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					Lecture 3. Hematopoiesis: The process to
          produce immune cells
CORE


         6-b.      Hematopoiesis
(1) 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
Hematopoiesis occurs in the adult bone marrow


           Figure 1-10
Hematopoiesis generates immune cells



                        Hematopoietic stem cells:
                               1. Self renewal
                               2. Totipotency

                        They are in bone marrow after
                        birth

                        They make immune cells,
                        platelets, and RBCs

                        T cell progenitors migrate to
                        thymus and generate T cells

                        B cell progenitors reside in
                        bone marrow to make naïve B
                        cells
                Myeloid vs. Lymphoid cells
Myeloid cells          Stem cells

                                     Lymphoid cells




                              T cells: T cell antigen receptor
                              B cells: B cell antigen receptor
                              NK cells: no antigen-specific receptor
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
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
Eosinophils: worm (parasites) killers
 Origin : bone marrow
 Antigen receptors: No

 Function: killing of antibody-coated parasites through
  release of killing mix (cytotoxic granules)
 IgE&IgG receptors
 Effector machinery: cytotoxic granules, lipid mediators,
  cytokines and chemokines
    T-cell-derived IL-5 induces eosinophil proliferation
Mast cells: parasite killers
 Origin : bone marrow; found in skin, lungs, and digestive tract
 Antigen receptors: No

 Function: to kill parasites
 Sensor: IgE receptor
 Effector machinery:cytotoxic granules, lipid mediators,
  cytokines and chemokines
 Found in connective tissues
       Stem cell factor (SCF) induces mast cells
Basophils: relatives of mast cells and eosinophils
 Origin : bone marrow
 Antigen receptors: No
 Circulating granulocytes

 Function: important effector cells in allergic disorders and
  immune responses to parasites

 Sensor: IgE receptor
 Effector machinery:cytotoxic granules, lipid mediators,
  cytokines and chemokines
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; Fc receptors
T lymphocytes: master regulators of the immune system

 Origin: Bone marrow
 Maturation: Thymus
 Differentiation to effector cells in secondary lymphoid
  tissues (lymph nodes, spleen, Peyer’s patch, and tonsils)

 Antigen receptors: Yes
 Function: regulate humoral and cell-mediated immune
  responses
 Mechanisms: cytokines, cell surface molecules,
                    cytotoxic granules.
B lymphocytes: antibody producers

 Origin and maturation: Bone marrow
 Differentiation to plasma B cells in secondary lymphoid
  tissues (lymph nodes, spleen, Peyer’s patch, and tonsils)
 CD19+CD20+
 Antigen receptors: B cell receptor (cell surface
  immunoglobulins)
 Function: Production of antibodies (IgM, IgE, IgA, and IgG)
 Regulated by T cells and DCs
B lymphocytes




                Antigens+ T cell help
Circulating blood cells

                  Figure 1-12
           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®
MHC molecules and antigen
  presentation to T cells
                          Lecture objectives
• Big questions?
How TCR recognizes antigens?
What are the autoimmune diseases that are associated with particular types of MHC genotypes?
Are MHC molecules 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
          A big picture:
How do T cells recognize antigens?




            MHC molecule   TCR
CORE




10. 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)
Figure 3-25
Genes in yellow: Functional MHC II genes

Genes in dark gray: pseudogenes (not expressed, so not functional)
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)
CORE



d. 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.
Figure 3-13 part 1 of 2
T CELL RECEPTOR


                  Figure 3-2
Figure 3-21
The peptide-binding groove of MHC molecules
             Figure 3-15
CORE


b. 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).
                                                            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
                                         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.
   Genetics of MHC gene expression:
both 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.

                         • Haplotype: The particular
                           combination of MHC alleles
                           found on a given chromosome 6.
Present Ag to
CD8 T cells




                Present Ag to
                CD4 T cells
Figure 3-28
Each MHC isoform binds a characteristic set of peptides

-Anchor residues in peptides are important for binding to MHC
-Not all residues are important Figure 3-29




Degenerate binding allows each MHC molecule handles many different peptides.
         Figure 3-26

Tom


Jane


Martin


John
            MHC: things to remember
•   MHC molecules are 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]
Different cell distribution of MHC class I and II
               Figure 3-22
                             • Almost all cells express MHC I
                               for comprehensive surveillance
                               by CD8 T cells
                             • Only some cells express high
                               levels of both 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
Processing and presentation of endogenous antigen via the MHC class
   I pathway (endogenous pathway):

1. 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.
Processing and presentation of exogenous antigen via the MHC class II
   pathway (exogenous pathway):

1. Professional antigen processing cells internalize antigens.
2. Antigens are internalized into endosome & degraded into peptide
   fragments.

3. 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).

4. Exocytic vesicles (from Golgi) containing MHC II proteins fuse with
   endosome containing peptides.

5. Ii is degraded, and CLIP is removed by HLA-DM protein.
6. MHC II proteins bind peptides, vesicles fuse with plasma membrane,
   and MHC II/peptides are expressed on cell surface and presented to
   CD4 cells.
 Two different types of antigens:
Extracellular for MHC II and intracellular for MHC I
          They are processed differently.
Antigen processing is required to present antigen peptides to TCR.
 TCRs bind short antigen peptides but not whole antigen proteins
                     Figure 3-9
Transport of Cytosolic Peptides into ER
Role of transporter associated with antigen processing, TAP

                         Figure 3-17 syndrome: No TAP
                                Bare lymphocyte
                                       expression  no MHC I expression 
                                       no CD8 T cells  defective cytotoxic
                                       activity against virus-infected cells
                                       chronic respiratory infection

                                       TAP=TAP1+TAP2

                                       Proteasome: 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
                 Assembly of antigen peptide/MHC class I complex.
                Molecular chaperons (calnexin, calreticulin and tapasin)
                   aid the folding of MHC I and loading of peptides
                                 Figure 3-18




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.
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
                           Figure 3-20
-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.
MHC class I molecules present antigens to CD8+ T cells, and
 MHC class II molecules present antigens to CD4+ T cells:

                     Figure 3-12
T cell receptor recognition of antigens is MHC-restricted
                   Figure 3-30
       TCR recognition of antigens induces T cell activation, functional maturation, and
       killing/activation of target cells

                                         Figure 3-11




The T cells cytokines are produced only when T cells are engaged with APC
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.
HLA typing: how?
Microcytotoxicity assay for detection of HLA antigens
Anti-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 ELISA
Monoclonal antibodies to different MHC alleles have been generated.
Using panels of these antibodies, HLA typing before transplantation is
possible.

RFLP: Restriction Fragment Length Polymorphism
Digestion 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 Reaction
PCR using MHC gene-specific primers
and DNA sequencing
Another (crude) method:
Mixed Lymphocyte Reaction (MLR) is used to test for HLA compatibility between
individuals
                             Figure 5-14


            Person A                              Person B
                           White blood cells




                                                 The higher the response
                                                 The higher the mismatch
CORE




(2) Diseases associated with specific HLA antigens
If you have the MHC allotypes (left), you have a higher chance of
    getting the following diseases (right):


(a) HLA-DR3/DR2 Systemic Lupus Erythematosus

(b) HLA-DR4  Rheumatoid Arthritis

(c) HLA-B7 and DR2  Multiple Sclerosis

(d) HLA-B8, DR3/DR4  Type 1 diabetes

(e) HLA-B27  Ankylosing Spondylitis

				
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