• 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

– 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

SBT CT

CD25

CD

4

Regulatory T cells (triple stain)

CD45RCS

SC

Regulatory T cell:CD45RClo

CD4+

CD25+

(courtesy of Dr. Lina Tian)

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 B cell 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

Mitogen (Con A)-induced T cell proliferation(measured by level of DNA synthesis)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0 0.625 1.25 2.5 5 10 15

Con A Concentration ( g/ml)

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

Visualizing T cell activation

CFSE intensity

Cel

l num

ber

SS

C (g

ranu

larit

y)

FSC (size)

Unstimulated Con A-stimulatedCFSE-labeled unlabeled

Visualizing & Phenotyping T cell Division

CFSE intensity

Cel

l num

ber

CD25-PE

AB

A

B

Mitogen-induced T cell proliferation, division vs IL-2R (CD25) expression

CFSE intensity

CD

25

Unstimulated Con A-stimulated

DC:T cell interaction

(Huang YG, unpublished data)

Isot

ype

IsotypeM

HC

Cla

ss II

CD

80

CD86

CD40

- LPS + LPS

67.214.0

DC maturity & APC activityin Mixed Lymphocyte Reaction (MLR)

0

50000

100000

150000

200000

250000

1:1 1:5 1:10 1:20 1:30 1:50 1:100 1:200

Responder (SpC) Ratio

3H-

Thym

idin

e Up

take

(cpm

)

Mature DCImmature DC

Key points for discussions Requirements for naïve T cell activation?

Key changes during DC maturation?

Association of DC maturity with APC activity?

Induction and measurement of T cell activation – the basis - cell proliferation assay? - indicators of T cell activation? - T cell mitogen?

MLR - peptides presented to T cells in: - allogenic responses? - semi-allogenic responses? - syngenic responses?

Isot

ype

Isotype

MH

C C

lass

IIC

D80

CD86

CD40

GM-CSF GM-CSF+IL-4 GM-CSF + IL-4 GM-CSF + IL-4 (TW) (TW + LPS)

7.2 67.248.714.0