Lecture 3. Hematopoiesis: The process to produce immune cells

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• Lecture 3. Hematopoiesis The process to produce
  immune cells

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6-b. Hematopoiesis
CORE

• Stem cells and cytokines
• Hematopoiesis in bone marrow is regulated by some
  cytokines such as stem cell factor, IL-1, IL-3,
  IL-6, IL-7, GM-CSF, EPO, G-CSF and M-CSF

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Figure 1-10
Hematopoiesis occurs in the adult bone marrow
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Hematopoiesis generates immune cells
Stem cells 1. Self renewal 2.
Totipotency They are in bone marrow after fetal
development. They make all myeloid and lymphoid
immune cells and RBCs T cell progenitors migrate
to thymus and generate T cells B cell
progenitors reside in bone marrow to make naïve B
cells
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Myeloid vs. Lymphoid cells
Stem cells
Myeloid cells
Lymphoid cells
T cells T cell antigen receptor B cells B cell
antigen receptor NK cells no antigen-specific
receptor
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• Neutrophils Phagocytes
• Origin and maturation Bone marrow
• Antigen receptors No
• Function Phagocytosis and killing of
  microorganisms
• Where in blood circulation
• Sites of function infection sites
• Short life span

Induced by the cytokines IL-3, GM-CSF and G-CSF
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• Monocytes Macrophage precursors
• Origin bone marrow
• Antigen receptors No
• Function to become macrophages
• Present in blood circulation

IL-3, GM-CSF and M-CSF induce monocytes
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• Eosinophils worm (parasites) killers
• Origin bone marrow
• Antigen receptors No
• Function killing of antibody-coated parasites
  through release of killing mix (cytotoxic
  granules)
• Effector machinerycytotoxic granules, lipid
  mediators, cytokines and chemokines

IL-5 induces eosinophils
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• Mast cells parasite killers
• Origin bone marrow
• Antigen receptors No
• Function to kill parasites
• Sensor IgE receptor
• Effector machinerycytotoxic granules, lipid
  mediators, cytokines and chemokines
• Found in connective tissues

Stem cell factor (SCF) induces mast cells
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• Basophils relatives of mast cells and
  eosinophils
• Origin bone marrow
• Antigen receptors No
• Function important effector cells in allergic
  disorders and immune responses to parasites
• Sensor IgE receptor
• Effector machinerycytotoxic granules, lipid
  mediators, cytokines and chemokines

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• NK cells natural killers
• Origin Bone marrow and thymus
• Antigen receptors No
• Function Kill tumor and virus-infected cells
• Effector machinery (weapons) perforins and
  granzymes
• Activating or inhibitory receptors

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• T lymphocytes master regulators of the immune
  system
• Origin Bone marrow
• Maturation Thymus
• Differentiation to effector cells in secondary
  lymphoid tissues (lymph nodes, spleen, Peyers
  patch, and tonsils)
• Antigen receptors Yes
• Function regulates humoral and cell-mediated
  immune responses
• Mechanisms cytokines, cell surface molecules,
• cytotoxic granules.

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• B lymphocytes antibody producers
• Origin and maturation Bone marrow
• Differentiation to plasma B cells in secondary
  lymphoid tissues (lymph nodes, spleen, Peyers
  patch, and tonsils)
• Antigen receptors B cell receptor (cell surface
  immunoglobulins)
• Function Production of antibodies (IgM, IgE,
  IgA, and IgG)
• Regulated by T cells

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B lymphocytes
Antigens T cell help
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Figure 1-12
Circulating blood cells
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Hematopoietic stem cell therapy cytokine therapy

• Bone marrow transplantation into immunodeficient
  patients or cancer patients after
  radiation/chemotherapy
• Recombinant G-CSF and GM-CSF for neutropenia
  (neutrophils and monocytes)
• G-CSF Neupogen and Neulasta
• GM-CSF Leukine
• Help prevent infection by boosting leukocyte
  production.
• Recombinant erythropoietin (EPO) for anemia (red
  blood cells)
• EPOGEN

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• MHC molecules and antigen presentation to T cells

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Lecture objectives

• Big questions?
• How TCR recognizes antigens?
• What are the autoimmune diseases that are
  associated with particular types of MHC
  genotypes?
• Why MHC molecules are the major antigens
  responsible for transplantation rejections?
• To know
• Names of human MHC I and II genes.
• How antigens are processed for presentation on
  MHC I and II?
• Endogenous antigens and exogenous antigens?
• MHC class I and II molecules present antigens of
  different origins. How?
• MHC polymorphism
• How many different MHCs a person can express?
  Why?
• Structures of MHC I and II
• HLA typing

