Basic Immunology and Multiple Sclerosis

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					Basic Immunology
       and
 Multiple Sclerosis



   Ben Thrower M. D.
 Shepherd MS Institute
      Atlanta, GA
        Why Immunology?
                         disease.
• MS is an autoimmune disease
• Existing and future therapies work by
  modulating or suppressing the immune
  system.
             References
1.        AK
1 Abbas AK, Lichtman AH AH.
   Basic Immunology: Functions and
                            System. W.B.
    Disorders of the Immune System W B
    Saunders Company. 2001.
2. Sospedra M M ti R A
2 S      d M, Martin R. Annu. R Rev.
    Immunol. 2005; 23: 683-747.
Immunology is like an onion …..
                it s
….. not because it’s stinky and makes
         you want to cry…..
                                  layers.
….. but because there are lots of layers
Overview of the Immune System
                             Immune System




                                                      Adaptive
             Innate
                                                      (Specific)
        (Nonspecific)                             o line of defense
                                                2
       1o line of defense                      Protects/re-exposure




  Cellular             Humoral            Cellular              Humoral
Components            Components        Components             Components
        Function of the Immune System
         (Self/Non-self Di
         (S lf/N              i i ti )
                      lf Discrimination)

•       To protect from pathogens
    •                 ( g
        Intracellular (e.g. viruses and some bacteria
        and parasites)
    •   Extracellular (e.g. most bacteria, fungi and
        parasites)


•   To eliminate modified or altered self
    Effects of the Immune System
• Beneficial:
     •   Protection from invaders
     •   Elimination of altered self

•   Detrimental:

     •   Discomfort and collateral damage

         (inflammation)
         (i fl    ti )
     •   Damage to self (hypersensitivity or
         autoimmunity)
Innate (Nonspecific) Immunity
      Innate Host Defenses Against
                Infection
                I f ti
• Anatomical barriers
  – Mechanical factors
  – Chemical factors
  – Biological factors

• Humoral components
  – Complement
    Coagulation system
  – C     l ti     t
  – Cytokines

• Cellular components
  –   Neutrophils
  –   Monocytes and macrophages
  –   NK cells
  –   Eosinophils
  The Adaptive Immune System


• Cell-mediated Immunity (Cytotoxicity)
  • T cells
     • CD4+ (Th1 & Th2)
     • CD8+


• Humoral Immunity (Antibody production)
  • B Cells
    Comparison of Innate and Adaptive
               Immunity
               I       it

    Innate Immunity             Adaptive Immunity

•    No time lag            •   A lag period


•    Not antigen specific   •   Antigen specific


•    No memory              •   Development
                                of memory
          Cells of the Immune System

                                      Immune System




          Myeloid Cells                                       Lymphoid Cells




Granulocytic          Monocytic                T cells           B cells       NK cells




Neutrophils         Macrophages             Helper cells
 Basophils
 B    hil            Kupffer ll
                     K ff cells           S
                                          Suppressor cells
                                                         ll    Pl       ll
                                                               Plasma cells
Eosinophils         Dendritic cells        Cytotoxic cells
       Immune Interactions
• Cell Receptors
       Immune Interactions
• Cell Receptors

                   Endothelial Lining
        Immune Interactions
• Cytokines – Powerful chemical substances
  that allow for interactions between cells of
               system
  the immune system.
        Immune Interactions
• Chemokines – Chemicals used by one
  immune cell to attract another cell to an
          inflammation
  area of inflammation.
    Cells of the Immune System
• Lymphocytes
     1) B cells, T cells,
                            cells.
        Natural killer (NK) cells
     2) Have receptors for specific
        antigens.
        antigens
     3) Key mediators of adaptive immunity.
     4) Nomenclature based upon surface
        proteins called CD (cluster of differentiation).
 Cells of the Immune System
• Antigen-presenting cells (APC)
      1) Dendritic cells, macrophages,
             ll    i     li (CNS).
         B cells, microglia (CNS)
      2) Capture of antigen for display to
         lymphocytes.
               Cytokines
                                 immunity,
• Mediators involved in cellular immunity
  including hormone-like glycoproteins
  released by activated T cells and
  macrophages.
              co-stimulators
• Some are co stimulators of T cells and T
  cell proliferation.
      T cells
      T-cells
                CD8
                T cell



