The role of hyaluronan and hyaladherins in T cell proliferation Mark Mummert, Ph.D. UNT Health Science Center.

The role of hyaluronan and hyaladherins in T cell

proliferation

Mark Mummert, Ph.D.UNT Health Science Center

I. Hyaluronan overview

II. Hyaluronan mediated T cell proliferation

III. CD44 regulated T cell proliferation

Seminar Outline

Biochemical Properties of Hyaluronan (HA)

Unbranched glycosaminoglycan

Repeating N-acetyl-glucosamineand glucuronic acid subunits

Molecular weight at least >300kDa

HA DegradationHAS1 HMW-HA polymer

HAS2 HMW-HA polymer

HAS3 Intermediate HA

Hyal-1 Tetra- and hexasaccharides

Hyal-2 HMW fragments

Hyal-3 Unknown

Hyal-4 Unknown

HA Synthesis

UDP-GlcA

UDP-GlcNAc

HA Synthase

Nucleus

Functions of HA

Tissue hydration

Structural integrity

Lubrication

Cell activation

Cell trafficking

Cell proliferation

WF/GM/OE-HABP

LYVE-1

CD54

CD38

BEHAB

Inter--trypsin inhibitor related proteins

LEC HA receptor

Neurocan

Hyaluronectin

Versican

Aggrecan

Link Protein

RHAMM

CD44Hyaluronan

WF/GM/OE-HABP

LYVE-1

CD54

CD38

BEHAB

Inter--trypsin inhibitor related proteins

LEC HA receptor

Neurocan

Hyaluronectin

Versican

Aggrecan

Link Protein

RHAMM

CD44HyaluronanCollagens

Fibronectin

Chondroitin sulfates

Heparin

Heparin sulfate

Serglycines

Osteopontin

Hyaluronan BindingProtein (HABP)

HA Receptor-Fc Proteins

Enzymatic digest of bovine nasal cartilage

Variation in quality of different batches

Expensive

Produced recombinantly

Not available commercially

Synthesis is typically low

Specificity is questionable

Available HA Probes Comments

Frequency of Sequences from Phage Clones

Peptide Designation

1

2

3

4

5

Frequency

13/19

2/19

1/19

2/19

1/19

* *

*

*

*

*** **

* β-counter

Metabolic LabelingWith 35S

HA-coated Plate Wash Plate

Lyse Cells

B16-F10

% Bound

0 5 10 15 20 25 30 35 40

Scrambled: WRHGFALTAVNQ

Scrambled: QVNLAHTWARFG

Scrambled: AGNHLATVFWQR

Scrambled: RNHALVGWQFTA

Pep-1: GAHWQFNLATVR

RP: S A T P A S APYPL A

No Inhibitor

Background

**

**

% Bound0 5 10 15 20 25 30 35

Pep-1 (V A)Pep-1 (T A)Pep-1 (L A)

Pep-1 (F A)Pep-1 (Q A)Pep-1 (W A)

Pep-1 (G A)RP

No InhibitorBackground

Pep-1 (WT)

Pep-1 (N A)

Pep-1 (H A)

Pep-1 (R A)

**

**

**

**

**

Functional roles of HA in DC-dependent Agrestricted T cell proliferation

Diana I. MummertDale Edelbaum

Francis HuiHiroyuki MatsueAkira Takashima

Derived from bone marrow precursors and are found in lymphoid and non-lymphoid tissues. They have a dendritic morphology and are potent stimulators of T cells.

A subset of lymphocytes defined by their development in the thymus and by heterodimeric receptors (α:β or γ:δ) associated with the CD3 complex (CD3γ,δ, ε, and dimeric ζ chains).

Dendritic cells (DC)

T cells

T cellsTCRLFA-1ICAM-3

DCAg/MHC IIICAM-1DC-SIGN

Adhesive Molecules and Antigen Presentation

CD44 Overview

Glycoprotein

Expressed by leukocytes

Major receptor for HA

Leukocyte trafficking

Signaling co-receptor

Stimulated T cells express functional CD44 isoforms

Exogenous HA enhances mitogen induced T cell proliferation

Anti-CD44 blocks HA augmented proliferation

CD44/ HA Molecular Interaction and T cells

Figure 3. CD44 in SignalingA. There is little evidence for direct CD44 signaling upon ligation with hyaluronan.B. CD44 can function as a co-receptor, physically linked to other classical signaling receptors.C. CD44 can serve as a docking protein for pericellular proteins, such as MMPs or, for cytoplasmic proteins such as kinases, Smad1, actin-binding proteins and other adapter proteins, which can subsequently activate signal pathways (pink arrows). D. A two-step proteolytic cleavage (red lightening bolts) releases the CD44 intracellular domain (ICD), which exhibits nuclear translocation and functions in transcriptional activation.