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A big pictureHow do T cells recognize antigens?
MHC molecule
TCR
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CORE

• Major Histocompatibility Complex (MHC Human
  Leukocyte Antigens HLA)
• a. Class I , II and III MHC genetic loci (short
  arm of chromosome 6)
• Major Class I genes HLA-A, B, C
• Minor Class I-like genes HLA-E, F, G, H, J, X
• Major Class II genes HLA-D region
• DP (A1, A2, B1, B2), DQ (A1, A2, B1, B2, B3),
  DR (A, B1, B2, B3)
• Major Class III genes Diverse (non-antigen
  presenting functions)

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Figure 3-25
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Genes in yellow Functional MHC II genes Genes
in dark gray pseudogenes (not expressed, so not
functional)
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CORE
c. Structure of Class I MHC proteins (1) a1, a2
and a3 domains of heavy chain (a1 a2 form
peptide binding site groove amino acid
differences account for polymorphism and antigen
specificity) (2) b2 - Microglobulin (invariant
but essential)
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CORE

• Structure of Class II MHC proteins
• (1) Composed of one a and one b chain
• (2) a1 and b1 domains comprise the peptide
  binding site (groove). Again, amino acid
  differences account for polymorphism and antigen
  specificity. a2 and b2 domains are constant.

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Figure 3-13 part 1 of 2
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Figure 3-2

T CELL RECEPTOR
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Figure 3-21
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Figure 3-15
The peptide-binding groove of MHC molecules
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CORE

• Inheritance
• Definition of haplotype,
• Example of inheritance pattern,
• Pseudogenes
• Cell surface expression (which cell types
  express Class I, Class II).
• Polymorphic nature of the MHC proteins (allotypes
  and gene polymorphism).

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CD8 T or NK cells
NK cells
Remains intracellular
NK cells
MHC CLASS I molecules form ligands to activate
CD8 cells and inhibit NK cells
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Polymorphism presence of multiple alternative
forms (alleles) of a gene.
Help peptide loading
Present antigen peptides to CD4 T cells
Polymorphism allows the population can handle a
variety of pathogens.
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Genetics of MHC gene expressionboth alleles are
expressed (co-dominant)

• In any mating, four possible combinations of
  haplotypes can be found in the offspring thus
  siblings are also likely to differ in the MHC
  allele they express.
• Halplotype The particular combination of MHC
  alleles found on a given chromosome 6.

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Present Ag to CD8 T cells
Present Ag to CD4 T cells
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Figure 3-28
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Figure 3-29
Each MHC isoform binds a characteristic set of
peptides-Anchor residues in peptides are
important for binding to MHC-Not all residues
are important
Degenerate binding allows each MHC molecule
handles many different peptides.
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Figure 3-26
Tom
Jane
Martin
John
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MHC things to remember

• MHC molecules in humans is also called HLA (human
  leukocyte antigen)
• Class I and II loci.
• HLA-DR alpha chain is monomorphic
• HLA-DRB1 is most polymorphic in MHC II genes
• HLA-DRB1 is always present in any individual
• HLA-DRB3/4/5 is present in some but not all
  people.
• A heterozygote person (most people) expresses two
  haplotypes.
• A person can express 3-6 class I and 3-8 class II
  isoforms.
• 2406 possible class II isoforms in the human
  population.
• 753 MHC I isoforms in the human population.
• MHC isoforms ? presentable antigen peptides

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Figure 3-22
Different cell distribution of MHC class I and II

• Almost all cells express MHC I for comprehensive
  surveillance by CD8 T cells
• Only some cells express high levels of MHC II
  and MHC I
• These are B cells, macrophages, dendritic cells
  and thymic epithelial cells.
• B cells, macrophages and dendritic cells are
  called professional antigen- presenting cells
  (APC).
• IFN-g increases the expression of MHC II in APC
  and induces the expression in non-APC cells at
  sites of infection

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• Processing and presentation of endogenous antigen
  via the MHC class I pathway (endogenous pathway)
• Cytoplasmic proteins (e.g. viral proteins) are
  ubiquitinated, hydrolyzed to peptide fragments in
  the proteasome, and the peptides are transported
  into the ER via TAP.
• 2. MHC I proteins are synthesized and assembled
  in ER and associated with TAP with the help from
  calnexin chaperone.
• 3. MHC I proteins bind peptides, vesicles fuse
  with plasma membrane, and MHC I/peptide complexes
  are expressed on cell surface and presented to
  CD8 cells.

