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)