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Loyola University Chicago Loyola University Chicago
Loyola eCommons Loyola eCommons
Dissertations Theses and Dissertations
2010
Phospholipase D Signaling in T Cells Phospholipase D Signaling in T Cells
Uma Chandrasekaran Loyola University Chicago
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Part of the Immunology and Infectious Disease Commons
Recommended Citation Recommended Citation Chandrasekaran, Uma, "Phospholipase D Signaling in T Cells" (2010). Dissertations. 162. https://ecommons.luc.edu/luc_diss/162
This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected].
Copyright by UMA CHANDRASEKARAN, 2010 All rights reserved
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my mentor, Dr. Makio Iwashima
for providing an excellent learning environment in the laboratory that has
facilitated my development as a scientist. I am forever grateful to Dr. Knight for
her mentoring and encouragement throughout my graduate school career.
I would also like to thank my dissertation committee, Dr. Christopher
Wiethoff, Dr. Herbert Mathews and Dr. Adriano Marchese for their helpful
suggestions. I am especially grateful to Medical College of Georgia for providing
me a solid foundation during the start of my graduate school career.
I would like to thank all present and past members of the Iwashima Lab for
their technical help and suggestions. Special thanks to Dr. Nagendra Singh,
Mayuko Takezaki, Dr. Yoichi Seki and Dr. Mutsumi Yamamoto; without their
technical expertise, my project would not have been possible. I would also like to
thank my friends Oana Maier, Mariko Takami and Kathleen Mishler for their
friendship and unwavering support.
Finally, I would like to thank my parents, my brother, my husband and the
rest of my family for their continued support, love and encouragement.
iv
TABLE OF CONTENTS ACKNOWLEDGEMENTS iii LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS xi ABSTRACT xv INTRODUCTION 1 CHAPTER ONE: LITERATURE REVIEW 5 The T lymphocyte in Immunology 5 Introduction 5 Establishing the role and function of Thymus 7 Delineation of two major lymphocyte subsets 8 Subdivision of T lymphocytes: CD4 and CD8 10 Heterogeneity of helper T cells 12 Introduction 12 Identification of Th1 and Th2 cells 12 Development and regulation of Th1 and Th2 cells 14 Effector functions and disease implications 16 Th17 cells: new member of effector T cells 16 Heterogeneity of Regulatory T cells 20 Introduction 20 Identification of distinct subsets of Treg cells 21 Phenotypic and functional characteristics of Treg cells 23 Immune homeostasis: population balance between 29 effector and regulatory T cell subsets Role of TCR mediated signaling pathways in altering 32 the immune balance Balancing act of Phospholipase D signaling in T cells 34 PLD: structure, function and expression 34 Regulation of catalytic activity of PLD 38 TCR signaling and PLD 40 Modulation of Phospholipase D signaling by 42 extra-cellular stimuli
v
Adenosine and PLD signaling 45 Conclusion 47 CHAPTER TWO: MATERIALS AND EXPERIMENTAL 48 METHODS Mice 48 Southern blot 49 Adoptive transfer in mice 49 Cell isolation and culture 50 Flow cytometry 53 T cell proliferation assay 54 ELISA 55 Phospholipase D (PLD) assay 56 cAMP measurement 57 Western blot 57 Generation of PLD YFP fusion constructs 58 Confocal microscopy 59 CHAPTER THREE: EXPERIMENTAL RESULTS 65 Regulation of Phospholipase D signaling by exogenous factors 65 Ethanol and CD4 T cell population dynamics 66 Effect of alcohol ingestion on the balance between 80 Foxp3+ and Foxp3- CD4 T cells Clostridium difficile toxin: Hijacking the immune system 82 through PLD signaling Regulation of phospholipase D signaling by endogenous factors 90 Adenosine and CD4 T cell population dynamics 97 Cross talk between adenosine receptor and 101 Phospholipase D signaling Adenosine A3 receptor signaling and cAMP 105 PLD signaling: synergy between ethanol and adenosine 111 Structure-function analysis of Phospholipase D 115 Determinants of PLD1 peri-nuclear localization 116 Determinants of PLD2 plasma membrane localization 122 Effect of PLD2 gene deletion on T cell response 125 CHAPTER FOUR: DISCUSSION 136 Introduction 136 Immune balance: effect of exogenous factors 137
