Innate Immunity and the Immune response II by ecj13059

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									                                                         Autoantibodies and Disease

                                                           Evidence for “pathogenic” role of autoantibodies
 Innate Immunity and the                                     IgM – only autoantibody of clinical importance is the
 Immune response II                                          RF – T-cell independent
                                                             IgG – many autoantibodies of clinical importance, I.e.
                                                             autoantibodies to Ro/SS-A and La/SS-B – T-cell
                                                             dependent (for affinity maturation and class switch)
         Dr. Newkirk 2006                                    IgA – several diseases associated with this
                                                             subclassT-cell dependent (for affinity maturation and
                                                             class switch)
                                                           Tolerance/autoimmunity




                                                        Rheumatoid Arthritis
IgM RFs

 Disease associations:
   Rheumatoid Arthritis
   Mixed cryoglobulinemia, associated with Hepatitis
   C infection (IC causes vasculitis, kidney disease)
   Sjogren’s syndrome




Rheumatoid Arthritis

 RA, a “new” disease
 Ethnic group with highest RA is North American
 Indians (NAI)
 Infectious etiology and/or genetic susceptibility?
 Caucasians RA 1%, often skips generations
 Higher in NAI, but varies with the tribe, present in
 every generation
 Female to male ratio of RA is 7:3



                                                            La Familia de Jordaens en un Jadin, by Jacob Jordaens (c 1630)




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Immune Response in RA                              Autoantigens in RA - Ubiquitous

 Germinal centers in the synovium                   IgG Fc
 Dendritic cells, Macrophages                       Citrulline containing peptides (CCP)
 Atypical T-cells (CD28 negative)                   Filaggrin, Keratin, Perinuclear factor, Sa
 B-cells, Plasma cells                              ANCA, ANA
   Antigens?                                        Glucose-6-phospnate isomerase (GPI)
                                                    Calreticulin
                                                    Others….but not DNA




B-cell depleting (anti-                            IgG Autoantibodies
CD20/Rituximab) therapy in RA
  24 weeks
                                                    High affinity
                                                      Target organs
                                                      Activate complement pathway at site
                                                      “Organ specific autoimmunity”
                                                      Immune complex mediated systemic diseases
                                                      (SLE)
 48 weeks




                   Panayi GS, J. Rheumatol. 2005




 Immune Complex Mediated Disease                   SLE

                                                    Butterfly rash            95% are female
                                                                              Onset in the “20’s”
                        SLE – the prototypic                                  Multiple autoantibodies
                        IC mediated disease                                   Arthritis, Hair loss
                                                                              common
                                                                              Those with kidney
                                                                              involvement most at
                                                                              risk




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Kidney - C3 deposits                            Pathogenic IgG Autoantibodies
                                                Neonatal lupus




Anti-Ro/SS-A, Anti-La/SS-B                      Fate of the Autoantibody Lesions
                                                in Child
 Detected in patients with Systemic Lupus        Damage to the heart is generally not
 Erythematosus or Sjogren’s syndrome             reversible
 IgG anti-Ro and anti-La crosses the placenta    Skin rash disappears as the antibodies clear
 and causes disease                              from the circulation (half-life of IgG is up to 1
  Can cause complete heart block in utero        month, depending on the isotype)
  Can cause a sun sensitive rash in the baby
  (neonatal lupus)




Autoantibodies Predate Disease                  Do IgG autoantibody positive B-cells
                                                require TLR activation?
                                                 To date the specificities examined have
                                                 been:
                                                   Anti-DNA (TLR9 not as important as predicted)
                                                   Anti-chromatin (TLR9 important)
                                                   Anti-Sm/RNP (TLR7 important)
                                                   Anti-phospholipid
                                                   Anti-nucleolar (TLR9 important)



         Arbuckle et al 2004 NEJM 349:1526




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Paper 1 - Lartigue                           Lartigue Fig 1   What is the phenotype?



 Mouse model of Lupus – on B6 background
 Focused on TLR9
 Double knock out compared with single
 knock out




                                               Fig 3 What are the
                                               specificities of the
                                               autoantibodies on the
    Fig 2 Are there                            different genetic
    abnormalities in                           backgrounds…any
    the T cell                                 surprises?
    populations in the
    spleen?




                                                        B6-lpr/lpr
                                                        B6-lpr/lpr-TLR9-/-




Is there polyclonal activation?
Strain               Total IgM   Total IgG   Fig 5 What is the
                                             impact of activation with
                     mg/ml       mg/ml       CpG in vivo?
                                             - Would these mice
B6 – lpr/lpr         5.8±0.7     5.4±1.4     ever see CpG in vivo
                                             even in the water
                                             control?
B6-lpr/lpr –         3.6±1.6     30.7±7.0
TLR9-/-




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                                                                         What would you do to improve the
             Fig 6 What is the                                           study
             phenotype?
             Why did they focus on                                         Housing of animals?
             the kidney?
                                                                           CpG oligo?
                                                                           Control oligo?
                                                                           Should they look at other TLRs?
                                                                           Look at other autoantibodies?
                                                                           What would you do next (i.e. what was their
                                                                           major finding and how would you advance
                                                                           that?)




    Lymphoproliferative disease in man                                   Paper 2 – YAA x FcR-/- model
    (defective Fas)
       Is “lpr” a good model for human SLE?                                Chimeric B-cells
       In man, lymphoproliferative disease is rare…about                   Lupus model
       100 people have been studied
       Autoantibodies:                                                     Why did they use different allotypes?
             Anticardiolipin (65%+)
             ANA (25%+)
       Diseases diagnosed in only 47%
             Autoimmune Hemolytic anemia (AHA) (29%), Idiopathic
             thrombocytopenia (ITP) (23%), glomerulonephritis (3%)
             None with SLE!




