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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. - PowerPoint PPT Presentation
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Summary of the last lecture:DC & 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
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, TNF (endo-pyrogen) & chemokinesAcute phase proteins, PG, histamineBacterial products: LPS
endogenous & exogenous “danger signals”
Immunological effects:vasodialation, vascular permeabilitycell adhesion, chemotaxis – cell mobilization & immobilizationDC activation
Dendritic cells & immune regulation
• Immune regulation & deviations
• DC heterogeneity
• DC & immune regulation
• Applications of flow cytometry in basic immunology
• Student presentation & essay topic
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
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+
CD4+ T cell subsets induced during infection or immunization with Leishmania Major in mice
(Liew FY 1986)
Cells Source Protection Ab IL-4 IFN• Tr cured + + - +• Ti i.v. immunized + + - +• Ts progressive - + + -• Tsc s.c. immunized - + + -
Refs.: Liew FY. Immunology Today 1989; 10:40-45Liew FY. Parasitol. Today 1986; 2:264-270
T helper clones distinct in cytokine production profiles(Mosmann TR, Coffman RL et al.,1986)
• TH1: IL-2, IFN-, GM-CSF, IL-3
• TH2: BSF1 (IL-4), IL-3 …
TH cell subsetsTH0
TH2TH1
IFN- IL-4
Cell-mediatedimmunity
Antibodyproduction
inhibition
TH3 TGF-
Type of immune effector mechanism induced may determine the outcome of an infection
TH0
TH2TH1
IFN- IL-4
Cell-mediatedimmunity
Antibodyproduction
inhibition
IL-12IL-18
IL-4
Factors driving TH cell differentiation
• Factors important for TH1 differentiation– IL-12 (MQ, DC)– IL-18 (MQ, Kupffer cells, DC) – IFN- (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: NK1.1+CD4+CD1+
DC are heterogenic cell populations
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– Langhan’s cells– Spleen, lymph node DC– Intestinal, Peyer’s patch, liver, lung DC– Migrating DC in the lymph
• Plasmacytoid DC– CD11C-, IL-3R+, B220+, GR-1+)
DC subsets and lineage origins
• DC lineage origins (mouse)– Myeloid markers: CD11b, CD11c, 33D1– Lymphoid markers: CD8, DEC205,
• Key DC subsets– Mouse: CD8- (myeloid)
CD8+ (lymphoid)– Human: DC1 (myeloid): e.g. PBMC-DC
DC2 (lymphoid?): plasmacytoid DC (IL-3R++)– Rat: OX62+OX41+ CD4+
OX62+OX41-CD4-
DC direct immune responses by releasing different cytokines
• Mouse DC– CD8- (myeloid): IL-10– CD8+ (lymphoid): IL-12, IFN-
• Human DC– DC1 (myeloid): IL-12, IL-18– DC2 (pDC, lymphoid?): IL-6, IL-10, IFN-, IFN-
Dendritic cell heterogeneity:
immunogenicity vs tolerogenicity
The “micro-anatomical model”(by B. Fazekas de St Groth, Imm.Today 1998; 19:448-54)
B
B
B
T
+ DC
GC
(B + FDC)F
T: T cell areaB: B cell area
F: B cell follicleGC: germinal centre
Missions & fate of DC migrating to the secondary lymphoid organs
Preparation ofPreparation ofintestinal lymphintestinal lymphdendritic cellsdendritic cells
Small Intestine
Afferent Lymphatics
Mesenteric nodes :-
Excision leads to joining of afferent and efferentlymphatics
Efferent lymphatic :-Joins thoracic duct
Peyer's Patch
0.2 - 0.5% DC
Centrifugation over NycoprepDeplete lymphocytes by MACS
90-95% DC
- A model for generating ‘authentic’ DC migrating in lymph
(G. Gordon MacPherson, Oxford)
Two DC subsets in rat
OX41+CD4+ 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
Presentation of KLH to sensitised T Presentation of KLH to sensitised T cells by DC subpopulationscells by DC subpopulations
0 2 4 6 8 100
10000
20000
30000
40000
50000
60000
Whole DC
OX41+ DC
OX41- DC
OX41+&- mix
DC added x10-3
Gro
ss c
pm
Intestinal DC Intestinal DC populationspopulations
OX41+ DC OX41- DC
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
Express high levels of MHC Class IIExpress CD80 & CD86 (B7.1, B7.2)Acquire enteric antigens
Features that change during DC maturation process(Banchereau J & Steinmain RM, Nature 1998)
“Ag uptake mode” “Ag presenting mode”
(danger signals)
Distribution of MHC Class II molecules on DC (Turley S. & Mellman I.)