XS106

0 20 40 60

Inhibitor

None

None

Anti-CD44

Control Ig

Pep-1

RP

HA

-

+

+

+

+

+

0 4 8 12 16

Murine T Cells

% Bound

Human T Cells

0 5 10 15 20

Not Tested

Not Tested

**

** **

**

**

****

**

HAS3

HAS2

HAS1

Hyal-1

Hyal-2

Hyal-3

-Actin

XS

52

DC

XS

106

DC

BM

-DC

T C

ells

, (+

)Co

n A

T C

ells

, (-)

Co

n A

100 101 102 103 104

FL2-Height

XS52 DC

Splenic T cells (+ ConA)

100 101 102 103 104

FL2-Height

Splenic T cells (-ConA)

100 101 102 103 104

FL2-Height

Splenic DC

100 101 102 103 104

FL2-Height

BM-DC

100 101 102 103 104

FL2-Height

In Vitro Antigen Presentation Assays

Bone Marrow DCDO11.10 T cellsOva Peptide

Cytokine Production

3H-thymidine uptake

CFSE fluorescence profile

DC number/well (x103)

Pro

life

rati

on

(cp

m x

104

)

0 5 100

5

10

15

20

25

****

**

*

Pep-1

RP

Impact of Pep-1 on Ag specific T cell proliferation

C

C

C

C

NN

N N

N C Monomer

TetramerPEG

Pep-1 (M)

% I

nh

ibit

ion

0 100 200 3000

20

60

100**

**

****

PEG-Pep-1

Pep-1

Multivalent Pep-1 enhances T cell inhibition

Prol

ifera

tion

(cpm

x 1

04 )

DC number/well (x103)

0 2 4 6 8 10

0

5

10

15

20

25

**

**

**

PEG-RP

PEG-Pep-1

PBS PBS PEG-Pep-1 PEG-RP

(+) OVA(-) OVA

T cell proliferation: CFSE staining

DC

+

-

-

-

+

+

+

T cells

-

+

-

+

+

+

+

OVA

-

-

+

+

+

+

+

Inhibitor

-

-

-

-

-

bPG

denat. bPG

Proliferation (cpm x 105)0 1 2 3 4 5 6

**

Inhibition of T cell proliferation by bPG

% I

nh

ibit

ion

mAb (g/ml)0 20 40 60

0

20

40

60

80

100

**

** ****

**

**

Cell Adhesion

Antigen Presentation

IL-2

0 1.0 2.0 0 0.5 1.0

IFN

Cytokine concentration (ng/ml)

DC+--+++

T cells-+++++

OVA--++++

Inhibitor----

PEG-Pep-1

PEG-RP

** **

Impact of PEG-Pep-1 on T cell cytokine secretion

Summary

DC and T cells express mRNAs for HA synthases and hyaluronidases

DC and activated T cells express HA polymers on their surfaces

DC induced T cell activation is blocked by two HA inhibitors

Exogenous HACD44

Proliferation

Concept #1

CD44

HA Synthesis

Proliferation

Concept #2

0.0 0.5 1.0 1.5

Proliferation (cpm x 105)

Anti-CD3

-

+

-

+

+

+

Anti-CD28

-

-

+

+

+

+

Inhibitor

-

-

-

-

Pep-1Pep-1

RPRP

Impact of endogenous HA on T cell proliferation

Christie Mahaffey

Inhibitor of HA synthesis

Natural product found in plantsCeleryParsleyManna AshGerman Chamomile

4-Methylumbelliferone (4-MU)

ConA 4-MU - -

+ -

+ + **

3H-glucosamine uptake

0 200 400 600 800 1000 1200 1400

0 20 40 60 80 1000

20

40

60

80

100

0 20 40 60 80 1000

1000

2000

3000

4000

5000

6000

7000

3 H-t

hym

idin

e u

pta

ke

% v

iab

ilit

y

4-MU,μg/ mL

**

****

Impact of 4-MU on ConA stimulated T cells

T cell proliferation by different stimuli

0 20 40 60 80 1000

5000

10000

15000

20000

25000

30000

0 20 40 60 80 1000

2000

4000

6000

8000

10000

12000

14000

4-MU,μg/ mL

3 H-t

hym

idin

e u

pta

ke

**

****

******

PMA / ionomycin Allogeneic Spleen Cells

Impact of 4-MU on CD69 expression

19% 67% 71%

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

A450

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Impact of 4-MU on cytokine secretion

-

- -

+

++

ConA 4-MU

** **

IL-2 IFNγ

0 2 4 6 8

-

+ -

+

++

ConA 4-MU

+

+

+

IL-2

3H-thymidine uptake

HA Synthesis

Proliferation

IL-2 Secretion

CD44

Concept #3

IL-2R

No HA

Control IgG

Anti-CD44

ConA ConA + 4-MU

* **

Adhesion of ConA stimulated T cells to HA

% Adhesion

0 2 4 6 8 10 12 0 2 4 6 8 10 12

Control IgG

Anti-CD44

No ConA

Impact of anti-CD44 on IL-2 secretion

A450

0.0 0.2 0.4 0.6 0.8

HA Synthesis

Proliferation

IL-2 Secretion

??

