Adhesion Molecules And Lymphocyte Homing - Buffalo, NY · Adhesion Molecules And Lymphocyte Homing Sharon S. Evans, Ph.D. Dept. Immunology, RPCI Jan. 29 & Feb. 3, 2015

Adhesion Molecules And Lymphocyte Homing

Sharon S. Evans, Ph.D. Dept. Immunology, RPCI Jan. 29 & Feb. 3, 2015

Research in Cell Adhesion

1982 – 1986 Studies proposing to study cell adhesion IRRELEVANT,

“…lacking overall functional relevance…” (NIH)

1987 Richard Hynes published landmark paper in Cell recognizing

that multiple disciplines (embryology, cell biology, neurobiology,

immunology, tumor biology) studying similar cell-to-cell adhesive

interactions. “Integrins: a family of cell surface proteins.”

1986 – 1992 Scramble to identify cell adhesion molecules/ligands

14 a, 8 b chains of integrins identified → 24 integrins

L, E, and P-selectin structure determined

1990 – present Adhesion molecules, chemokines and signal transduction

b2, b1 integrins

L-selectin

homeostatic & inducible chemokines

1990s-present Identification of 50 chemokines and 19 chemokine receptors

Reading Assignments

Lecture 1:

Girard JP, Moussion C, Förster R. HEVs, lymphatics and

homeostatic immune cell trafficking in lymph nodes. Nat Rev

Immunol. 12(11):762-73, November 2012. (Required

reading – for background).

Lecture 2:

Moussion C, Girard JP. Dendritic cells control lymphocyte

entry to lymph nodes through high endothelial venules.

Nature 13;479(7374):542-6, November 2011. (Required

reading – primary paper)

Be able to read and understand field of adhesion and

leukocyte trafficking.

Working knowledge of experimental approaches used

to dissect role of adhesion molecules/chemokines in

leukocyte trafficking.

Understand structure/function relationship of trafficking

molecules that direct leukocyte migration under

homeostatic versus inflammatory conditions.

Objectives

Outline

General introduction to lymphocyte trafficking – homeostatic

recirculation vs active recruitment.

Homeostatic trafficking of lymphocytes across specialized high

endothelial venules (HEVs)

Structure/function of adhesion molecules involved in initial tethering/rolling

(L-selectin/PNAd)

Structure/function of chemokine/chemokine receptors that mediate activation

(CCR7/CCL21)

Structure/function of adhesion molecules required for firm

arrest/transendothelial migration (LFA-1/ICAM-1 & α4β7 integrin/MAdCAM-1)

Leukocyte recruitment in acute and chronic inflammation.

Thymus Spleen

Lymph nodes

Gut-associated lymphoid tissue

Bone marrow

T cell

Antibodies

B cell

Macrophage

Cellular Mediators

of the Adaptive

and Passive

Immune Response

Peripheral

Blood

Lymphocytes

Infection Inflammation

Injury Autoimmune

Diseases

Peripheral

Lymph Nodes Peyer’s

Patches

Spleen Skin

Pathways of Lymphocyte Trafficking

Tumor

Sites

Pregnant

Uterus

Outline






(L-selectin/PNAd)


(CCR7/CCL21)




Lymphocyte Extravasation Occurs Primarily

Across Post-Capillary High Endothelial Venules

High Endothelial

Venules (HEV)

Post-Capillary

Venules

Pathways of Lymphocyte Recirculation

Pathogen (virus, bacteria,

parasites)

HEV

Tenants of T Cell-Based Immune Response

1 Generate pathogen-

specific effector T cells

Draining

Lymph Node

Afferent lymphatics

Efferent lymphatics

Deliver T cells to peripheral sites

• Infected, injured tissue

Peripheral Site • Skin

• Gut

• Lung

• Genital

• Urinary

von Andrian and Mempel

Nature Reviews / Immunology 2003

2

Entry from peripheral tissues:

• Dendritic cells

• Lymphocytes (activated)

• Antigen

• Soluble factors


parasites)

HEV


1 2 Generate pathogen-


Draining

Lymph Node

Afferent lymphatics

Efferent lymphatics




• Gut

• Lung

3x106 cells

recruited/day

Major site of naïve

lymphocyte entry

(~1-10 in 106 T cells

recognize a specific Ag)




parasites)

HEV




Draining

Lymph Node

Afferent lymphatics

Efferent lymphatics




• Gut

• Lung

Lymphocyte migration through

LN parenchyma is active process




parasites)

HEV




Draining

Lymph Node

Afferent lymphatics

Efferent lymphatics




• Gut

• Lung

Exit from peripheral LN:

• Naïve lymphocytes

• Activated lymphocytes

EXIT: S1P/S1P1



Architecture and Vascular Organization of

Lymph Node (H & E)

T cell-rich paracortex

Morphological Comparison of Squamous/Flat vs.

