Summary of the last lecture: DC

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Summary of the last lectureDC the initiation
of immune responses

• Lymphocyte activation the requirements
• Signal 1
• Signal 2
• Models of T-B, APC-T-B cell cooperation
• DC the initiator of immunity
• Activator of naïve T cells
• The special Ag processing presenting machine
• The Self vs Non-self and Danger models

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Inflammation danger signals Immune System
Turned on by Danger (P Matzinger)

• Clinical signs of inflammation
• erythema, swelling, heat pain
• Mediators
• Cytokines IL-1, IL-6, TNFa (endo-pyrogen)
  chemokines
• Acute phase proteins, PG, histamine
• Bacterial products LPS
• ? endogenous exogenous danger signals
• Immunological effects
• vasodialation, vascular permeability
• cell adhesion, chemotaxis cell mobilization
  immobilization
• DC activation

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Dendritic cells immune regulation

• Immune regulation deviations
• DC heterogeneity
• DC immune regulation
• Applications of flow cytometry in basic
  immunology
• Student presentation essay topic

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Immune regulation homeostasis

• Antigens and antibodies
• Feedback and intracellular signaling
• Apoptosis and program cell death
• Neuro-endocrine-immune regulation
• Immune deviations and cytokines
• TH1 versus TH2 responses

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T helper cell functional heterogeneity

• Liew FY Parish CR. (1972,1974)
• Distinct T helper functions for
• Ab response to hapten-carriers
• DTH responses to the carrier protein
• Tada T et al. (1978)
• T helper types for Ab responses to different
  hepten-carrier conjugates
• TH1 nylon wool non-adherent, Ia-
• TH2 nylon wool adherent, Ia

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CD4 T cell subsets induced during infection or
immunization with Leishmania Major in mice(Liew
FY 1986)

• Cells Source Protection Ab IL-4 IFNg
• Tr cured -
• Ti i.v. immunized -
• Ts progressive - -
• Tsc s.c. immunized - -

Refs. Liew FY. Immunology Today 1989
1040-45 Liew FY. Parasitol. Today 1986
2264-270
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T helper clones distinct in cytokine production
profiles(Mosmann TR, Coffman RL et al.,1986)

• TH1 IL-2, IFN-g, GM-CSF, IL-3
• TH2 BSF1 (IL-4), IL-3

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TH cell subsets
TH0
TH3
TGF-b
TH2
TH1
inhibition
IFN-g
IL-4
Cell-mediated immunity
Antibody production
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Type of immune effector mechanism induced may
determine the outcome of an infection
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TH0
IL-12 IL-18
IL-4
TH2
TH1
inhibition
IFN-g
IL-4
Cell-mediated immunity
Antibody production
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Factors driving TH cell differentiation

• Factors important for TH1 differentiation
• IL-12 (MQ, DC)
• IL-18 (MQ, Kupffer cells, DC)
• IFN-g (T, NK, DC)
• Factors important for TH2 differentiation
• IL-4 (NK-T/mast cells? T, DC)
• IL-6 (T, MQ)
• IL-10 (T, MQ, DC)

NK-T NK1.1CD4CD1
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DC are heterogenic cell populations
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Types and source of DC

• Bone marrow-derived (BM-DC)
• Can be generated in the presence of GM-CSF and
  IL-4
• Peripheral mononulear cell-derived DC (PBMC-DC)
• Can be generated in the presence of GM-CSF and
  IL-4
• DC isolated from tissues and organs
• Thymic DC
• Langhans cells
• Spleen, lymph node DC
• Intestinal, Peyers patch, liver, lung DC
• Migrating DC in the lymph
• Plasmacytoid DC
• CD11C-, IL-3R, B220, GR-1)

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DC subsets and lineage origins

• DC lineage origins (mouse)
• Myeloid markers CD11b, CD11c, 33D1
• Lymphoid markers CD8aa, DEC205,
• Key DC subsets
• Mouse CD8aa- (myeloid)
• CD8aa (lymphoid)
• Human DC1 (myeloid) e.g. PBMC-DC
• DC2 (lymphoid?) plasmacytoid DC (IL-3R)
• Rat OX62OX41 CD4
• OX62OX41-CD4-

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DC direct immune responses by releasing
different cytokines

• Mouse DC
• CD8aa- (myeloid) IL-10
• CD8aa (lymphoid) IL-12, IFN-g
• Human DC
• DC1 (myeloid) IL-12, IL-18
• DC2 (pDC, lymphoid?) IL-6, IL-10, IFN-a, IFN-b

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Dendritic cell heterogeneityimmunogenicity vs
tolerogenicity
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The micro-anatomical model (by B. Fazekas de St
Groth, Imm.Today 1998 19448-54)
B
B
T
B
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Missions fate of DC migrating to the secondary
lymphoid organs
F
DC
(B FDC)
GC
T T cell area B B cell area F B cell
follicle GC germinal centre
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- A model for generating authentic DC migrating
in lymph
(G. Gordon MacPherson, Oxford)
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• Two DC subsets in rat
• OX41CD4 DC
• Smaller with short/fine processes
• Contain low/no NSE reactivity

• (Liu L.M. et. al. J. Immunol. 1998)
• OX41-CD4- DC
• larger with long/big processes
• Contain strong NSE reactivity