APC    Naïve
       T cell

                 CD4
                 T cell
         CD4 T Helper Cells
• Activation of macrophages
• Activation, proliferation and differentiation
  of T and B lymphocytes
 Major Histocompatibility Complex
              (MHC)
• Proteins expressed on the cell surface
  which play a role in the immune system
       autoimmunity.
  and autoimmunity
• Referred to as HLA (human leukocyte
              humans.
  antigen) in humans
 Major Histocompatibility Complex
              (MHC)
• MHC class I – present on all nucleated
  cells. Present antigen fragments to CD8
  cells
  cells.
• MHC class II – present on antigen
                   (APC).
  presenting cells (APC) Present antigen to
  CD4 cells.
      CD4+ T Helper Cells

      MHC II – Major Histocompatibility Complex*



                     CD4
APC                TCR

                      CD8




            Immature CD4+/CD8+ T Cell


      * MHC = HLA (Human Lymphocyte Antigen)
CD4+ T Helper Cells



         CD4
       TCR

          CD8




      Mature CD4+ Cell
        CD4 / APC Receptors



             CD4
             TCR        Class II MHC

             CD3
             CD28   B7-1/B7-2 (CD80/CD86)
            LFA-1         ICAM -1
VLA-4



CD4 T Helper Cell              APC
        Immunological Synapse”
   The “Immunological Synapse
• The interaction
  between the TCR and
  MHC molecules is not
  strong
• Accessory molecules
  stabilize the interaction
   – CD4/Class II MHC or
     CD8/Class I MHC
   – CD2/LFA-3
     LFA 1/ICAM 1
   – LFA-1/ICAM-1
        Immunological Synapse”
   The “Immunological Synapse
• Specificity for antigen
  resides solely in the
  TCR
• The accessory
  molecules are
  invariant
• Expression is
  increased i response
  i         d in
  to cytokines
         Immunological Synapse”
    The “Immunological Synapse
     g g
• Engagement of TCR and
  Ag/MHC is one signal
  needed for activation of T
  cells
• Second signal comes from
  costimulatory molecules
   – CD28 on T cells interacting
     with B7-1 (CD80) or B7-2
     (CD86)
   – Others
• Costimulatory molecules
  are invariant
             CD4 / APC Receptors



                  CD4
Costimulatory     TCR    Class II MHC
      p
  Receptor
                  CD3
                  CD28    B7-1/ B7-2
                 LFA-1     ICAM -1
     VLA-4



     CD4 T Helper Cell                 APC
            CD4 / APC Receptors



                CD4

Adhesion        TCR    Class II MHC
Receptors
R    t
                CD3
                CD28    B7-1/ B7-2
               LFA-1     ICAM -1
   VLA-4



   CD4 T Helper Cell                 APC
        CD4 / APC Receptors



             CD4
             TCR    Class II MHC
    g
Integrins
             CD3
             CD28    B7-1/ B7-2
            LFA-1     ICAM -1
VLA-4



CD4 T Helper Cell                 APC
       Costimulation is Necessary for T Cell
                    Activation
•   Engagement of TCR and
    Ag/MHC in h b             f
    A /MHC i the absence of co-
    stimulation can lead to anergy

•   Engagement of co-stimulatory
    E            t f    ti l t
    molecules in the absence of
    TCR engagement results in no
        p
    response
•   Activation only occurs when
    both TCR and co-stimulatory
    molecules are engaged with
    their        ti li   d
    th i respective ligands
•   Downregulation occurs if CTLA-
    4 interacts with B7
     – CTLA 4 sends an inhibitory
        CTLA-4            inhibitor
        signal
Key Steps in T cell Activation
            p
• APC must process and p          peptides to T cells
                          present p p
• T cells must receive a costimulatory signal
   – Usually from CD28/B7
• Accessory adhesion molecules help to stabilize binding
  of T cell and APC
   – CD4/MHC-class II or CD8/MHC class I
     LFA-1/ICAM-1
   – LFA 1/ICAM 1
   – CD2/LFA-3
• Signal from cell surface is transmitted to nucleus
   – Second messengers
• Cytokines produced to help drive cell division
   – IL-2 and others
    Naïve CD4 T cells differentiate into TH1 and TH2
                          cells
                                  Naïve
                                  CD4
                                  T cell
               Th1                                         Th2

