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Analysis of anti-lymphocyte autoimmune
antibody responses |
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Principal Investigator : Rahul Pal Co-Investigator Ph D Student Systemic Lupus Erythematosus (SLE) is a prototypic
non-organ specific autoimmune disease. Anti-lymphocyte antibodies are often
found in 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
seeks to investigate these phenomena by immortalizing B cells that secrete
antibodies with lymphocytic specificity. Monoclonal antibodies isolated from
such cells will be used to investigate anti-lymphocyte autoimmune phenomena
and its relationship with other autoimmune responses. 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. Various techniques were employed to further
characterize the human monoclonal anti-lymphocyte antibodies generated during
the previous year. The observation of specific recognition of human apoptotic
cells by the antibody RN86 (IgMk) was confirmed and extended. Upon activation
of normal PBMC with phorbol ester and ionomycin, the antigen was expressed on
both B and T cells. Time-kinetic FACS studies revealed consistent dual
staining patterns with Annexin-V, and variable co-localization with the
activation antigen CD69. In addition, dexamethasone treated mouse thymocytes
were also recognized by RN86, thus implicating an evolutionarily conserved
death related antigen. Using confocal microscopy, RN86 as well as another
apoptotic cell specific antibody Pr8 (IgMl), exhibited a characteristic
speckled localization pattern on the surface of apoptosing cells, whereas
minimal recognition of healthy cells was demonstrated. In contrast, the
antibody Pr5 (IgMk, previously shown to recognize healthy and apoptosing cells
equally) demonstrated more homogenous cell surface associated staining. Several cytoplasmic autoantigens are expressed on the
cell surface in characteristic “blebs” during apoptosis. In many cases,
these antigens are highly homologous across species. This fact, coupled with
the characteristics of RN86 outlined above, led us to investigate whether the
antibody recognized well-known auto-antigens. Reactivity against recombinant
Ro60, Ro52, La, SmB, SmD, the U1RNP A and 70kD proteins, and against double
stranded DNA was assessed. Predominant recognition of RN86 was directed
towards Ro52, with Ro60 and the 70kD also being recognized. These observations
reconciled with western blot and immuno-precipitation data, which revealed
reactivity to antigens in the 50-70kD range. Interestingly, appreciable
recognition was also observed towards double stranded DNA. The other apoptotic
cell specific antibody, Pr8, also recognized the three auto-antigens albeit to
a lesser degree. Pr5, on the other hand, did not recognize any of these
auto-antigens. Thus, poly-reactivity towards protein and nucleic acid
auto-antigens, previously known to transmigrate to the cell surface during
apoptosis, was demonstrated by death-specific human monoclonal antibodies.
These observations have implications for the pathogenesis and etiology of
lupus. Studies to assess idiotypic connectivity of
anti-lymphocyte antibodies with other autoimmune humoral responses were
initiated. Antisera generated against RN86 in rats inhibited the interaction
of the antibody with permeabilized MOLT-4 cells. The specificity of this
inhibition is being evaluated. Genetic analysis was carried out for anti-lymphocyte
antibodies generated in the previous year (BPG70, 228, 295 and 378). Briefly,
total RNA was isolated from hybridoma cells, and reversed transcribed into
cDNA. PCR amplifications were then carried out for variable region genes,
using heavy chain (IgM) and light chain (lambda or kappa) sequence specific
primers. Amplified fragments were gel-purified and either sequenced directly,
or cloned into appropriate vectors and then sequenced. Comparisons were
carried out to the closest germ line sequences in the database. All heavy
chains belonged to the VH4 family. The light chain family of BPG70 was Vk 4,
of BPG228 was Vl1, of BPG295 was Vl 2 and of BPG378 was Vk 3. Though changes
in the sequences were observed, they were not significant enough to
characterize the antibodies as being somatically mutated. These studies show
that pathogenic (complement-fixing) anti-lymphocyte autoantibodies can be
essentially germ-line encoded. Publications Original peer-reviewed articles 1.
Mandokhot A, Pal R, Nagpal S, Chauhan VS, Ahlawat S and Om Singh
(2000) Humoral hyporesponsiveness to a conjugate contraceptive vaccine and its
bypass by diverse carriers using permissible adjuvant. Clin Exp Immunol
122:101-108. 2.
Pal R and Om Singh (2001) Absence of corpus luteum rescue by
chorionic gonadotropin in women immunized with a contraceptive vaccine. Fertil
Steril (in press). 3.
Gupta A, Pal R, Ahlawat S, Bhatia P and Om Singh (2001) Enhanced
immunogenecity of a contraceptive vaccine using diverse synthetic carriers.
Vaccine (in press). 4.
Gupta A, Chandrasekhar S, Pal R, Ahlawat S and Om Singh (2001)
High expression of human chorionic gonadotropin b-subunit using a synthetic
vaccinia virus promoter. J Mol Endocrinol (in press). |