vi
Immune balance: effect of endogenous factors 140 AB-MECA and Ethanol: Potential use as a 146 Tolerogenic adjuvant PLD localization: interaction between multiple structures 147 PLD2 and T cell functioning 149 Concluding remarks 151 REFERENCES 152 VITA 191
vii
LIST OF TABLES
Figure Page
1. PCR primers 64
viii
LIST OF FIGURES
Figure Page
1. A schematic presentation of the hypothesis proposed 4
2. Heterogeneity of CD4 T cells 19
3. Different subsets of regulatory T cells 28
4. Schematic representation of catalytic reaction of PLD 36 and domain structure of PLD isoforms found in mice 5. Western blot analysis of PLD1 deletion mutant constructs 60
6. Western blot analysis of PLD2 deletion mutant constructs 61
7. Confocal microscopic analysis of YFP fusion constructs 63
8. Effect of ethanol on CD4 T cells in vitro 67
9. Effect of ethanol on CD4 T cells in the presence of 69 antigenic stimulation in vitro
10. Proliferation kinetics of CFSE labeled CD4 T cells 70 in the presence of ethanol
11. Flow cytometry analysis and quantification of cell 73 numbers of CD4 T cells in mice injected with ethanol 12. Effect of ethanol on the proliferation kinetics of Foxp3 74 positive and Foxp3 negative CD4 T cells in vivo in the presence of antigen
ix
13. Effect of ethanol on homeostatic proliferation of 77 CD4 T cells 14. Effect of alcohol containing diet on mice CD4 T cells 81
15. Analysis of proliferation kinetics of Foxp3+ and Foxp3- 83 T cells in the presence of C.difficile toxin supernatants 16. Flow cytometric analysis of activation markers expressed 86 by CD4 T cells following antigenic stimulation in the presence of C.difficile supernatants 17. Flow cytometric analysis of Foxp3+ T cell percentage in 88 mice infected with C.difficile spores 18. Western blot analysis of adenosine A3 receptor 92 expression on CD4 T cells 19. Analysis of activation markers on T cells following 93 antigenic stimulation in the presence of A3 receptor signaling 20. Comparison of proliferation and cytokine secretion profile 95 of DMSO and AB-MECA treated CD4 T cells 21. Effect of adenosine A3 receptor signaling on CD4 T 98 cell population dynamics 22. Flow cytometric analysis of Foxp3 induction in CD4+CD25- 100 T cells following adenosine A3 receptor signaling
23. Effect of A3 receptor signaling on the proliferation kinetics of 102 Foxp3 positive and Foxp3 negative CD4 T cells in vivo 24. Assay of PLD activity in AB-MECA treated CD4 T cells 104 25. Assay for cAMP levels and activity in CD4 T cells 106 26. Western blot analysis of phosphorylation patterns of 110 PKA substrates in AB-MECA treated CD4 T cells 27. Combinatorial effect of ethanol and adenosine on CD4 112 T- cell population dynamics.
x
28. Western blot analysis of phosphorylation patterns of PKA 114 substrates in CD4 T cells treated with a combination of ethanol and AB-MECA 29. Confocal microscopic analysis of the sub-cellular localization 117 of PLD1 loop deletion mutant 30. Confocal microscopic analysis of the sub-cellular localization 118 of PLD1 loop alone or loop and full length PLD1 combinations 31. Schematic representation of the mutant PLD1 constructs 120 and confocal microscopy of their sub-cellular localization 32. Schematic representation of the mutant PLD2 constructs 123 and confocal microscopy of their sub-cellular localization 33. Phenotypic analysis of conditional PLD2 deficient mice 126 34. Characterization of CD4 T cell function in PLD2 128 deficient T cells 35. Cytokine profiles of PLD2 deficient CD4 T cells 130 36. Differentiation of PLD2 CD4 T cells into Th1, Th2 132 and Th17 effector cells 37. Effect of A3 receptor agonist on PLD2 deficient 133 CD4 T cells 38. Effect of ethanol on PLD2 deficient CD4 T cells 134
39. Schematic representation of A3 receptor signaling 145 in CD4 T cells
observed very little, if any, increase in phosphorylation of PKA substrates in
samples treated with the combination of AB-MECA (50μM) and 0.5% ethanol
compared to 0.5% ethanol alone (Fig 28). Since there are many potential bands
that are not separated by 1D gel analysis, further analysis is needed to determine
whether ethanol and AB-MECA have synergistic effect on PKA activity.
