000 by langkunxg


									      Immune Cells

             Paul Zhou
Institute Pasteur of Shanghai, CAS
Overview of lymphatic system

1. Consist of organs,
nodes and vessels
2. Distribution throughout
of body
3. Interface with
4. Circulating throughout
body (uni-directional,
regional, connected to
blood circulation system)
5. Immune privilege sites
Organ and tissues in immune system

1. primary lymphoid organs: bone marrow and thymus
- ontogeny and development of lymphocytes and some
non-lymphocyte cells (acquisition of capability of
antigen recognition, becoming effector/memory cells,
and homing)
2. secondary lymphoid organs: spleen, lymph nodes,
tonsils and Peyer’s patches
- antigen encounter and immune activation (and
effector response)
3. tertiary lymphoid tissues: skin and mucosa
- sites of antigen acquisition and processing and
effector response
Cells in immune system

1. Lymphoctyes: T, B and NK cells
- determine the immune specificity and orchestrate the
effector response (T and B cells); both innate and
adaptive immunity (NK cells)
2. Non-lymphocytes: monocytes/macrophages,
dendritic cells and neutrophils, basophils, eosinophils
and mast cells
- interact with lymphocytes and play a critical role in the
antigen presentation and the mediation of immunologic
3. Specialized epithelial and stromal cells
- provide the anatomic environment in which immunity
Important features in immune cells
1. Developed from hematopoietic stem cells
- ontogeny and lineage differentiation
2. Replenished throughout life
- proliferation, differentiation and maturation,
circulation and migration
3. Have great potential in response to various insults
in antigen specific or non-specific manner
- activation, proliferation, maturation, circulation and
4. Activate locally and then migrate (or circulate)
5. Expand and contract during immune response
6. Cross talk among immune cells and between
immune and non-immune cells
          Leukocytes in blood
                  Cell number/μl    Range
Whole blood cells      7,400     4,500 - 11,000

Neutrophils          4,000     1,800 - 7,000
Eosinophils            200         0 - 450
Basophils               40         0 - 200
Lymphocytes          2,500     1,000 - 4,800
Monocytes              300         0 - 800
   Topics to be covered in this lecture

1. Primary lymphoid organs: ontogeny of
   immune cells
2. Secondary lymphoid organs: sites of
   immune activation
3. Tertiary lymphoid tissues: the sites of
   antigen acquisition and effector immune
4. Migration and homing of immune cells
Primary lymphoid organs:
ontogeny of immune cells
Bone marrow
1. Cellular content of
bone cavity
2. A reservoir for
3. A site of B cell
throughout life
4. sIgM expressing B
cells generated in
bone marrow migrate
to spleen for further
B cell differentiation in bone marrow
                          B-2 cells            B-1 cells
Adult vs. fetal             adult               fetal

Percentages                  95                  5

Surface markers           CD19/CD45RA         CD19/CD45RA

Ig repertoire             highly diverse      limited diverse

Antigens                  T dependent         T independent
                          protein in nature   variety including

Requirement of Th cells      yes                 no

Ig class switching           yes                 no
Affinity maturation          yes                 no
Thymic architecture in human and mouse
Trafficking of thymocytes in T cell development
                  and selection
Maturation of T lymphocytes in thymus
Positive and negative selection
Secondary lymphoid organs: sites
     of immune activation
           Lymphocyte distribution
              (% of total)
         Blood Lymph nodes Spleen

B cells    10 - 15    20 - 25   40 - 45
Th cells   50 - 60    50 - 60   40 - 50
Tc cells   20 - 25    15 - 20   10 - 15
NK cells   about 10    rare     about 10
Structure and histology of spleen
 Follicle B cells
Marginal zone B
Histology of
lymph node

1. capsule
2. HEV
3. cortex – para-
cortex (T zone) and
4. germinal center
5. sinus
6. in and out lymph
Lymph node structure with or without immunization
B cell activation and humoral immune response
T cell activation and cellular immune response
• Two major subsets: α/β TCR and γ/δ TCR
• Two lineages in α/β TCR T cells: CD4 and CD8
   - differ in antigen recognition as well as regulatory and
        effector functions
• Two subtypes in CD4 T cells: Th1 and Th2
  - differ in cytokine secretion and helper functions
• Naïve, effector (activated), and memory T cells
• Recognize processed antigen/MHC complex on the surface
  of APCs
• T cells become activated through signals 1 and 2
• Activated (effector) T cells become proliferative through IL-2
  and IL-2R autocrine and paracrine loop and migrated to
  infection site(s)
• Activated T cell contraction
• Central memory and effector memory T cells
The Dendritic Cells and lymphocytes on
                T Cell Receptors
• TCR consists of two subsets: α/β and γ/δ.
• Through somatic gene arrangement, the TCR
  displays extreme sequence diversity.
• TCR expresses on the surface of T cells in a clonal
  fashion, in which to a given T cells only one pair of
  α/β or γ/δ TCR is expressed.
• α/β or γ/δ TCR itself does not mediate signaling.
  Instead it is intimately associated with CD3
  complex (γδ2ε2ζ). It is the latter that mediates TCR
• The signal mediated through TCR/CD3 complex is
  called the signal one.
Human TCR gene organization
TCR/CD3 complex
Co-stimulatory molecules between T cells and
           antigen presenting cells
         Effector functions of T cells
• Early changes
  – Changes in pH
  – Changes in membrane potential (MTT assay)
  – Fluxes in cyclic nucleotides and calcium (Ca influx assay)
• Late changes
  – Cyto-skeletal changes (morphologic change)
  – Activation of the cytolytic mechanism (51Cr and enzyme
    release assays)
  – Gene regulation: CD25, CD69, IL-2, IL-3, IFNγ, GM-CSF,
    CTLA-4, MHC class II, VLA-2, 4F2, transferrin receptors,
    and insulin receptors, etc. (Elisa, Elispots, intracellular
    cytokine staining, activation markers)
  – T cell proliferation (3H-thymidine and CFSE assays as
    well tetramer staining)
Tertiary lymphoid tissues: the sites
of antigen acquisition and effector
         immune response
Mucosal tissues
Mucosal sites   Lymphocyte subsets     Distribution (%)   Possible functions
                CD3+ T cells           35–40
                CD4+, CD8-             65                 Major T-helper cells
                                                          for mucosal
                CD4-, CD8+             30                 CTL precursors;
                CD4-, CD8-             2–4                Express gd TCRs