Cuboidal/High Endothelial Venules (HEV)

HEV Cuboidal

endothelial cells

Squamous

endothelial cells

Squamous

Leukocyte – HEV Interactions

Gowans and Knight, 1964

EC

EC

Leukocyte

Thome (1889) – described HEV

structure

Schumacher (1899) – noted

presence of lymphocytes in

walls of HEV

Direction of lymphocyte

migration (from blood → tissue

or from tissue → to blood) not

known until 1964 (Gowans and

Knight)

Gowans & Knight injected

radiolabeled lymphocytes into

mouse (i.v.). 1 hour later,

recovered radioactivity in

tissues – supporting blood →

tissue route of migration.

Rosen and Stoolman, 1987

Anderson and Anderson, 1976

Lymphocyte Adhesion to Peripheral Lymph Node HEV

A. Anderson

HOMING

Free flowing ~ 500 mm/sec

Tethering & Rolling

< 25 mm/sec (1-3 sec)

Firm Sticking 0 mm/sec (10 min)

Dynamic Regulation of Leukocyte-Endothelial Adhesion

Transmigration (10-15 min)

Lymphocyte

HEV

Adapted from Immunol. Rev. 2002

In vivo Imaging of Lymphocyte – HEV Interactions

in PLN by Intravital Microscopy (IVM)

Femoral Artery

(injection of Calcein-

labeled lymphocytes)

Inguinal LN

VCR

PC

von Andrian Cur Opin Immunol 2004

Chen et al Cancer Immunol Immunoth 2006

Chen et al Nature Immunol 2006

Lymphocyte – Endothelial Interactions Observed

Under IVM in Inguinal LN

Vein

Art

Chen et al, Canc. Immun. Immunoth. 2006; Nature Immunol. 2006

Post-Capillary

Venules

HEV

Outline






(L-selectin/PNAd)


(CCR7/CCL21)




Ig-CAMs

mucins

Cytoplasm

Classes of Cell Adhesion Molecules

mucins selectins

Lymphocyte Endoth.

Cell

Tethering/

rolling

Firm

arrest

1

2

3

Tethering/Rolling • L-selectin/PNAd

• a4b7/MAdCAM-1

Activation • CCL21/CCR7

Sticking • LFA-1/ICAM1

Transmigration

4

Adapted from Immunol. Rev. 2002

Dynamic Regulation of Leukocyte-Endothelial Adhesion

HOMING

HEV

Complement-binding

repeats

Transmebrane

(Cleavage site)

17 A.A.

Cytoplasmic

Tail

Seed 1989

Tedder 1989

Rosen 1989

Protein Motifs of L-Selectin PLN Homing Receptor

Extracellular

Lectin

Immunogold Labeling of L-Selectin

on Lymphocyte Microvilli

Microvilli Cell Body

Im

mu

no

go

ld P

art

icle

s/1

00

ce

lls

Immunogold-labeled

L-selectin mAb

Evans et al., J. Immunol. 1998. 1999

800

400

Endothelial Cell

Leukocyte

COOH

NH2

L-selectin; CD62L

(MEL-14/LAM-1/

DREG-56)

E-selectin; CD62E

(ELAM-1)

NH2

COOH COOH

NH2

P-selectin; CD62P

(GMP-140/PADGEM)

Endothelial E-selectin, lectin

and EGF repeats

Ligand-binding loop

EGF

Module

Lectin

Module

Selectin Family

Remove PLN

Stain with anti-

PNAd mAb

(MECA-79 mAb,

green)

5 min.

TRITC-

labeled

cells

Lymphocytes Traffic Across PNAd+ (MECA-79

mAb reactive) HEV in Peripheral Lymph Nodes

MECA-79+ HEV

Lymphocyte

MECA-79 / Lymphocyte

Chen et al., Nature Immunol. 2006

MECA-79+ HEV

Lymphocyte

50 µm

MECA-79 / Lymphocyte

Remove PLN

Stain with anti-

PNAd mAb

(MECA-79 mAb,

green)

1 h

TRITC-

labeled

cells


Lymphocytes Traffic Across PNAd+ (MECA-79

mAb reactive) HEV in Peripheral Lymph Nodes

LN HEV

PNAd is the Major L-Selectin Ligand in

Peripheral Lymph Node HEV

Mucin Stalk

~ 30 nm

O-linked carbohydrate (glycosyl transferases

substitute CHO on serines and threonines)

Fucose (Fucosyl transferase

(FucT-IV, FucT-VII)

Sialylated polysaccharides

(Sialyl Lewis X [sLex]; sialyltransferase 3

Gal IV; ST3GalIV))

Sulfated saccharide (GlcNAc-6-O-

sulfotransferase, 6-O-ST). This enzyme

restrictively expressed in HEV. 6-O-SO4 is

the recognition epitope of MECA-79 mAb.