Concept #4

IL-2R

Pep-1 blocked T cell proliferation and IL-2 secretion

4-MU inhibited HA synthesis by T cells

4-MU inhibited T cell proliferation in a dose dependent fashion but did not globally impair T cell activation

4-MU inhibited the secretion of IL-2 and IFNγ

Addition of IL-2 to T cells treated with ConA and 4-MU reversed the block in proliferation

Blocking the HA binding module of CD44 did not recapitulate the effects of 4-MU on IL-2 production or T cell proliferation

SUMMARY

Endogenously synthesized HA plays a role in IL-2 mediated T cell proliferation. Furthermore, HA inhibitors (e.g., Pep-1) or HA synthase inhibitors (e.g., 4-MU) may have utility in regulating T cell responses.

CONCLUSION

CD44 and the Proliferation of Jurkat T cells

Li-Shu ZhangHe-Wen MaWeilan Zuo

Henry Greyner

Immortalized line of T cells originally derived from the peripheral blood of a 14 year old boy with T cell leukemia. they are CD44 negative.

Expression of CD44 in stably transfected Jurkat cells

RT-PCR Flow Cytometry

Vector CD44

Impact of CD44 on Jurkat cell morphology

Cell density-dependent proliferation of Jurkat cells

25,000 / well 5,000 / well 1,000 / well

Up- and down-regulated genes (2-fold cut-off)

Accession No.

SymbolFold up- or down-regulation

Gene Name Description

Down-regulation:

NM_001964 EGR1 -6.81 AT225/G0S30 Early growth response 1

NM_021784 FOXA2 -4.17 HNF3B/TCF3B Forkhead box A2

NM_002982 CCL2 -3.40 GDCF-2/GDCF-2 HC11

Chemokine (C-C motif) ligand 2

NM_020182 TMEPAI -2.85 PMEPA1/STAG1 Transmembrane, prostate androgen induced RNA

NM_002228 JUN -2.61 AP1/c-Jun Jun oncogene

NM_000639 FASLG -2.41 APT1LG1/CD178 Fas ligand (TNF superfamily, member 6)

NM_003202 TCF7 -2.21 TCF-1 Transcription factor 7 (T-cell specific, HMG-box)

NM_000589 IL4 -2.15 BSF1/IL-4 Interleukin 4

Up-regulation:

NM_000584 IL8 15.98 3-10C/AMCF-I Interleukin 8

NM_004591 CCL20 10.94 CKb4/LARC Chemokine (C-C motif) ligand 20

NM_000450 SELE 5.75 CD62E/ELAM Selectin E (endothelial adhesion molecule 1)

NM_000598 IGFBP3 5.14 BP-53/IBP3 Insulin-like growth factor binding protein 3

NM_015869 PPARG 5.07 NR1C3/PPARG1 Peroxisome proliferator-activated receptor gamma

NM_000230 LEP 3.68 OB/OBS Leptin

NM_000418 IL4R 3.45 CD124/IL4RA Interleukin 4 receptor

NM_005551 KLK2 3.21 KLK2A2/hK2 Kallikrein-related peptidase 2

NM_002416 CXCL9 2.62 CMK/Humig Chemokine (C-X-C motif) ligand 9

Transcription FactorNuclear PhosphoproteinZinc Finger MotifBinds GCGGGGGCG promoter sequencesTransiently expressed in lymphocytes

EGR-1 (Early Growth Response Gene-1)

EGR-1 transcript levels in Jurkat cells

EGR-1 protein expression in Jurkat cells

SB239063: Inhibits p38 activation

UO126: Inhibits ERK 1/2 activation

Wortmannin: Inhibits Akt activation

LY294002: Inhibits Akt activation

Pharmacological Inhibitors to Study Cell Proliferation

Impact of inhibitors on cell proliferation and EGR-1 expression

Akt is hypophosphorylated in CD44 expressing Jurkat cells

3H-Thymidine

0 2000 4000 6000 8000 10000

Vector

CD44

CD44 + Akt

Impact of myristolated Akt on Jurkat cell proliferation

% Inhibition

0 20 40 60 80 100

-

- +

+

CD44 EGR-1 siRNA

Impact of EGR-1 siRNA on Jurkat cell proliferation

CD44 knock in inhibited Jurkat cell proliferation

EGR-1 expression was significantly reduced in CD44knock in Jurkat cells

EGR-1 expression was correlated with Akt activation

EGR-1 siRNA could partially block Jurkat cell proliferation

SUMMARY

CD44 inactivates Akt and subsequent EGR-1 expression

CONCLUSION