STRUCTURE-FUNCTION ANALYSIS OF PHOSPHOLIPASE D
The data presented above show that various exogenous and endogenous
factors influences the immune outcome by regulating PLD signaling. However,
the contribution of PLD to T cell signaling is not clearly understood. As described
previously, the catalytic action of PLD on phosphatidyl choline (PC) results in the
formation of phosphatidic acid (PA) and soluble choline. The formation of PA
constitutes the crucial contribution of PLD to cellular signaling and function. PA is
an important lipid second messenger that can also be synthesized by several
other routes and thus the contribution of PLD to the overall mass of PA in cells is
quite miniscule (Ktistakis et al., 2003). Therefore, PLD is thought to only
influence the local levels of PA. Thus, delineation of the spatial regulation of PLD
isoforms in T cells will help us understand its function in T cells.
As mentioned previously, the two isoforms of PLD localize at different
locations in the cell. Over expression and epitope tag studies reveal that PLD1 is
found mainly in punctate structures around perinuclear regions while PLD2 is
primarily found in the plasma membrane. To determine the region that controls
the localization of PLD1 and PLD2, a series of mutants of both PLD1 and PLD2
116
were generated. In order to assess the localization of these mutants in the cell,
these mutants were tagged with YFP in the N-terminal and transfected in Jurkat
T antigen cells, which express SV40 large T antigen to allow high level of
expression of transfected genes (Northrop et al., 1993) . After transfection (24
hrs), live cells were separated using Ficoll gradient, mounted on glass slides
coated with lysine, fixed and covered with cover slip. These slides were then
analyzed using confocal microscopy.
Determinants of PLD1 perinuclear localization
At first, we addressed the region responsible for the differential localization
of PLD1 and PLD2. The primary sequence of PLD1a revealed the presence of
116 amino acid ‘loop region’ inserted between catalytic sites II and III (Frohman
et al., 1999). Previous studies in which the loop region of PLD1 was deleted
have reported modest increase in the basal activity of PLD1 (Sung et al., 1999).
Based on this observation, we hypothesized that deletion of the loop region
redistributes PLD1 similar to PLD2 and this results in increased catalytic activity
of PLD1. To test this idea, mutant human PLD1b with loop (aa. 504-585) deletion
was constructed and its localization was assessed using confocal microscopy.
The loop deletion mutant of PLD1 lost its ability to localize in punctate structures
surrounding the nucleus and localized to the plasma membrane, similar to PLD2
(Fig 29). These data suggested that the loop region is a required component for
PLD1 localization in vesicular structures.
117
PLDc
5851 1036
Fig 29. Confocal microscopic analysis of the sub-cellular localization of
PLD1 loop deletion mutant. PLD1 and PLD2 full length and loop deletion
mutant are tagged with YFP. Green indicates the localization of the respective
construct and red in propidium iodide staining of the nucleus. These constructs
were transfected in jurkat T antigen cells. LIve cells were isolated 24 hours later
by ficoll gradient and plated on lysine coated glass slides. Cells were then
imaged using the confocal microscope and images were processed using LSM
image examiner software.
