Analysis of anti-lymphocyte autoimmune antibody responses

Collaborators
Rohini Handa, AIIMS, New Delhi

Project Associates/Assistants
Roopali Gandhi (till Jan 2004)
Smriti Kala (since Jan 2004)

Ph D Students
Joy Das
Harshita Bhatnagar

Humans are plagued by a large variety of autoimmune diseases. The causes of these diseases have not been clearly delineated and the diversity of immune responses generated is the subject of intense investigation. Anti-lymphocyte antibodies are often found in systemic lupus erythematosus (SLE) and other diseases of persistent immune stimulation. Serum titres of such antibodies exhibit broad correlation with disease severity, implicating them in the immune dysfunction that accompanies SLE. This study investigated these phenomena by immortalizing B cells that secrete antibodies with anti-lymphocytic specificity; the monoclonal antibodies generated by such cells were employed to study divergent aspects of the anti-lymphocyte antibody response. Using these reagents as probes, the study sought to determine the physiological implications of anti-lymphocyte responses, specifically in relation to disease-associated immune abnormalities.

SLE is a prototypic non-organ specific autoimmune disease. Immune dysfunction is a hallmark of the disease, and abnormalities are observed in cells belonging to both the B and T cell lineages. A generalized hyper-gammaglobulinemia is observed and is often associated with the presence of specific humoral responses to several self-proteins and to double-stranded DNA. Lymphocytes are also often the targets of autoantibody responses; such responses are believed to contribute towards pathogenesis. This study aims to investigate the nature of the anti-lymphocytic autoimmune responses observed in SLE and to draw correlations with antibody-mediated biological effects. Human and mouse monoclonal anti-lymphocyte antibodies would be generated towards this effort; cell-type and antigenic specificity will be determined. Antibody variable regions will be sequenced. The effects of monoclonal antibodies on lymphocyte function will be analyzed in vitro. The long-term objectives of the project would include understanding the observed temporal relationships between anti-lymphocyte antibodies and autoantibodies of diverse specificity. Cross-reactivities, as well as idiotypic connectivity, with known and well-characterized autoantigens would be investigated. Using appropriate animal models, the postulate that anti-lymphocyte antibodies could arise via epitope spreading would be studied.

Work on two human monoclonal antibodies generated from SLE patients (PR5 and RN86) was extended. PR5 exhibited broad cellular specificity, and mediated cytotoxic and anti-proliferative effects. RN86 appeared to preferentially recognize apoptotic cells, and enhanced their phagocytosis by macrophages. The sequences of the light and heavy chain variable region genes for both antibodies had also been determined.

It was of importance to determine the autoantigen(s) recognized by PR5. Reactivity towards a panel of autoantigens frequently targeted in SLE (Ro52, Ro60, SmB, SmD, U1RNP-70kD, A Protein, and La) was first ascertained. No significant reactivity was observed. Next, Western blots were conducted using lysates from a wide spectrum of cell lineages, but no conclusive or reproducible results were obtained. In immunoprecipitation assays, employing both surface and cytoplasmic biotin labeling strategies and a variety of detergents, no specific reactivity could be discerned.

The presence of lymphocytotoxic antibodies (like PR5) has been associated with neuropyschiatric manifestations in SLE. Therefore, in further attempts at antigenic characterization, a human brain cDNA library was screened, using the antibody as probe. In keeping with the polyreactive nature of the antibody, two distinct proteins were identified. On comparison with the NCBI database, one of the proteins was found to be ‘CRIPT’; recently, CRIPT has been found to associate with the Post Synaptic Density (PSD) 95 protein (a membrane associated guanylate kinase), possibly linking it to tubulin. CRIPT is ubiquitously expressed; its presence in the brain appears to be concentrated in the hippocampus and cortex, regions that appear to undergo degenerative changes in pathological states such as Alzheimer’s Disease, which are characterized by cognitive impairment. Significantly, in extension of results documenting complement-mediated cytotoxicity by PR5 on other cell lineages, the antibody was shown capable of inducing the killing of both glial cells and neurons. The second protein recognized by PR5 was a nuclear ribonucleoprotein; these classes of proteins constitute prominent autoantigens, some of which are targeted to the cell membrane when cell undergo apoptosis.