                                                       Anti-
      Pro-inflammatory
                                                       inflammatory
      Cytokines
          • IL-2
                                                       Cytokines
          • Il-12
                          Reciprocal Inhibition             • IL-4
                                                            • IL-5
          • IFN-γ
                                                            • Il-10
          • TNF
                                                            • IL-13
                                                            • TGF-β (TH3)


                                                  B cell
             Macrophage


Yong, W
                   CD4 Subsets
                        CD4
                        T cell

           IL-12
           IL 12
                                  IL-6/IL-23
                      IL-4


 CD4                                      CD4
Th1 cell                CD4             Th17 cell
                       Th2 cell
                            Cells
                   CD4 Th17 C ll

• Recently described subset of CD4 cells.
                       IL-17     TNF
• Cytokines produced = IL 17 and TNF-
  alpha.
  Stimulated by IL-6 d IL-23 t ki
• Sti l t d b IL 6 and IL 23 cytokine.




 Bettelli E, Carrier Y, Gao W, et al. Nature 2006; 441(7090):235-238.
      T cells
      T-cells
                CD8
                T cell



APC    Naïve
       T cell

                 CD4
                 T cell
             CD8 T Cells
• Cytotoxic CD8 cells are the only T cells
  capable of killing other cells by direct
  contact.
  contact
• CD8 cells may also serve a regulatory or
               role.
  suppressive role
           CD8+ T Cells

      MHC I – Major Histocompatibility Complex*



                     CD4
APC                TCR

                      CD8




            Immature CD4+/CD8+ T Cell


      * MHC = HLA (Human Lymphocyte Antigen)
CD8+ T Cells
                       TC
                    Cytotoxic


    CD4
  TCR

     CD8
                        TS
                    Suppressor

 Mature CD8+ Cell
        CD8 / APC Receptors



               CD8
               TCR     Class I MHC

               CD3
               CD28    B7-1/ B7-2
               LFA-1    ICAM -1
VLA-4



  CD8 T Cell                      APC
              Cytotoxic T Cell (Tc)
          ,                  ,            y
• TC cells, or killer T cells, are the only T cells that
  can directly attack and kill other cells
• They circulate throughout the body in search of
  body ll h display h               i        hi h h
  b d cells that di l the antigen to which they
  have been sensitized
• Their targets include:
   –   Virus-infected cells
   –                                        p
       Cells with intracellular bacteria or parasites
   –   Cancer cells
   –   Foreign cells from blood transfusions or transplants
      Mechanisms of Tc A ti
      M h i       f    Action
• In some cases, TC cells:
  – Bind to the target cell and release perforin into
    its membrane
     • In the presence of Ca2+ perforin causes cell lysis
       by creating transmembrane pores
• Other TC cells induce cell death by:
  – Secreting lymphotoxin, which fragments the
    target cell’s DNA
  – Secreting gamma interferon, which stimulates
    phagocytosis by macrophages
                B Cells
• B cells may mature into antibody
  producing plasma cells.
                          APC s.
• B cells may function as APC’s
B Cell – CD4 Th Interactions



          CD40                CD 40 Ligand

                                             CD4 Th
 B Cell                       TCR             C ll
                                              Cell
          MHCII


                                    CD28
           B7




          Cytokine Receptor
B Cell Proliferation    CD4 Activation
and Differentiation




          CD40         CD 40 Ligand

                                      CD4 Th
 B Cell                TCR             Cell
                                       C ll
          MHCII