HEC-GlcNAc6ST-deficient mice (Rosen).

Posttranslational Modifications of

Endothelial L-Selectin Ligands

*Peripheral Lymph Node Addressin [PNAd] CD34

S

*HEC-GlcNAc6 sulfotransferase

(HEC-GlcNAc6ST)

*Requirement for sulfation during

tethering/rolling shown in HEC-

GlcNAc6ST-knock-out mice; Rosen &

Fukuda’s group Nature Immunol 2005

SO3 -

6-sulfo sialyl

Lewis X

MECA-79

epitope

SO3 -

SO3 -

Fucosyl Transferase VII

(FUT IV/VII)

Sialyl Transferase 3 Gal IV

(ST3GalIV)

Modified from Girard JP et al.,

Nat Rev Immunol. 12:762-73, 2012

PNAd

L-Selectin – PNAd Interactions in

Peripheral Lymph Node HEV

LN HEV

PNAd

Glycocalyx - 10 nm

L-selectin Lectin

Ca++

Lymphocyte

SO3 -

SO3 -

SO3 -

SO3 -

SO3 -

SO3 -

SO3 -

PNAd

Input Output (L-selectin+ cells)

Ca++ & EDTA 2.5 dynes/cm2

Ca++ Ca++ Dependent L-selectin/PNAd

Interactions Support Lymphocyte

Tethering and Rolling Under Shear

Analysis of Lymphocyte Homing In Vivo

Inject i.v.:

3x107 PKH-26-labeled

Wild-type Cells

3x107 FITC-labeled

L-selectin-/- (knock-out) Cells

or with

L-selectin blocking mAb

(DREG-56 or Mel-14)

Collect: Sacrifice mouse one hour

after injection of mixed cell

populations

Analyze:

Determine ratio of

FITC-/PKH26-labeled cells in

tissues

PP, LN, MLN, SP,

Blood

Homing Capabilities of L-Selectin-Deficient Lymphocytes

Arbones, Tedder et al.

Immunity 1:247:247-260, 1994

Spleen PP MLN PLN

Wildtype

L-selectin-/-

Rela

tiv

e H

om

ing

1

1 hour

Count the numbers of

rolling, sticking and

non-interacting cells in

certain time period

Measure the

velocity of rolling

cells (order IV)

Quantifying Lymphocyte – Endothelial

Interactions in PLN

II II

III III

III III

IV

IV

V

V

V

I 100 µm Vein

von Andrian et al, J Exp Med. 2003

HEV

Art

III-V

III-V

Vein

I I

II

LOV

Chen et al, Nature Immunol. 2006

I

II II

III III

IV IV

V V

Low Order

Venules

(LOV)

High

Endothelial

Venules

(HEV)

Rolling

Fraction =

Sticking

Efficiency =

0

20

40

60

80

Venular order

Sti

ckin

g e

ffic

en

cy (

%)

Ro

llin

g f

racti

on

(%

)

0

20

40

60

80

I II III V IV

I II III V IV

HEV

1

3

2

4

Firm

Adhesion

Transmigration

Activation

CCL21

Tethering/

Rolling

Rolling cells

Total cells

Sticking cells

Total cells


Interactions in PLN

LOV HOV (HEV)

LOV HOV (HEV)


Blood flow velocity at level

IV venules: 500 µm/sec

Rolling

Fraction =

Ro

llin

g f

racti

on

(%

)

0

20

40

60

80

I II III V IV

HEV

1

3

2

4

Firm

Adhesion

Transmigration

Activation

CCL21

Tethering/

Rolling

Rolling cells

Total cells


Interactions in PLN

LOV HOV (HEV)

= Sticking cells

Total cells

Sticking

Efficiency

IV Venular order

0

20

40

60

80

Sti

ckin

g e

ffic

ien

cy (

%)

I II III V

LOV HOV (HEV)

L-selectin Ab

Venular order

L-Selectin

L-S

ele

cti

n

Protein Kinase C

Zn

+2-d

ep

en

de

nt

me

tallo

pro

tease

L-Selectin (CD62L) Shedding

Lymphocyte

Activation

Naïve Effector

CD62Lhi CD62Llo

ADAM-17

Lymphocyte Extravasation in LN HEV

L-Selectin/PNAd-1

Chemokines (CCL21)

LFA-1/ICAM-1/2 1

2

3

Initial

Attachment Rolling Firm

Adhesion Transmigration

Outline






(L-selectin/PNAd)


(CCR7/CCL21)




Adhesion Molecules And Lymphocyte Homing - Buffalo, NY · Adhesion Molecules And Lymphocyte Homing Sharon S. Evans, Ph.D. Dept. Immunology, RPCI Jan. 29 & Feb. 3, 2015

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