The role of hyaluronan and hyaladherins in T cell proliferation

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

1. Hyaluronan overview
2. Hyaluronan mediated T cell proliferation
3. CD44 regulated T cell proliferation

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Biochemical Properties of Hyaluronan (HA)
Unbranched glycosaminoglycan Repeating
N-acetyl-glucosamine and glucuronic acid
subunits Molecular weight at least gt300kDa
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(No Transcript)
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HA Degradation
HA Synthesis
HAS1 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
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HA Synthase
Nucleus
UDP-GlcA
UDP-GlcNAc
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Functions of HA
Tissue hydration Structural integrity Lubrication
Cell activation Cell trafficking Cell
proliferation
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WF/GM/OE-HABP LYVE-1 CD54 CD38 BEHAB Inter-a-tryps
in inhibitor related proteins LEC HA
receptor Neurocan Hyaluronectin Versican Aggrecan
Link Protein RHAMM CD44
Hyaluronan
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WF/GM/OE-HABP LYVE-1 CD54 CD38 BEHAB Inter-a-tryps
in inhibitor related proteins LEC HA
receptor Neurocan Hyaluronectin Versican Aggrecan
Link Protein RHAMM CD44
Hyaluronan Collagens Fibronectin Chondroitin
sulfates Heparin Heparin sulfate Serglycines Osteo
pontin
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Available HA Probes
Comments
Hyaluronan Binding Protein (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
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Frequency of Sequences from Phage Clones

Peptide Designation 1 2 3 4
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Frequency 13/19 2/19 1/19 2/19 1/19
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Metabolic Labeling With 35S
B16-F10





Wash Plate
HA-coated Plate


ß-counter
Lyse Cells
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Background
No Inhibitor
RP S
A
T
P
A
S
APYPL
A








Pep-1 GAHWQFNLATVR
Scrambled RNHALVGWQFTA
Scrambled AGNHLATVFWQR
Scrambled QVNLAHTWARFG
Scrambled WRHGFALTAVNQ
0
5
10
15
20
25
30
35
40
Bound
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Background
No Inhibitor

Pep-1 (WT)
RP
Pep-1 (G A)

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

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

Pep-1 (R A)
0
5
10
15
20
25
30
35
Bound
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Functional roles of HA in DC-dependent
Ag restricted T cell proliferation Diana I.
Mummert Dale Edelbaum Francis Hui Hiroyuki
Matsue Akira Takashima
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Dendritic cells (DC)
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.
T cells
A subset of lymphocytes defined by their
development in the thymus and by heterodimeric
receptors (aß or ?d) associated with the CD3
complex (CD3?,d, e, and dimeric ? chains).
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Adhesive Molecules and Antigen Presentation
T cells TCR LFA-1 ICAM-3
DC Ag/MHC II ICAM-1 DC-SIGN
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CD44 Overview

• Glycoprotein
• Expressed by leukocytes
• Major receptor for HA
• Leukocyte trafficking
• Signaling co-receptor

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CD44/ HA Molecular Interaction and T cells

• Stimulated T cells express functional CD44
  isoforms
• Exogenous HA enhances mitogen induced T cell
  proliferation
• Anti-CD44 blocks HA augmented proliferation

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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.
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Inhibitor None None Anti-CD44 Control Ig Pep-1 RP
HA -
XS106
Murine T Cells
Human T Cells
Not Tested
Not Tested
0
20
40
60
0
4
8
12
16
0
5
10
15
20
Bound
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T Cells, ()Con A
T Cells, (-)Con A
XS106 DC
XS52 DC
BM-DC
HAS1
HAS2
HAS3
Hyal-1
Hyal-2
Hyal-3
b-Actin
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XS52 DC
Splenic DC
BM-DC
Splenic T cells ( ConA)
Splenic T cells (-ConA)
FL2-Height
FL2-Height
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In Vitro Antigen Presentation Assays
Bone Marrow DC DO11.10 T cells Ova Peptide
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Impact of Pep-1 on Ag specific T cell
proliferation
25
20
RP

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Proliferation (cpm x 104)

Pep-1
10


5
0
0
5
10
DC number/well (x103)
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Monomer
N
C
C
C
N
N
Tetramer
PEG
N
N
C
C
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Multivalent Pep-1 enhances T cell inhibition


100
PEG-Pep-1
Inhibition
60


Pep-1
20
0
0
100
200
300
Pep-1 (mM)
30
25
20
PEG-RP
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Proliferation (cpm x 104)