                             CD28
           B7
                Antibodies
• Also called immunoglobulins
  – Constitute the gamma globulin portion of
    blood proteins
  – Are soluble proteins secreted by activated B
    cells and plasma cells in response to an
    antigen
           p              g p         y
  – Are capable of binding specifically with that
    antigen
• There are five classes of antibodies:
  IgD, IgM, IgG, IgA, and IgE
    Natural Killer (NK) Cells
                   antibody coated cells.
• Bind to and kill antibody-coated cells
• Called “natural killers” because they do not
                              first.
  have to recognize antigen first
               g
       Immunological tolerance
• Definition:
  – Specific immune unresponsiveness to an antigen that
    is induced by exposure of lymphocytes to that antigen
    (tolerogen vs immunogen)
• Significance:
  – All individuals should be tolerant of their own
    antigens (self-tolerance); breakdown -->autoimmunity
                                             p
  – The induction of tolerance could be exploited to treat
    autoimmune diseases
  – Mechanisms of tolerance must first be understood
 Mechanisms of unresponsiveness
         to lf ti
         t self antigens
• Central tolerance
   – Immature self-reactive T lymphocytes that recognize self
         g              y           g   g
     antigens in the thymus undergo negative selection
     (deletion).
• Peripheral tolerance
     Mature self-reactive T lymphocytes th t escape central
   –M t          lf   ti     l    h   t that              t l
     tolerance and recognize self antigens in peripheral tissues
                          (anergy),     (         )    g
     can be inactivated (anergy killed (deletion) or regulated
                               gy),
                               gy
     (suppressed).
• “Clonal ignorance”
   – Mature self-reactive lymphocytes do not respond to self
     antigens in non-inflamed settings.
Multiple Sclerosis

What went wrong?
  Key Steps in the MS Process
1.
1 A breakdown in the ability to distinguish
   self from non-self.
2.
2 Upregulation of myelin reactive CD4 Th1
   cells.
3. Increased permeability of the BBB.
3 I         d          bilit f th BBB
4. The role of B cells and CD8 cells.
Requirements for the development
   of an autoimmune disease




     Nature Immunology (9): 759-761 (2001)
     HLA Type and MS Risk
• Many autoimmune diseases in humans
  are linked to particular HLA alleles.
• HLA DR2 and DR4 haplotypes are
  associated with an increased risk for MS.
     Environmental Factors
• Many pathogens have been proposed to
  have a role in the pathogenesis of MS.
  HHV-6, EBV,
• HHV 6 EBV Chlamydia pneumonia
        Molecular Mimicry
• The pathogen contains similar peptide
  sequences to peptides found in myelin
 (MBP, MAG           others).
 (MBP MAG, MOG others)
• The pathogen may have similar three-
  di      i  l t t       th t      t
  dimensional structures that promote cross-
  reactive immunity.
   Immune Dysfunction in MS
• Myelin autoreactive CD4 cells may have
  less of a need for co-stimulation to
  become activated in a person with MS MS.
• Downregulation of CD4 cells via CTLA-4
                      MS.
  may be impaired in MS
Innate and Adaptive Immune
         Responses


           TLR (Toll Like               Autoreactive
                            Cytokines
             Receptor)
                                         CD4/CD8

Pathogen




           Innate Immune Cell
           I   t I       C ll
    Naïve CD4 T cells differentiate into TH1 and TH2
                          cells
                                  Naïve

                                  T cell
               Th1                                         Th2

                                                       Anti-
      Pro-inflammatory
                                                       inflammatory
      Cytokines
          • IL-2
                                                       Cytokines
          • Il-12
                          Reciprocal Inhibition             • IL-4
                                                            • IL-5
          • IFN-γ
                                                            • Il-10
          • TNF
                                                            • IL-13
                                                            • TGF-β (TH3)


