Title: The role of hyaluronan and hyaladherins in T cell proliferation
1The role of hyaluronan and hyaladherins in T cell
proliferation Mark Mummert, Ph.D. UNT Health
Science Center
2Seminar Outline
- Hyaluronan overview
- Hyaluronan mediated T cell proliferation
- CD44 regulated T cell proliferation
3Biochemical Properties of Hyaluronan (HA)
Unbranched glycosaminoglycan Repeating
N-acetyl-glucosamine and glucuronic acid
subunits Molecular weight at least gt300kDa
4(No Transcript)
5HA 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
6HA Synthase
Nucleus
UDP-GlcA
UDP-GlcNAc
7Functions of HA
Tissue hydration Structural integrity Lubrication
Cell activation Cell trafficking Cell
proliferation
8WF/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
9WF/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
10Available 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
11(No Transcript)
12Frequency of Sequences from Phage Clones
Peptide Designation 1 2 3 4
5
Frequency 13/19 2/19 1/19 2/19 1/19
13Metabolic Labeling With 35S
B16-F10
Wash Plate
HA-coated Plate
ß-counter
Lyse Cells
14Background
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
15Background
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
16 Functional roles of HA in DC-dependent
Ag restricted T cell proliferation Diana I.
Mummert Dale Edelbaum Francis Hui Hiroyuki
Matsue Akira Takashima
17Dendritic 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).
18Adhesive Molecules and Antigen Presentation
T cells TCR LFA-1 ICAM-3
DC Ag/MHC II ICAM-1 DC-SIGN
19CD44 Overview
- Glycoprotein
- Expressed by leukocytes
- Major receptor for HA
- Leukocyte trafficking
- Signaling co-receptor
20CD44/ 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
21(No Transcript)
22Figure 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.
23Inhibitor 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
24T Cells, ()Con A
T Cells, (-)Con A
XS106 DC
XS52 DC
BM-DC
HAS1
HAS2
HAS3
Hyal-1
Hyal-2
Hyal-3
b-Actin
25XS52 DC
Splenic DC
BM-DC
Splenic T cells ( ConA)
Splenic T cells (-ConA)
FL2-Height
FL2-Height
26In Vitro Antigen Presentation Assays
Bone Marrow DC DO11.10 T cells Ova Peptide
27Impact of Pep-1 on Ag specific T cell
proliferation
25
20
RP
15
Proliferation (cpm x 104)
Pep-1
10
5
0
0
5
10
DC number/well (x103)
28Monomer
N
C
C
C
N
N
Tetramer
PEG
N
N
C
C
29Multivalent Pep-1 enhances T cell inhibition
100
PEG-Pep-1
Inhibition
60
Pep-1
20
0
0
100
200
300
Pep-1 (mM)
3025
20
PEG-RP
15
Proliferation (cpm x 104)
10
5
PEG-Pep-1
0
0
2
4
6
8
10
DC number/well (x103)
31T cell proliferation CFSE staining
() OVA
(-) OVA
PBS
PBS
PEG-Pep-1
PEG-RP
32Inhibition of T cell proliferation by bPG
DC - - -
T cells - -
OVA - -
Inhibitor - - - - - bPG denat. bPG
Proliferation (cpm x 105)
33100
Cell Adhesion
80
60
Inhibition
40
Antigen Presentation
20
0
0
20
40
60
mAb (mg/ml)
34Impact 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)
35Summary
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
36Concept 1
CD44
Exogenous HA
Proliferation
37HA Synthesis
Concept 2
CD44
Proliferation
38Anti-CD3 - -
Anti-CD28 - -
Inhibitor - - - - Pep-1 RP
0.0
0.5
1.0
1.5
Proliferation (cpm x 105)
39Impact of endogenous HA on T cell
proliferation Christie Mahaffey
404-Methylumbelliferone (4-MU)
- Inhibitor of HA synthesis
- Natural product found in plants
- Celery
- Parsley
- Manna Ash
- German Chamomile
41ConA 4-MU - -
-
0
200
400
600
800
1000
1200
1400
3H-glucosamine uptake
42Impact of 4-MU on ConA stimulated T cells
viability
3H-thymidine uptake
4-MU,µg/ mL
43T 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
44Impact of 4-MU on CD69 expression
19
71
67
45Impact 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
46ConA
4-MU
IL-2
-
-
3H-thymidine uptake
47HA Synthesis
Concept 3
CD44
IL-2 Secretion
Proliferation
IL-2R
48Adhesion of ConA stimulated T cells to HA
ConA
ConA 4-MU
No HA
Control IgG
Anti-CD44
49Impact of anti-CD44 on IL-2 secretion
Control IgG
Anti-CD44
No ConA
50HA Synthesis
Concept 4
?
IL-2 Secretion
Proliferation
IL-2R
51SUMMARY
- 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
52CONCLUSION
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.
53 CD44 and the Proliferation of Jurkat T
cells Li-Shu Zhang He-Wen Ma Weilan Zuo Henry
Greyner
54Immortalized line of T cells originally derived
from the peripheral blood of a 14 year old boy
with T cell leukemia. they are CD44 negative.
55Expression of CD44 in stably transfected Jurkat
cells
RT-PCR
Flow Cytometry
Vector
CD44
56Impact of CD44 on Jurkat cell morphology
57Cell density-dependent proliferation of Jurkat
cells
25,000 / well
5,000 / well
1,000 / well
58(No Transcript)
59Up- and down-regulated genes (2-fold cut-off)
60Accession 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
61EGR-1 (Early Growth Response Gene-1)
Transcription Factor Nuclear Phosphoprotein Zinc
Finger Motif Binds GCGGGGGCG promoter
sequences Transiently expressed in lymphocytes
62EGR-1 transcript levels in Jurkat cells
63EGR-1 protein expression in Jurkat cells
64Pharmacological Inhibitors to Study Cell
Proliferation
SB239063 Inhibits p38 activation UO126
Inhibits ERK 1/2 activation Wortmannin Inhibits
Akt activation LY294002 Inhibits Akt activation
65Impact of inhibitors on cell proliferation and
EGR-1 expression
66(No Transcript)
67Akt is hypophosphorylated in CD44 expressing
Jurkat cells
68Impact of myristolated Akt on Jurkat cell
proliferation
69Impact of EGR-1 siRNA on Jurkat cell proliferation
70SUMMARY
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
71CONCLUSION
CD44 inactivates Akt and subsequent EGR-1
expression