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• Processing and presentation of exogenous antigen
  via the MHC class II pathway (exogenous pathway)
• Professional antigen processing cells internalize
  antigens.
• Antigens are internalized into endosome
  degraded into peptide fragments.
• MHC class II proteins are synthesized in ER and
  the peptide binding site is protected by Ii
  (invariant chaperone CLIP region of Ii protects
  the site).
• Exocytic vesicles (from Golgi) containing MHC II
  proteins fuse with endosome containing peptides.
• Ii is degraded, and CLIP is removed by HLA-DM
  protein.
• MHC II proteins bind peptides, vesicles fuse with
  plasma membrane, and MHC II/peptides are
  expressed on cell surface and presented to CD4
  cells.

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Two different types of antigensExtracellular
for MHC II and intracellular for MHC IThey are
processed differently.
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Figure 3-9
Antigen processing is required to present antigen
peptides to TCR.TCRs bind short antigen peptides
but not whole antigen proteins
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Figure 3-17
Transport of Cytosolic Peptides into ERRole of
transporter associated with antigen processing,
TAP
Bare lymphocyte syndrome No TAP expression ? no
MHC I expression ? no CD8 T cells ? defective
cytotoxic activity against virus-infected cells?
chronic respiratory infection TAPTAP1TAP2 Prot
easome a barrel shaped protein complex composed
of 28 subunits 8 or more AA-long polypeptides
are transported MHC I cannot leave ER without
loaded peptides
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Figure 3-18
Assembly of antigen peptide/MHC class I
complex.Molecular chaperons (calnexin,
calreticulin and tapasin)aid the folding of MHC
I and loading of peptides
In the absence of infection, self peptides are
presented on MHC, but do not activate T cells. A
HSV protein inhibits TAP function, and an Adeno
virus protein inhibits MHC I expression.
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Figure 3-20
Assembly of antigen peptide/MHC class II
complex-Extracellular microorganisms are taken
up by macrophages via phagocytosis and by B cells
via cell surface Ig-mediated endocytosis-MHC II
molecules bind peptides in the fused vesicles,
not in ER-Invariant chain, CLIP and HLA-DM guide
the peptide loading-After losing CLIP, MHC II
must bind peptides or gets degraded.-Certain
pathogens (e.g. mycobacteria), when engulfed,
prevent the fusion of phagosomes and lysosomes,
and persist in phagosomes.
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Figure 3-12
MHC class I molecules present antigens to CD8 T
cells, and MHC class II molecules present
antigens to CD4 T cells
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Figure 3-30
T cell receptor recognition of antigens is
MHC-restricted
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Figure 3-11
TCR recognition of antigens induces T cell
activation, functional maturation, and
killing/activation of target cells
The T cells cytokines are produced only when T
cells are engaged with APC
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CORE
f. Clinical importance of HLA antigens (1)
Transplantation and organ rejection (a) HLA
typing (i) Flow cytometry for HLA typing with
antibodies specific for each allotype. (ii)
RFLP analysis of class II genes (DNA digestion
with RE). (iii) PCR/sequencing-specific
oligonucleotide probes for HLA typing.
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HLA typing how?Microcytotoxicity assay for
detection of HLA antigensAnti-HLA serum, or
monoclonal antibody, is mixed with live
lymphocytes. Specific antibody binds to the
polymorphic protein moiety of the HLA molecule
expressed on the cell surface. Exogenous
complement is added to the well which will result
in lysis of cells to which antibody has been
bound. Cell death is determined by ethidium
bromide vital stain exclusion. Flow cytometry
or ELISAMonoclonal antibodies to different MHC
alleles have been generated.Using panels of
these antibodies, HLA typing before
transplantation is possible.RFLP Restriction
Fragment Length PolymorphismDigestion of genomic
DNA with certain restriction enzymes followed by
hybridization with radio-labeled MHC gene probes
gives MHC isotype-specific digestion
patterns.PCR Polymerase Chain ReactionPCR
using MHC gene-specific primersand DNA
sequencing
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Figure 5-14
Another (crude) method Mixed Lymphocyte Reaction
(MLR) is used to test for HLA compatibility
between individuals
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CORE

• Diseases associated with specific HLA antigens
• If you have the MHC allotypes (left), you have a
  higher chance of getting the following diseases
  (right)
• HLA-DR3/DR2 ?Systemic Lupus Erythematosus
• HLA-DR4 ? Rheumatoid Arthritis
• HLA-B7 and DR2 ? Multiple Sclerosis
• HLA-B8, DR3/DR4 ? Type 1 diabetes
• (e) HLA-B27 ? Ankylosing Spondylitis