Ref: Banchereau J & Steinman RM, Nature 1998
Early Intermediate Late
Lam
p-2
M
HC
cla
ss I
I
DC maturity &immuno-adjuvantivity
Immature DCMainly intracellular MHC
Lack co-stimulatory molecules
Mature DCHigh surface MHC
High B7 (CD80, CD86) …
TT
T
T
T
T
T
T
“Tolerogenic” “immunogenic”
Treg
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
Tolerogenic DC?
• Immature DC?
• DC “licensed to kill”– Thymic medullary DC mediated negative selection– CD8+ DC in mouse (Fas/FasL mediated killing)– OX41-CD4- DC in rat (NKR-P1)
• DC associated tolerogenic molecules– Cytokines: IL-10, TGF-– 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?
– 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
Applications of Flow Cytometry in Basic Immunology
• FSC (forward scatter) - Size• SSC (side scatter) - Granularity• FL (flurochrome) – FL-1, FL-2, FL-3 etc.
Flow cytometry - the basic parameters
(1) FSC – the size
Forward (parallel) lights
(2) SSC – the granularity
Side (angled) lights
(3) Fluorescent-labeled markers
Laser light
BandFilters
mirr
ors
FL-1 (green)
FL-3 (ultra red)
FL-2 (red)
Fluorescent detectors
Fluorochromes & wave length
Cell types of the immune system
Human blood cells analyzed by their FSC and SSC properties
FSC (size)
SSC
(gra
nula
rity)
RBC lysedTotal blood
Cell phenotypes & surface markers
T B NK
Cluster of Differentiation (CD) markers
Immuno-staining of cell surface markers
Direct Indirect
Human blood leukocytes (back-gating analysis)C
D14
-FIT
C
CD45-PE FSC
SS
C
Human PBL phenotyping
Isotype-FITC
Isot
ype-
PESS
C
Anti-CD3-FITCAnti-CD3-FITCFSCA
nti-C
D19
-PE
Ant
i-CD
8-PE
Ant
i-CD
4-PE
Ant
i-CD
56/1
6-PE
Anti-CD3-FITC Anti-CD3-FITC
Markers of T cell differentiation
CD4 and CD8 expression on thymocytes, splenocytes and PBL
Thymocytes Splenocytes PBL
CD8-FITC
CD
4-C
yt5
FP090502.015-021
GM GM/IL-4
MHC-Class II-PE
Immature DC Mature DC
+LPSDC maturity & MHC class II expression
Cell activation and proliferation analyzed by the FSC and SSC properties
FSC (size)
SSC
(g
ranu
larit
y)
Unstimulated Con A-stimulated
Dot plots
Density plots
T cell activation analyzedby CD25 (IL-2R) expression
CD25-PE
Cel
l num
ber
Unstimulated Con A-stimulated
SSC
FSC
Th1 & Th2 Phenotypes Determined By Cytokine Expression Profile
CFSE: Carboxyfluorescein diacetate Succinimidyl Ester
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: 488nmEmitting wave length: 519nm
1
½
¼...
Visualizing lymphocyte division
CFSE intensity
• Applications of flow cytometry in basic immunology
• Generation and characterization of DC
• Assays for T cell activation– Cell proliferation – Cell division – Cell activation marker
Laboratory demonstration - DC and T cell activation
Student Presentation & Essay TopicTopic: Regulatory T cells
Autoimmunity Tumor immunity Transplantation
Tasks:1. 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
2. Write an essay on the topic All students 1000 -1500 words Essay submission: by January 18, 2006
Contact:: Dr. FP Huang (Tel:2855 4864; e-mail: fphuang@hkucc.hku.hk)
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