10


5
PEG-Pep-1
0
0
2
4
6
8
10
DC number/well (x103)
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T cell proliferation CFSE staining
() OVA
(-) OVA
PBS
PBS
PEG-Pep-1
PEG-RP
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Inhibition of T cell proliferation by bPG
DC - - -
T cells - -
OVA - -
Inhibitor - - - - - bPG denat. bPG
Proliferation (cpm x 105)
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100


Cell Adhesion

80

60
Inhibition
40

Antigen Presentation
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0
0
20
40
60
mAb (mg/ml)
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Impact of PEG-Pep-1 on T cell cytokine secretion
IL-2
IFNg
DC - -
T cells -
OVA - -
Inhibitor - - - - PEG-Pep-1 PEG-RP


0
1.0
2.0
0
0.5
1.0
Cytokine concentration (ng/ml)
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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
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Concept 1
CD44
Exogenous HA
Proliferation
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HA Synthesis
Concept 2
CD44
Proliferation
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Anti-CD3 - -
Anti-CD28 - -
Inhibitor - - - - Pep-1 RP

0.0
0.5
1.0
1.5
Proliferation (cpm x 105)
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Impact of endogenous HA on T cell
proliferation Christie Mahaffey
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4-Methylumbelliferone (4-MU)

• Inhibitor of HA synthesis
• Natural product found in plants
• Celery
• Parsley
• Manna Ash
• German Chamomile

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ConA 4-MU - -
-

0
200
400
600
800
1000
1200
1400
3H-glucosamine uptake
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Impact of 4-MU on ConA stimulated T cells
viability
3H-thymidine uptake



4-MU,µg/ mL
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T cell proliferation by different stimuli
14000
Allogeneic Spleen Cells
30000
PMA / ionomycin
12000
25000
10000
20000

8000
3H-thymidine uptake

15000
6000

10000
4000



5000
2000
0
0
0
20
40
60
80
100
0
20
40
60
80
100
4-MU,µg/ mL
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Impact of 4-MU on CD69 expression
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71
67
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Impact of 4-MU on cytokine secretion
ConA
4-MU
IFN?
IL-2
-
-
-





0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
A
450
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ConA
4-MU
IL-2
-


-





3H-thymidine uptake
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HA Synthesis
Concept 3
CD44
IL-2 Secretion
Proliferation
IL-2R
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Adhesion of ConA stimulated T cells to HA
ConA
ConA 4-MU
No HA
Control IgG
Anti-CD44


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Impact of anti-CD44 on IL-2 secretion
Control IgG
Anti-CD44
No ConA
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HA Synthesis
Concept 4
?
IL-2 Secretion
Proliferation
IL-2R
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SUMMARY

• 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 IFN?
• 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

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CONCLUSION
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.
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CD44 and the Proliferation of Jurkat T
cells Li-Shu Zhang He-Wen Ma Weilan Zuo Henry
Greyner
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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.
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Expression of CD44 in stably transfected Jurkat
cells
RT-PCR
Flow Cytometry
Vector
CD44
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Impact of CD44 on Jurkat cell morphology
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Cell density-dependent proliferation of Jurkat
cells
25,000 / well
5,000 / well
1,000 / well
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Up- and down-regulated genes (2-fold cut-off)
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Accession No. Symbol Fold up- or down-regulation Gene Name Description
Down-regulation 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
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EGR-1 (Early Growth Response Gene-1)
Transcription Factor Nuclear Phosphoprotein Zinc
Finger Motif Binds GCGGGGGCG promoter
sequences Transiently expressed in lymphocytes
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EGR-1 transcript levels in Jurkat cells
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EGR-1 protein expression in Jurkat cells
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Pharmacological Inhibitors to Study Cell
Proliferation
SB239063 Inhibits p38 activation UO126
Inhibits ERK 1/2 activation Wortmannin Inhibits
Akt activation LY294002 Inhibits Akt activation
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Impact of inhibitors on cell proliferation and
EGR-1 expression
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Akt is hypophosphorylated in CD44 expressing
Jurkat cells
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Impact of myristolated Akt on Jurkat cell
proliferation
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Impact of EGR-1 siRNA on Jurkat cell proliferation
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SUMMARY
CD44 knock in inhibited Jurkat cell
proliferation EGR-1 expression was significantly
reduced in CD44 knock in Jurkat cells EGR-1
expression was correlated with Akt
activation EGR-1 siRNA could partially block
Jurkat cell proliferation
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CONCLUSION
CD44 inactivates Akt and subsequent EGR-1
expression