                                                                          Fig 2
   Fig 1 (Pisitikun)
                                                                         •What is the explanation for the increased levels of the
    Is the specificity of the anti-nuclear antibodies different          Autoantibodies of both specificities when the Yaa is present?
    when Yaa is present? (Homogenious – anti-chromatin;
    Speckled – anti-nucleolar)                                           •Why did the authors look at Btk?

                                                                         •When Btk is absent, what is the consequences on antibody levels
                                                                         And on disease?




CD69=an
activation
marker




                                 Chimeric mice: “a” allotype is FcR-/-
                                 “b” allotype is Fcr-/- Yaa




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                                                                          Fig 4    Why did they focus on TLR7?
                                                                                           Expressed on Y chromosome
                                                                                           Expressed in B cells
    Fig 3   FISH studies…Why did they focus on the X and Y chromosomes?                    Binds Btk
               Whole chromosomes          X and Y only                                     Ligand is ssRNA (part of nucleolar antigen)




                                                                           In C,D black=B6; white B6 Yaa; grey B6 Btk-/-; E no imiquimod, F with imiquimod




Questions?                                                                                       Scleroderma – means hard skin
                                                                                                  Autoantibodies: anti-centromere, anti-nucleolar,
                                                                                                  Anti-PDGF, topoisomerase etc.
 Any problems with this study? (phenotype of
 systemic sclerosis?)
 How would you make this study more
                                                                          Scleroderma, which is often known as systemic sclerosis, is a progressive and
 relevant to patients with autoimmunity? Are                              chronic connective tissue disorder, and is often classified as a rheumatic
                                                                          disease. Some unknown factor triggers the over-production of collagen (body
 there known polymorphisms, gene                                          protein) causing thickening, hardening and scarring of the skin and other organs.
 duplications or deletions in TLRs in man?                                Normally collagen keeps the skin soft, but the overproduction makes the affected
                                                                          tissue thick and hard. This, in turn, affects the amount of blood the small vessels
 What would you do next?                                                  carry to many parts of the body.




Paper 3 – Common Variable                                                      Cunningham-Rundles Fig 1: Does CpG stimulation cause the induction
Immunodeficiency                                                               of CD86 in the patients or controls?


 32 patients
  Age 18-73, both sexes
  Only 6 of them with slightly low % of CD19+ B-                                                                                            Normal
  cells (most within normal range)
  Low CD27+ and CD27+/IgM- (switched memory)
  Lymphocyte count low in only 8                                                                                                            CVID

  Antibody deficiency in IgM, IgG and IgA
 Had seen that there was an impaired
 response to a HIV rev gene which is a CpG                                             CD86 – B7.2
 of the “B” class (why look at HIV?)




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Fig 2: Can the B-cells from the patients be activated either through          Fig 3: Responsiveness to different doses of agonistic and control oligos
a TLR and/or the BCR? Why did they use anti-IgM?                              (ODN) as measured by cytokine production



                                                         Box is 10% of the
                                                         range, top and
                                                         bottom lines are
                                                         25 percentile




                                                                               Fig 5: Permeabilized cells – expression of TLR9 (protein) in B cells of
  Fig 4: Surface expression of TLR9 on memory B-cells when                     CVID patients or normal controls
  activated with CpG




      Fig 6: mRNA copy number in patients and normal controls
      (remember expression goes up in memory B-cells)
                                                                             Plasmacytoid DCs – high expression
                                                                             of TLR9 (Means et al 2005)




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     Fig 7: Intracellular TLR9 in plasmacytoid DCs – note similar levels in the
     CIVD patients and controls!
                                                                                  What is the reason the TLR9 does
                                                                                  not respond to the CpG?
                                                                                   Gene sequencing – no defects in TLR9 found
                                                                                   What could be another explanation?




            Despite normal expression, the TLR9 in CVID doesn’t appear
            to be responsive! (XLA another control – X-linked
            agammaglobulinemia)




                                                                                  Question

                                                                                   How would you improve this paper?
                                                                                   TLR9 expression in subsets of B-cells?
                                                                                   Other TLRs…ie TLR7. Is it non-functional
                                                                                   too? If so, maybe there is a problem in the
                                                                                   endosome.
                                                                                   If none of the B-cell expressed TLRs can be
                                                                                   stimulated perhaps a protein in the signal
                                                                                   transduction cascade is defective.




CVID and Autoimmunity                                                               Summary – TLR – Autoimmunity - CVID

Autoimmune states that can co-exist in                       Defect if
20-30% of patients with CVID                                 known
Autoimmune cytopenias, especially ITP and AHA (8%)           Low switched
                                                             memory
Sarcoidosis-like lung disease (27%)                          Low switched
                                                             memory, TNF
                                                             polymorphism
Inflammatory bowel disease (6%)                              Unknown
Rheumatoid arthritis, juvenile rheumatoid arthritis          Unknown                                                  Defective
                                                                                                                        TLR
Anti-IgA antibody, pernicious anemia                         Unknown                                                  response

Rare: Primary biliary cirrhosis, autoimmune thyroid          Unknown
disease, alopecia totalis, sicca syndrome, vitilago,
psoriasis, systemic lupus eythematous, vasculitis




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Discussion Point

 Based on these papers, how would you
 compare research using animal models
 versus humans with disease?




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