Previous work had demonstrated that the antibody RN86 was non-reactive towards healthy cells in the periphery, and specifically recognized cells undergoing apoptosis. When healthy (non-apoptotic) cells of central nervous system were employed, very different results were obtained. On neuronal cells, discrete membrane-associated staining was observed, with many other cells in the field remaining unstained. Immunoflourescence analysis on a glial cell line revealed a similar staining pattern; discrete staining was apparent, with certain areas of the cell body intensely stained, and the majority of the cell membrane unrecognized by antibody. Propidium iodide was used as a counter-stain to assess whether all plasma membrane regions recognized by antibody also contained nucleic acid, as would be expected if the areas recognized by RN86 constituted apoptotic blebs; membrane areas solely recognized by antibody were observed, discounting this possibility. Permeabilization of cells led to uniform cytoplasmic staining by RN86, as was observed in the case of Jurkat cells. Thus, it appears that an antibody specifically targeting apoptotic cell associated molecular patterns in the periphery, was capable of recognition of healthy cells belonging to the central nervous system. Dysfunctional apoptosis in the periphery (as has been documented in SLE) therefore leads to the generation of antibodies that specifically recognize (cross-reactive?) surface antigens on cells of the central nervous system, and may influence their viability. These findings assume relevance, given the frequent occurrence of psychiatric symptoms in SLE.

Studies characterizing the anti-idiotypic response to the apoptotic cell specific antibody RN86 were extended. Pooled anti-RN86 rat sera were loaded onto an RN86 affinity column, and adherent antibodies were passed through an IgM affinity column in order to purify the anti-idiotypic fraction. The postulate that these antibodies would also be self-reactive (as was the immunizing antibody RN86) was tested by a variety of assays. Self-reactivity was apparent upon FACS analysis on permeabilized human cells. Such reactivity was also evident on Western blots, where a recognition pattern distinct from the RN86 was observed; the 30-40kDa region was targeted, as opposed to the characteristic 50-70KDa region of recognition by RN86. Furthermore, a wider spectrum of auto-antigenic recognition was observed in comparison with RN86, when purified recombinant antigens were used as targets on ELISA. Interestingly, despite their enhanced specificity, anti-idiotypic antibodies also appeared to be preferentially directed towards apoptotic cells, as evidenced by dual colour FACS analysis with Annexin-V. Finally, immunofluorescence studies on glial cells revealed staining profiles reminiscent of RN86, with discrete, punctate areas of the membrane being recognized.

Experiments have been initiated on two autoimmune strains of mice (C57lpr-/- and NZBxNZW (F1)) as well. Hybridomas have been generated, using spleen cells from aged animals. Two hundred and fifty independent hybrids have been generated thus far, and two monoclonal antibodies (2H8, 2C11; both IgMk) specifically reactive towards apoptotic cells have been isolated. Upon induction of apoptosis, both antibodies exclusively recognized Annexin-V reactive, Trypan Blue negative cells. Antigenic and genetic analysis has been initiated on one (2H8) of the antibodies. Western blots using either isolated apoptotic blebs or whole cells as substrate revealed dominant recognition of 30 kDa and 45 kDa antigens; further characterization is in progress. The variable heavy chain of 2H8 belongs to the VH1 family, with closest germ line gene being J558.33. The CDR2 region showed extensive changes with respect to the germ line gene, with five replacement mutations observed. The D segment was not identifiable, probably due to extensive deletions and/or mutations. The JH3 segment was employed, with a 14-nucleotide deletion. The nearest light chain gene was found to be 19-23; Jk5 was employed. Unlike the heavy chain, the light chain did not exhibit significant mutations.

Many antibodies of the IgM isotype are known to exhibit enhanced antigen binding at temperatures that are lower than the physiological. The temperature dependence of the human and mouse monoclonal antibodies was therefore studied. PR5, the human monoclonal antibody that was non-discriminatory towards healthy and apoptotic cells, exhibited a classical cold reactive pattern; maximum binding was observed at 4°C, while no interaction was detectable at 37°C. The two mouse and one human apoptotic cell-specific monoclonal antibodies, however appeared equally reactive at 4°C and 37°C. This preliminary analysis will be extended by using different cell types as target, and will be carried out on additional antibodies and at intermediate temperatures, in order to ascertain whether such warm reactivity is a general characteristic of anti-apoptotic cell specific antibodies of the IgM isotype.