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
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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.
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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
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
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 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