                                                  B cell
             Macrophage


Yong, W
Th1 / Th2 Imbalance


Th1             Th2




      Normal
Th1 / Th2 Imbalance




       MS
   Increased BBB Permeability
                                increased,
• Some chemokine levels are increased
  leading to increased adhesion and
  attraction of autoreactive T cells into the
  CNS.
• Increased production of matrix
  metalloproteinase leads to increased BBB
  permeability.
  permeability
    The Role of B Cells in MS
• Increased immunoglobulin (Ig) production
  in the CSF, but not the serum.
• CSF Ig in MS shows an oligoclonal
  distribution, i.e. a limited number of B cell
                                production.
  clones contribute to the Ig production
How Do B Cells and Ab Contribute
      h Pathogenesis i MS?
  to the P h        i in
                        APC’s
• B cells may serve as APC s for
  autoreactive T cells.
• B cells provide costimulation for
  autoreactive T cells.
       ll       help     it t       ti
• B cells may h l recruit autoreactive T
  cells into the CNS.
• B cells produce myelin-specific
  autoantibodies that destroy myelin.
  The Role of CD8 Cells in MS
• Cells in the CNS express MHC I much
  more so than MHC II.
  Myelin-reactive
• Myelin reactive CD8 cells are more
  common than myelin-reactive CD4 cells in
             tissue
  MS brain tissue.
• Myelin-reactive CD8 cells secrete
   h      ki     for li      ti        ll
  chemokines f myelin-reactive CD4 cells.
      Mechanism of Action of
        Current Therapies
• Glatiramer acetetate
• Beta interferons
• Natalizumab
 MOA: Glatiramer acetate (GA)
• Anti-inflammatory effects
   – Th2/ T         ll
            Treg cells
       • GA promotes the development of Th2 and Treg cells
       • Release anti-inflammatory cytokines reducing the
         activity of Th1 cells in the periphery and CNS
   – Innate immune effects
       • Direct effect on monocytes/APCs yielding anti-
         inflammatory cytokine producing phenotype
       • Polarizes CD4+ T cell development down a Th2 and
         Treg path
                                    CD8
   – Possible role for regulatory CD8+ cells
       • Induces a population of GA-reactive CD8+ T cells that
         can suppress or even kill CD4+ T cells



   Weber MS, et al. Neurotherapeutics. 2007;4:647-53., Arnon R, et al. Mol Neurobiol. 2007;36:245-53.
  MOA: Glatiramer acetate (GA)
               p
• Enhanced reparative p  processes
   – Neurotrophic factors
      • GA reactive cells and resident CNS cells produce
        BDNF, NT-3/4, IGF-1, and GDNF
   – Remyelination
      • Increases proliferation, differentiation, and survival of
        OPCs
   – Neurogenesis
      • Augments neuronal progenitor cell proliferation,
        differentiation, and migration to sites of damage; axonal
        sprouting noted




   Weber MS, et al. Neurotherapeutics. 2007;4:647-53., Arnon R, et al. Mol Neurobiol. 2007;36:245-53.
MOA: GA
MOA: GA
MOA: GA
IFN β may shift T cells from a pro-inflammatory to
                       y profile in the p p y
   an anti-inflammatory p               periphery
     IFN β                                            IL-4, IL-10, IL-131,2
                                 Th2



     IFN-α/βR



     T cells                    T suppressor                          Activity3




                X
                                                                       IFNγ4
                                       Th1


                                        1. Mei E, et al. J Neurol Sci 2006; 246(1-2): 71-7
                          2. Wiesemann E, et al. J Neuroimmunol 2002; 123(1-2): 160-9
                                    3. Noronha A, et al. Ann Neurol 1990; 27(2): 207-10
                             4. Sega S, et al. Clin Neurol Neurosurg 2004; 106(3): 255-8
      IFN β decreases adhesion molecule and MMP
   expression, blocking T-cell migration across the BBB
     p                g          g
                                         Periphery                         BBB                             CNS

                     Increase in soluble                  IFN β
                    adhesion molecules
                          sVCAM-11
                                                                                                  MMP-9 is required
                                                        VLA4/VCAM-1
                                                        VLA4/VCAM 1                               for migration of T
                                                                                                  cells through the
                                                                   X                                  basement
                                                                                                  membrane of the
                                                                                                        BBB2-4
    IFN β
                                                      Activated
                                VLA4                   T cell
                                                                                            Decreased
                                                                                             MMP-9

                                                                                  1. Trojano M, et al. Neurology 1999; 53(7): 1402-08
                                                                                        j       ,               gy      ; ( )
Downregulation of                                                                   2. Nelissen I, et al. Brain 2003; 126(Pt6): 1371-81
 VLA4 in T cells5,6                    sVCAM-1 (decoy)                             3. Stone LA, et al. Ann Neurol 1995; 37(5): 611-19
                                                                                    4. Stuve O, et al. Ann Neurol 1996; 40(6): 853-63
VCAM-1=vascular cell adhesion molecule ; VLA-4=very               5. Soilu-Hanninen M, et al. J Neuroimmunol 2005; 167(1-2): 175-82
  late antigen 4; MMP-9=matrix metalloproteinase 9                        6. Murano PA, et al. J Neuroimmunol 2000; 111(1-2): 186-94
Natalizumab MOA




           Natalizumab
Thanks!