Inductive tissues
Peyer’s patches Naive                  30–40              Circulate within the
                                                          mucosal system
                Effector (activated)   30–35              Stimulated through
                                                          M-cell pathways
                Memory                 30–40              Homing to effector
                B220+ B cells          45–47              Include germinal
                                                          center where >60%
                                                          are sIgA+B cells

                sIgA+                  ~8–10              Committed to IgA
                CD3+ T cells           40–50
                CD4+ CD8-              ~60–65             Difficult to activate
                                                          via TCR
Mucosal sites      Lymphocyte subsets   Distribution (%)   Possible functions

Lamina propria     CD4-, CD8+           ~30–35             Mature CTLs; other
                                                           subset functions?
                   CD4-, CD8-           ~2–5               Express gd TCRs
                   Memory               >90
                   sIgA+ B cells        30–50
                   IgA plasma cells     10–15              Highest numbers of
                                                           plasma cells in the
                                                           mammalian immune
Effector tissues
                   CD3+ T cells         85–95
                   CD4+, CD8-           ~5–8               All express ab TCRs
lymphocytes        CD4-, CD8+           ~75–80             2/3 are CD8 aa; 80%
                                                           gd+; 50% ab+
                   CD4+, CD8+           ~7–10              All express of ab
                   CD4-, CD8-           ~5–8               All express gd TCRs
                                                           No B cells/plasma
Skin tissue
Primary immune site in skin tissue
    Background information on DCs
• Initial discovery of Langerhans cells in skin by Paul
  Langerhans in 1868
• Dendritic cells (DCs) first described by Steinman
  and Cohn in mouse spleen in 1973
• Special properties of DCs in initiating immunity (i.e.
  antigen and pathogen recognition, uptake, process
  and presentation as well as pathogen
  dissemination) were discovered after depletion of
  monocytes, macrophages and B cells
• Reside in most peripheral tissues, especially at sites
  interface with environment; but migrate to draining
  lymphoid nodes to activate T cells
• Can be generated from their precursor cells ex vivo
                LC    DDC-IDC   moDC       pDC

CD1a             +      +         +         -
CD11c            +      +         +         -
CD208, CD-LAMP +        +         +         -
CD123            +      +         +        ++
BDCA-2,4         nr     nr       -/+        +
e-cadherin       +      -         -         -
CD207, Langerin +       -         -         -
CD1d             -      +         +        nr
CD11b            -      +         +         -
CD52             -      -         +        +/-
CD83             +      +         +         +
LC, DDC-IDC and moDC are also termed myeloid DCs or conventional DCs.
pDCs (plasmacytoid DCs) are also termed lymphoid DCs.
            Immature DC    Mature DC
            (steady state)

CD80                 Low         High
CD86                 Low         High
HLA-DR               Low         High
Ag capture           Efficient   not
Ag processing        Efficient   not
Ag presentation      not         Efficient
Allo-stimulation     Weak        Strong
Tolerance maint.     Yes         No
Immuno-stimulation   No          Yes
 Signals that induce DCs to mature

• Receptors for direct pathogen signals: TLRs

• Receptors for indirect sensing of infection
  through inflammatory cytokines, internal
  cellular compounds and on-going immune
  responses: cytokine receptors, TNF receptor
  family molecules, CD40, FcR, and sensors
  for cell death
    TLRs recognize pathogen-associated molecular

TRL1 (surface, mDC)     Soluble bacterial factor (heterodimer with TRL-2)
TRL2 (surface, mDC)     bacterial peptidoglycans, lipoproteins
TRL3 (intra, mDC)       viral dsRNA, poly(I:C)
TRL4 (surface, mDC)     LPS
TRL5 (surface, mDC)     flagellin
TRL6 (surface, mDC)     diacyl lipopeptides
TRL7 (intra, pDC)       viral ssRNA
TRL8 (intra, mDC)       viral ssRNA
TRL9 (intra, pDC)       unmethylated CpG motifs
TRL10                   unknown
TRL11                   unknown

Most of TLR-ligand interactions results in intracellular signaling through
  MyD88, which in turn up-regulates CD83, co-stimulatory molecules and
  CCR7 and leads to the secretion of IFNα by plasmacytoid DCs or IL-6,
  IL-10, TNFα and IL-12 by conventional DCs.
Signaling through TRL7/9 in pDCs
Link innate and adaptive immunity through
              DC maturation
Differentiation of mononuclear phagocytes
Migration and homing of
     immune cells
• List three examples in nature that
  demonstrate there is an immune system that
  protects a mammalian organism from
  foreign insults.

• Why are immune cells migratory cells?
  Why is it so important to understand the
  migratory pattern of immune cells in
  developing a vaccine against a foreign
                  NK cells
• Initial discovery
• Missing self theory
• Inhibitory receptors
• Stimulatory receptors
• Effector functions
• Role in immune surveillance and anti-
  microbial pathogens
• Ontogeny

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