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Analysis of anti-lymphocyte autoimmune antibody responses |
| Principal Investigator : Rahul Pal
Co-Investigator Project Associates
/ Assistants Ph D Students Systemic
lupus erythematosus (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. Monoclonal anti-lymphocyte antibodies would be generated
towards this effort; cell-type and antigenic specificity will be determined.
The effects of monoclonal antibodies on lymphocyte function will be analyzed
in vitro and molecular characterization of reactive antigens will be carried
out. The
long-term objectives of the project would include investigations aimed at
understanding the observed temporal relationships between anti-lymphocyte
antibodies and autoantibodies of diverse specificities. 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. Two
human monoclonal antibodies PR5 and RN86, generated from peripheral B cells of
SLE patients, were subjected to further biological and genetic analysis. The
antibodies were purified from culture supernatant in order to permit
unambiguous characterization. In all subsequent assays, human IgM similarly
purified from peripheral blood was used as the negative control. In
addition to previously documented reactivity on T cells and monocytes, PR5 was
observed to bind granulocytes and erythrocytes as well. It thus appears that
the antibody exhibits very wide polyreactivity, binding to several cell types
present in the blood. PR5 induced a dose dependent decrease in antigen-induced
proliferation of human PBMC, and a variable degree of complement-mediated
toxicity was demonstrated against erythrocytes and granulocytes, confirming
and extending work reported last year. Immunoprecipitations and Western blots
carried out to ascertain the nature of reactive antigen(s) upon the surface of
erythrocytes revealed dominant reactivity towards a 30KDa membrane protein,
the identity of which is being ascertained. Genetic analysis of the heavy and light chain variable regions of PR5 was completed. The heavy chain demonstrated closest homology with the 08 germline (GL) gene, belonging to the VH1 family; a six base deletion was observed at the 3’ end of the gene (see below). Heavy chain sequences revealed very few mutations, and these were not sequestered in the CDR1 or CDR2 regions. The antibody demonstrated an essentially germline configuration. However, extensive non-templated nucleotide modifications were noted at both V-D and D-J junctions, as diagrammed below. For the J chain, complete identity was observed beyond the region shown. In comparison with closest germline segments (top line), bases in bold typeface refer to deletions and the underlined bases to non-templated additions. The arrow indicates a point mutation:
In the case of the PR5 light chain, the entire L6 germline gene (VkIII family) was used (not shown). The Jk2 segment was employed with a single base pair deletion at the 5’ end and a point mutation, as indicated by the arrow Jk2 GL PR5 Light Chain Near
germline-encoded (but heavy and light chain CDR3 region-modified) polyreactive
antibodies directed at ubiquitous antigens present on the cell surface of
healthy cells can therefore mediate immune dysfunction in multiple ways.
Establishing the identity of reactive antigens could be important in explaining
some aspects of disease etiology. It is known that cells undergoing apoptosis are preferentially recognized and internalized by phagocytic cells; these latter cells have recently been shown to express the IgM receptor. We therefore determined the influence the apoptotic-cell specific antibody RN86 on this process. MOLT-4 cells were labeled with 3H-thymidine and apoptosis was induced by treatment with Camptothecin, a DNA topoisomerase I inhibitor. It was observed that the presence of RN86 in the culture caused an enhancement in the engulfment of apoptotic cells. This data assumes significance, since some specificities present in normal human IgM have recently been shown to bind to apoptotic cells. Coupled with the fact that serum IgM knockout animals display features of systemic autoimmune disease, these results may indicate a disease-ameliorating role for RN86-like antibodies. Last
year, studies to investigate the possible role of the idiotypic network in the
phenomenon of epitope spreading were initiated, since idiotypic connectivity has
been previously demonstrated in SLE-related autoreactive specificities, and one
of the RN86-reactive antigens (Ro60) exists in complex with La, another
ribonucleoprotein. Antibodies generated in rats against RN86 were capable of
preventing the interaction of the monoclonal antibody with its cellular
target(s). To assess if epitope spreading had indeed occurred, the reactivity of
these antibodies was assessed on the panel of autoantigens (Ro60, Ro52, La, SmB,
SmD, the U1RNP A protein and the 70kDa protein). In consonance with our
hypothesis, the antibodies recognized La (a ribonucleoprotein associated with
Ro60), reactivity not apparent in the original antibody. Interestingly, antisera
also recognized Ro60, as did the immunogen RN86; this data was reminiscent of
results with the 16/6 anti-DNA idiotype described in the literature. Efforts are
underway to determine if discrete or cross-reactive antibodies are responsible
for this recognition. Sequencing
of the RN86 heavy and light chain variable regions was also carried out. A
comparison of the RN86 heavy chain gene (Accession Number: AJ548507) with the
NCBI database revealed closest homology with the V3-53 heavy chain germline (GL)
gene, belonging to the VH3 family. The D3-10 and JH6 segments appeared to be
employed; both were extensively modified. Heavy chain sequences revealed very
few mutations, and these were not sequestered in the CDR1 or CDR2 regions. The
antibody demonstrated an essentially germline configuration. The entire V3-53
gene was employed (not shown), without 3’ deletions. Once again, for the J
chain, complete identity was observed beyond the region shown; with respect to
respective germline segments, bases in bold indicate deletions, while underlined
bases indicate non-templated additions:
Publications Original
peer-reviewed articles 1. *Kathuria S, Sriraman R, Nath R, Sack M, Pal R, Artsaenko O, Talwar GP, Fischer R and Finnern R (2002) Efficacy of plant produced recombinant antibodies against hCG. Human Reprod 17:2054-2061 (*in press last year, since published). 2.
Kathuria S, Nath R, Pal R, Om Singh, Fischer R, Lohiya NK and Talwar GP (2002)
Functional recombinant antibodies against human chorionic gonadotropin
expressed in plants. Curr Sci 82:1452-1457. |