Major Histocompatibility Complex

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Major Histocompatibility ComplexDr.R.Premechandra
n,MD.,MICROBIOLOGY
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HISTORY

• Gorer in 1930
• The antigen responsible for allograft rejection
  in mice that led to discovery of the major
  histocompactability complex.

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• Gorer identified two blood group antigen
• Antigen1-Common to all the strains
• Antigen2-Some strainsH2-Antigen
• The histocompatability antigens are cell surface
  antigen that induce an immune response leading to
  rejection of allografts.
• The major histocompatibility antigen closely
  linked multiallelic cluster of genes which was
  called the major histocompatibility complex.

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• Dausset studies on human leucocyte antigen,later
  on major histocompatibility antigen in human
  beings.
• The genetic basis of immune response proved by
  Benacerraf and colleagues.
• Three different classes of protein
• Class1protein that determine histocompatibility
• Class2protein that regulate the immune response
• Class3proteinthat include complement system

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HLA Human Leucocyte Antigen

• HLA forms part of the Major Histocompatibiblity
  Complex (MHC)
• Found on the short arm of chromosome 6
• MHC antigens are integral to the normal
  functioning of the immune response.
• Essential role of HLA antigens lies in the
  control of self recognition and thus defence
  against micro-organisms and surveillance.

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• HLA comprises two classes Class I
• Class II
• . Class I A,B,C most significant (other loci
  eg E,F,G,H etc are not so important in
  transplantation)
• . Expressed on most nucleated cells
• . Have soluble form in plasma
• . Are adsorbed onto platelets (some antigens more
  readily than others)
• . HLA-A contains 24 different antigenic
  specificities,
• HLA-B contains 52 and HLA-C contains 11

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• HLA Class II five loci DR, DQ, DP, DM and DO
• HLA DR, DQ, DP most significant
• Expressed on B lymphocytes, activated T
  lymphocytes, macrophages, endothelial cells ie
  immune competent cells.
• Comprise 2 chains encoded by HLA genes, alpha and
  beta each with 2 domains.
• Hypervariable region is in the beta 1 domain
• HLA-DP contain 6 different antigenic
  specificities, HLA-DQ contains 9 and HLA-DR
  contains 20.

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• Class 3 or the complement region containing for
  complement components C2 and C4 of the classical
  pathway ,properdin factor B of the alternative
  pathway,heat shock protein and tumor necrosis
  factor alpha and beta.

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MHC I Molecules

• Membrane bound glycoprotein (Ig super family)
• Alpha chain
• Three domains
• Encoded by HLA complex
• Anchored in cell membrane
• Cytoplasmic tail
• Beta chain
• A single domain
• Encoded by a different gene on another chromosome
• Not anchored in cell membrane
• Bound to alpha chain non covalently
• Necessary for MHC I expression

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MHC I Molecules

• Antigen presenting features
• Alpha 1 and alpha 2 domains form peptide-binding
  cleft
• 8-10 amino acid peptides can bind to MHC I
  molecule
• Alpha 3 interacts with CD 8 on T cytotoxic cells

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Classical class 1 gene

• soluble antibodies recognized by B cells are
  effective only
• against some extracellular pathogens
• pathogens rarely leave traces of them on the
  surface of the
• cell -gt cells exhibit on their surface a sample
  of peptides
• the function of the classical MHC proteins is to
  bind the peptides
• and display them
• hence the name antigen-presenting proteins
• in addition, T cells only recognize antigens,
  that are
• associated with the same individuals MHC
  proteins
• T cells scan cells all the time to ensure there
  are no foreign
• motifs on their surface
• MHC proteins express both foreign and self
  peptides
• immune reaction depends on the T cells antigen
  recognition

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Nonclassical class 1 gene

• MHC class I-like genes HLA-E, HLA-F, HLA-G
• encode proteins similar to class I molecules in
  sequence and
• structure
• no such polymorphism
• may be encoded outside the MHC
• fulfill a variety of roles, often
  specialized antigen-presenting
• features
• some present lipids and bacterial cell wall
  components
• some NK-receptors recognize only HLA-E molecules
• HLA-G expressed at high level on maternal/fetal
  interface, role
• remains unclear
• even less known about HLA-F
• MHC class II-like genes HLA-DM and HLA-DO
• regulate peptide loading onto classical MHC class
  II molecules

