This document discusses autoantibodies known as antinuclear antibodies (ANA) that are directed against nuclear antigens. It covers the prevalence of ANA, various nuclear antigens they may be directed against like DNA and histones, mechanisms of autoimmunity and autoantibody production, roles of B cells and T cells, and methods of detecting ANAs. It also describes different patterns of ANA fluorescence and their associations with various autoimmune diseases.

1. ANGAN KARMAKAR
(JUNIOR RESIDENT)
CHAIRPERSON: PROF. R.N.SARKAR

2. LE cell phenomenon

3. Anti nuclear antibodies
 (auto)antibodies reactive with antigens in nucleoplasm
 POSITIVE when titre above normal level
 Most ANAs – directed against nucleic acid or
associated proteins
 First demonstration- in 1957 by Holborow et al using
indirect immunofluoroscence
 In SLE- predominant antigen is nucleosome

4. Prevalence of ANA positivity

5. ANA
Physiological
 Low titer
 IgM
 Secreted by CD5+B cells
 Resides in peritoneum
 Weak avidity for self Ag
 Widely cross-reactive with
multiple Ag
Pathological
 High titer
 IgG
 Secreted by CD19+B cells
 Resides in hematopoietic
system
 High avidity for self Ag
 Have restricted specificity

6. ANANUCLEAR LINE ANTIGEN ???EXTRACTABLE NUCLEAR ANTIGEN ???

7.  Anti-DNA and nucleosome autoantibodies
recognize histone-DNA complexes
 Autoantibodies against chromatin and nucleosomes in
SLE are diverse, recognizing the H2A-H2B-DNA
complex, as well as individual histones, ssDNA, and
dsDNA
 About 70% of SLE patients have anti-dsDNA
antibodies at some time in their disease course

8.  Anti-Sm and RNP autoantibodies recognize the
U1 ribonucleoprotein
 proteins U1-70K, A, B’/B, C, D1/2/3, E, F, and G
associated with U1 sn RNA
 B’/B, D, E, F, and G assemble into Sm core particle
 Anti sm- SLE
 Anti 70K, A, C – PM, Scleroderma, SLE
 High levels of anti-RNP without anti-Sm - MCTD

9. Anti-Ro60 and La antibodies recognize cytoplasmic
ribonucleoproteins
Associated with sicca syndrome
Anti-Ro60:60-80% of primary Sjogren syndrome and
10-50% SLE patients
 Anti Ro60 bind to 60-kDa protein associated with Y1,
Y3, Y4, or Y5 RNA.
 47-kDa La antigen is a termination factor for RNA
polymerase III

11. Development of autoimmunity
 defects in the Fas-Fas ligand (FasL) pathway
 defects in endosomal DNA degradation
 defects in degradation of misfolded RNA
 defects in the clearance of apoptotic cells by
phagocytes
 over-expression of TLRs
MECHANISM OF SLE

12. Autoantigenesis
Mechanisms:
 Defective apoptosis
 Impaired removal of apoptotic cells
 Mutation
 Genetic polymorphisms
 Alternate splicing
 Post translational modification
 Molecular mimicry
 Defective sensing and uptake of autoAg

13. How does auto-Ag produce
auto-antibody
 Induction of effector T cells
 Reduced activation of suppressor cells
 Activation of TLRs
Others-
 Acting as chemoattractants
 Altered recognition of auto-antigens

14. Role of T cells
 T-cell-independent response-rapid antibody
production by short-lived plasma cells that
develop extrafollicularly and produce low-affinity
IgM antibodies
 T-cell-dependent response– in secondary
lymphoid organs e.g. spleen or lymph nodes,
which provide an optimal milieu for interactions
between T cells, B cells, and APCs

16. IMPORTANCE OF CO-STIMULATORY SIGNALS

17.  IFN-1 rescues autoraective T cells from apoptosis
 Newer therapy directed at co-stimulation:
CTLA4-Ig: potent inhibitor of co-stimulation and thus
T-cell activation

18. Role of B cells
 B1- IgM-high, IgD-low, CD23-neg
T cell independent
produces low affinity Ab
 B2 (conventional)-IgM-low,IgD-high, CD23-high,CD5- neg
requires CD 4+ T cells
high affinity Ab production
 Deletion (apoptosis), anergy, receptor editing, and
immunological ignorance -determines whether B cells
become activated or are tolerized

20.  High-affinity B cells tend to develop into short-
lived plasma cells, whereas lower-affinity B cells
enter germinal centers and are more likely to become
long-lived plasma cells and memory cells.
 Maintenance of long-lived plasma cells requires
continuous expression of Blimp-1 ,IL-6 and BAFF
produced by stromal cells

21. Extra follicular generation of
autoantibodies
 anti-DNA and rheumatoid factors, can develop
extrafollicularly without T cells
 T-cell independent
 exhibit significant somatic hyper mutation and class
switching

22. Role of BAFF/APRIL
 interact differentially with three receptors: BAFF-R ,TACI,
and BCMA.
 BAFF/BAFF-R interactions regulate the differentiation and
survival of B-2 and MZ B cells
 APRIL regulates CD40-independent class-switching and
plasma cell survival
 TACI regulates TI-2 responses.

23. CD40-CD40 ligand signalling
 CD40-CD40L interactions are important for generating
high-affinity autoantibodies.
 Newer therapeutic options:
1. human monoclonal anti-BAFF antibody : BELIMUMAB
2.anti-CD40L reduces anti-dsDNA antibodies

25. Basic mechanism of ANA production
Breakdown of tolerance
Transformation of autoreactive B cells
into long lived plasma cell & memory B
cells

27. Sensitivity Specificity
IIF 80% 90-97%
ELISA 99% 36-94%
IIF- gold standard
ELISA
Immunodiffusion
CIEP
Multiple immunobead assay
Methods of detection of ANA

28. Patterns of ANA
Homogeneous/Rim
SLE
Drug induced SLE
Positive ANA
Speckled
SS/SSC/PM
SLE/MCTD
Centromere
Scleroderma
Limited
Nucleolar
Systemic Sclerosis

29. Pattern Antigen
Homogenous histone
Rim dsDNA
Speckled Ro, La, Sm, RNP, SCL70
Nucleolar RNA polymerase III
Centromere Centromere B