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Presentation of KLH to sensitised T cells by DC
subpopulations
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Intestinal DC populations
OX41 DC
OX41- DC
Express high levels of MHC Class II Express CD80
CD86 (B7.1, B7.2) Acquire enteric antigens
Strong APC Weak APC Not present in T cell
areas Continuously transport under steady state
self-Ag derived from apoptotic IEC to T
cell areas under steady non-danger
state
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Features that change during DC maturation process
(Banchereau J Steinmain RM, Nature 1998)
(danger signals)
Ag uptake mode Ag presenting mode
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Distribution of MHC Class II molecules on DC
(Turley S. Mellman I.)
Early
Intermediate Late

Lamp-2 MHC class II
Ref Banchereau J Steinman RM, Nature 1998
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DC maturity immuno-adjuvantivity
Immature DC Mainly intracellular MHC Lack
co-stimulatory molecules
Mature DC High surface MHC High B7 (CD80, CD86)
T
T
T
T
T
T
T
T
Treg
Tolerogenic immunogenic
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DC-based immunotherapies

• DC-based tumor vaccines for cancer treatment
• Immunodeficiencies and tumors
• Graft-versus-leukemia (GVL) responses
• Anergized TAA-specific T cell clones
• DC immune adjuvanticity
• DC subsets and maturity
• Immunotherapeutic potential of tolerogenic DC
• Tolerance induction in autoimmune diseases
• Tolerance induction in transplantation

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Tolerogenic DC?

• Immature DC?
• DC licensed to kill
• Thymic medullary DC mediated negative selection
• CD8aa DC in mouse (Fas/FasL mediated killing)
• OX41-CD4- DC in rat (NKR-P1)
• DC associated tolerogenic molecules
• Cytokines IL-10, TGF-b
• IDO (Indoleamine 2,3-dioxygenase)
• DC and the generation of T regulatory cells
• DC confers tolerogenic memory and specificity
  through the induction of T regulatory cells?

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Applications of Flow Cytometry in Basic
Immunology

• Basic parameters
• Applications
• Cell phenotyping
• Measurement of cell activation
• Visualization of cell division
• Detection of intracellular molecues (e.g.
  cytokine)
• Analysis of specific ligand binding
• Analysis of intracellular calcium concentration
• Analysis of DNA content
• Cell sorting

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Flow cytometry - the basic parameters

• FSC (forward scatter) - Size
• SSC (side scatter) - Granularity
• FL (flurochrome) FL-1, FL-2, FL-3 etc.

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(1) FSC the size
Forward (parallel) lights
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(2) SSC the granularity
Side (angled) lights
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(3) Fluorescent-labeled markers
Laser light
Band Filters
Fluorescent detectors
FL-1 (green)
mirrors
FL-2 (red)
FL-3 (ultra red)
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Fluorochromes wave length
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Cell types of the immune system
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Human blood cells analyzed by their FSC and SSC
properties
RBC lysed
Total blood
SSC (granularity)
FSC (size)
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Cell phenotypes surface markers
NK
T B
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Cluster of Differentiation (CD) markers
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Immuno-staining of cell surface markers
Direct Indirect
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CD14-FITC
Human blood leukocytes (back-gating analysis)
SSC
CD45-PE
FSC
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Human PBL phenotyping
SSC
Anti-CD19-PE
Anti-CD4-PE
Anti-CD3-FITC
Anti-CD3-FITC
FSC
Isotype-PE
Anti-CD56/16-PE
Anti-CD8-PE
Isotype-FITC
Anti-CD3-FITC
Anti-CD3-FITC
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Markers of T cell differentiation
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CD4 and CD8 expression on thymocytes, splenocytes
and PBL
Thymocytes Splenocytes
PBL
CD4-Cyt5
CD8-FITC
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FP090502.015-021
GM GM/IL-4
DC maturity MHC class II expression
LPS
Immature DC Mature
DC
MHC-Class II-PE
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Cell activation and proliferation analyzed by the
FSC and SSC properties
Unstimulated Con A-stimulated
Dot plots
SSC (granularity)
Density plots
FSC (size)
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T cell activation analyzedby CD25 (IL-2Ra)
expression
Unstimulated Con A-stimulated
Cell number
CD25-PE
SSC
FSC
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Th1 Th2 Phenotypes Determined By Cytokine
Expression Profile
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Analysis of cell division by CFSE labeling
Cell incubation with CFSE (serum free, 37 0C,
10mins. wash) CFSE crosses cell membrane
attaches to cytoplasmic proteins Removal of
carboxyl groups of CFSE by intracellular
esterase Fluorescein Analyed by FC Excitation
wave length 488nm Emitting wave length 519nm
1 ½ ¼ . . .
CFSE Carboxyfluorescein diacetate Succinimidyl
Ester
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Visualizing lymphocyte division
CFSE intensity
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Laboratory demonstration - DC and T cell
activation

• Applications of flow cytometry in basic
  immunology
• Generation and characterization of DC
• Assays for T cell activation
• Cell proliferation
• Cell division
• Cell activation marker

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Student Presentation Essay Topic

• Topic Regulatory T cells
• Autoimmunity
• Tumor immunity
• Transplantation
• Tasks
• Class presentation
• December 7, 2005 (5-9pm)
• 3 per group
• 20 25 minutes for presentation (per group)
• 10 minutes for questions discussions
• Optional for MMedSc students
• Write an essay on the topic
• All students
• 1000 -1500 words
• Essay submission by January 18, 2006
• Contact Dr. FP Huang (Tel[phone] e-mail
  fphuang_at_hkucc.hku.hk)