrane was rapid, but transient. A nadir was reached after approximately 120 min of 37 degrees C incubation, followed by essentially complete reexpression of antigen several hours later. Although modulation occurred spontaneously at 37 degrees C in the absence of SLE serum, loss of antigen was enhanced by IgM anti-lymphocyte autoantibodies, despite their low thermal amplitude. Modulation was inhibited by sodium azide, by fixation of cells with paraformaldehyde, and by low incubation temperatures. Colchicine and cytochalasin D had no effect on this process, suggesting that the integrity of the cytoskeleton was not essential. Cycloheximide did not prevent loss of antigen, but inhibited its reexpression. In experiments to determine the fate of modulated antigen, both intracytoplasmic accumulation and shedding from the cell surface were demonstrated. Only shedding was increased by the presence of anti-lymphocyte antibodies, however. These studies delineate modulation of T cell membrane antigen as a new mechanism for anti-lymphocyte autoantibody action in SLE. The occurrence of modulation at physiologic temperatures in vitro suggests that a similar phenomenon of potential relevance to T cell dysfunction may obtain in patients with this disorder.

Monoclonal antibodies to known surface antigens on B cells and on resting and activated T cells of various types were used in several approaches to examine the specificity of IgM antilymphocyte antibodies in systemic lupus erythematosus (SLE). Surface determinants that were sought included: T3, T11, Leu-1, Leu-8 (pan-T); T4, T8 (T subset); beta 2-microglobulin (beta 2m); L243, Leu-10 (DR and DS/DC framework, respectively); anti-Tac (interleukin-2 receptor); 5E9 (transferrin receptor); and 4F2, AA1 (other activation antigens). The first strategy was based on inhibition of rosette formation between mouse monoclonal antibody-coated targets and anti-mouse IgG-coated erythrocytes by SLE sera, either directly at 4 degrees C or after modulation of IgM antilymphocyte antibody-reactive target cell antigen at 37 degrees C. Significant rosette inhibition, defined as greater than 2 standard deviations from the mean value for 10 control sera, was seen only for beta 2m (13 of 20 SLE sera were positive; inhibition = 15-58%). Next, relative fluorescence intensity of lymphocyte staining by monoclonal antibodies was assessed by flow microfluorometry after preincubation of cells with SLE serum at 4 degrees C or after modulation of SLE antibody-reactive antigen. Modulation markedly reduced or eliminated SLE antilymphocyte antibody IgM staining. Except for beta 2m, neither cold nor warm temperature preincubations altered the relative fluorescence intensity for the known surface antigens. These data confirm anti-beta 2m as a common antibody specificity in SLE and suggest that antilymphocyte antibodies in this disorder are not directed to Ia or to certain other defined lymphocyte antigens of functional interest.

Three cell lymphocyte antigens of m.w. 55,000, 70,000, and 105,000 to 110,000 were identified by Western blotting as targets of IgM autoantibodies in serum from a group of 49 patients with systemic lupus erythematosus. The 55- and 70-kDa antigens were well expressed on unstimulated peripheral T cells, whereas the 105- to 110-kDa target was demonstrable only on mitogen-activated T cells and lymphoblastoid T cell lines. Localization of these molecules to the plasma membrane was established by cytoabsorption experiments in which IgM antibody staining of blotted antigens was specifically absorbed from systemic lupus erythematosus serum during 4 degrees C incubations with intact lymphocytes, and by their detection in purified lymphocyte plasma membranes. While the identity of these target antigens vis a vis known surface determinants was not defined, their expression on peripheral T cells from multiple donors and on cell lines of both undifferentiated (HSB-2) and phenotypically mature (Jurkat; HUT 78) types excluded alloantigens, major histocompatibility complex-encoded determinants, and most T cell differentiation antigens as candidates in this regard. Expression of the IgM autoantibody targets on HSB-2 cells argues against discrete T subset specificities as well. IgM reactivity with the 55-, 70-, and 105- to 110-kDa antigens by blotting was highly correlated with antilymphocyte antibody activity in complement-dependent cytotoxicity assays (Fisher's p less than 0.001), and paralleled flow microfluorimetric and microcytotoxicity quantitation of IgM antibody activity in serial observations of individual patients studied during different phases of disease activity. Taken together, these data suggest that IgM lymphocytotoxic antibodies in systemic lupus erythematosus are directed predominantly against a limited number of non-T cell subset-specific antigens.