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MHC II Molecules

• Membrane bound glycoprotein (Ig super family)
• Alpha chain
• Two domains
• Encoded by HLA complex
• Anchored in cell membrane
• Cytoplasmic tail
• Beta chain
• Two domains
• Encoded by HLA complex
• Anchored in cell membrane
• Cytoplasmic tail

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MHC II Molecules

• Antigen presenting features
• Alpha 1 and beta 1 domains form peptide-binding
  pocket
• 13-18 amino acid peptides can bind to MHC II
  molecule
• Alpha 2 and beta 2 interact with CD 4 on T helper
  cells

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MHC I Peptide Interaction

• Stable associations
• Endogenously processed antigens
• Up to six allelic variants
• 100,000 MHC molecule variants per cell
• 100 MHC-peptide complexes required for T
  cytotoxic cell recognition

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MHC II Peptide Interaction

• Stable associations
• Exogenously processed antigens
• Up to 12 allelic variants

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MHC Diversity

• Genetically determined
• Polygenic characteristic
• Different alleles
• Genes with overlapping functions
• 12 million haplotypes
• Diversity in HLA type

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MHC I Expression

• Varies by cell type
• Lymphocytes high
• Hepatocytes low
• Regulated by cytokines
• Interferons
• TNF
• Suppressed by viruses
• Herpes family
• Hepatitis B

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MHC Restriction

• Endogenously processed cytosolic peptides in
  virus infected cells or tumor cells are
  transported to the surface of the cells
• They bind to MHC I molecules to be recognized
  by cytotoxic T-cells which then kill these cells
• In other words
• T-cells are only activated when they recognize
  both antigen and class I MHC molecules in
  association

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Inheritance of MHC(HLA) Type

• Polymorhisms
• Many allelic types within a species
• Haplotype
• The genetic loci of MHC are closely linked
• Inherited as a group (haplotype)
• One haplotype from each parent
• Co-dominant expression

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Inheritance of MHC

• Inbred mice
• Homozygous parents
• F 1 generation has MHC loci from both
  (heterozygous)
• F1 generation can accept grafts from either
  parent
• Neither parent can accept graft from F1 offspring
• WHY?

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Inheritance of MHC

• Humans
• Heterozygous parents
• F 1 generation has MHC loci from both
  (heterozygous)
• Four combinations
• F1 generation can not accept grafts from either
  parent
• Neither parent can accept graft from F1 offspring
• 1 in 4 F1 generation are HLA compatible

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• Syngeneic-Identical at all genetic loci.
• Polygenic-Similar gene but not identical
  structure and function.
• Congenic-Gentically identical except at a single
  genetic locus or region.
• Codominantly expressed-Both maternal and paternal
  gene products are expressed in the same cells.
• Linkage disequilibrium-Difference between the
  frequency observed for a particular combination
  of alleles and expected from the frequency of the
  individuals alleles.

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• Transplantation and Graft
  Rejection

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Types of grafts

• 1) Autografts
• The transfer of an individuals own tissues
• from place to place
• e.g. Skin grafts (regularly accepted)
• 2) Isografts
• Transfer of tissues between genetically
• identical persons
• e.g. Identical twins ( accepted permanently)

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• 3) Allografts (homograft)
• - Transfer of a graft between genetically
  different
• members of same species
• e.g from one human to another
• - Rejection occur if donor and recipient are
  not matched
• 4) Xenograft (heterograft)
• - Transfer of tissues between different
  species
• - Always rejected

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Mechanism Of Graft Rejection

• 1) Both TH and TC are activated
• - TC cells destroy graft cells by direct
  contact
• TH cells secrete cytokines that attract and
  activate macrophages, NK cells and polymorphs
  leading to cellular infiltration and destruction
  of graft (Type IV)
• - B cells recognize foreign antigens on the
  graft and produce antibodies which bind to graft
  cells and
• . Activate complement causing cell lysis
• . Enhance phagocytosis, i.e. opsonization
  (Type II)
• . Lead to ADCC by macrophages, NK,PML
• - Immune complex deposition on the vessel
  walls induce platelets aggregation and
  microthrombi leading to ischemia and necrosis of
  graft (Type II)

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Types Of Graft Rejection

• !) Hyperacute rejection
• - It occurs hours after transplantation
• - In individual with preformed antibodies
  either due to - blood groups incompatibility or
  previous sensitization
• by blood transfusion, previous
  transplantation
• 2) Acute Rejection
• - It occurs 10 to 30 days after
  transplantation
• - It is mainly T-cell mediated
• 3) Chronic or late rejection
• - It occurs over a period of months or years
• - It may be cell mediated, antibody mediated
  or both

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Graft Versus Host (GVH) Reaction

• An immunologically competent graft is
  transplanted into an immunologically suppressed
  recipient (host)
• The grafted cells survive and react against the
  host cells
• i.e instead of reaction of host against the
  graft,
• the reverse occurs
• GVH reaction is characterized by fever,
  pancytopenia, weight loss, rash , diarrhea,
  hepatsplenomegaly

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HLA and diseases (1)

• HLA complex has been associated with over a 100
• diseases, many of them autoimmune diseases
• includes diseases such as asthma, type I
  diabetes,
• rheumatoid arthritis, psoriasis, MS
• among all genes studied for possible association
  with
• autoimmune diseases, the best candidate genes are
• MHC genes
• most autoimmune diseases associated with class II
  MHC
• genes, for example diabetes mellitus associated
  with the
• combination of certain HLA-DR and HLA-DQ alleles
• ankylosing spondylitis associated with HLA-B27
  allele
• individuals expressing it have 90-100 times
  bigger risk of
• developing AS than those who dont express HLA-B27

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HLA and diseases (2)

• disease-associated HLA alleles found in healthy
• individuals as well
• expressing a certain HLA allele seems to be a
• necessary but not a sufficient condition
• according to one of the models, both genetic
• predisposition determined by MHC genes and
• exposure to certain environmental agents are
  needed
• in order to develop an autoimmune diseases
• certain HLA alleles encode proteins that present
• autoantigens with greater efficiency than
  healthyones?

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LAB DIAGNOSIS

• Complement Dependent Cytotoxicity (CDC)
• Viable peripheral blood lymphocytes are obtained
  by discontinous density gradient centrifugation
  using Ficoll / Tryosil or Ficoll / Sodium
  Metrizoate at a density of 1.077 at 19º - 22ºC.
• Microlymphocytotoxic test 3 stages

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• 1.Viable lymphocytes are incubated with HLA
  specific antibodies.
• If the specific antigen is present on the cell
  the antibody is bound.
• 2.Rabbit serum as a source of complement is
  added, incubate. If antibody is bound to the HLA
  antigen on the cell surface it activates the
  complement which damages the cell membrane making
  it permeable to vital stains.

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• 3.Results are visualised by adding dye usually a
  fluorochrome eg Ethidium Bromide although both
  Trypan Blue and Eosin have been used in the past.

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Cellular typing

• Not / Rarely used by laboratories these days.
• Requires panels of homozygous typing cells.
• Cell culture method therefore takes a long time.
  Labour intensive involves use of radioisotopes.

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Molecular Methods

• Southern Blot technique was used to identify
  restriction fragment length polymorphisms
  (RFLPs)
• polymerase chain reaction (PCR)
• Electrophoresis
• Luminex technology SSOP based. Just beginning
  to be introduced into laboratories for routine
  use on non urgent samples.

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• TREATMENT

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Immunosuppresseive agents
corticosteroids, prednisone
Anti-inflammatory, altering T-cell and PMN traffic
organ transplant, hypersensitivity, autoimmunity
inhibition of IL-2 production by T cells
Cyclosporin, FK-506
organ transplant
Inhibition of T cell activation by IL-2
rapamycin
organ transplant
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Immunosuppresseive agents
azathioprine, 6-MP
purine metabolism
organ transplant
folate metabolism
organ transplant
methotrexate
alkylation of DNA, RNA and proteins
autoimmune diseases, organ transplant
cyclophosphamide, melphalan
malignancy/marrow transplantation
x-irradiation
Lymphopenia
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• Thanks