PX PH PLDc
loop
C-term
504
PLDc
5861 1036
PX PH C-ter
503
PLDc
PLDc
1 933
PLDcPX PH C-term
Full length PLD1b
Full length PLD2
Loop deleted PLD1b
Full length PLD1
Full length PLD2
Loop deleted
118
Full length PLD1
+ Full length PLD1
58loop
50FLAG
58loop
50FLAG
Fig 30. Confocal microscopic analysis of the sub-cellular localization of
PLD1 loop region alone or loop region and full length PLD1 combinations.
PLD1 full length is tagged with YFP. Green indicates the localization of the full-
length PLD1 construct. Loop alone construct was tagged with FLAG. Blue is the
DAPI staining of the nucleus. Loop alone construct was detected using anti-
FLAG conjugated with flurochrome. PLD1 and loop construct were transfected in
jurkat T antigen cells either alone or together. Live cells were isolated 24 hours
later by ficoll gradient and plated on to lysine coated glass slides. Cells were then
imaged using the confocal microscope and the images were processed using
LSM image examiner software.
119
Next, we determined if the loop region is sufficient to determine the
localization of PLD1 into vesicular structures. Furthermore, if loop interacts with
unknown target molecules in the cell that dictate the localization of PLD1 then
expression of the loop alone construct could interfere with PLD1-target molecule
interaction. Therefore, flag tagged ‘loop alone’ construct was co-transfected with
YFP fusion full length PLD1. Full length PLD1 localized in punctate structures
and when co-transfected with the ‘loop alone” construct, there were no
observable differences in its localization (Fig 30). The loop alone construct was
mainly detected in the plasma membrane. The data suggest that the loop region
is required for PLD1 sub-cellular localization in the vesicular structures but loop
alone (aa 504-585) is not sufficient to create the structure of PLD1 that interacts
with the cellular target that determines the localization of PLD1.
Also, we determined the contribution of PX, PH and C-terminal regions to
the peri-nuclear localization of PLD1 by deletion of the corresponding regions
from full length PLD1. These constructs were transfected into Jurkat T antigen
cells and their localization was determined by confocal microscopy. The
construct lacking PX and PH domain (a.a 1-333 deletion) localized entirely in the
cytoplasm (Fig 31). The mutant with only PX and PH domain (a.a 1-342) had
diffuse localization in both nucleus and cytoplasm, similar to vector alone.
Interestingly, the construct with only the C-terminal (aa. 933 -1036) localized
entirely in the nucleus. This suggests that the C-terminal (aa-933-1036) has
nuclear import property. Indeed, PLD1b localization in the nucleus has been
reported before (Freyberg et al., 2001).
120
PLDc
5851 1036
PLDcPX PH loop
C-term
504
PLD1b Full length
C-term
PLDc
585334 1036
PLDc loop
504
N-terminal deletion of PLD1
1
PX PH
342PX+ PH only
C-terminal only
1036 933
C-termina
Full length PLD1b
N-terminal deletion PX+PH only C-terminal only
Merged
YFP
DAPI
121
Fig 31. Schematic representation of the mutant PLD1 constructs and
confocal microscopy of their sub-cellular localization. All the constructs are
tagged with YFP. Green indicates the localization of these constructs. Blue is the
DAPI staining of the nucleus. These constructs were transfected in jurkat T
antigen cells. Live cells were isolated 24 hours later by ficoll gradient and plated
on lysine coated glass slides. Cells were then imaged using the confocal
microscope and images were processed using LSM image examiner software.
122
Together, these data suggest that the interaction of PX, PH domain with the rest
of PLD1 domains determines the perinuclear localization of PLD1. It is evident
that interactions between different domains rather than a single domain are key
to the sub cellular localization of PLD1.
Determinants of PLD2 plasma membrane localization
PLD2 has strikingly restricted distribution to the plasma membrane. Thus,
YFP fusion constructs of PLD2 deletion mutants were made to determine the
domain necessary for its plasma membrane localization. The construct
containing PX+PH+ C-terminal localized to the cytoplasm (Fig 32). Similar to
PLD1, 'C-terminal only’ construct of PLD2 localized to the nucleus. This
observation is in agreement with previous studies indicating highly homologous
C-termini of both PLD isoforms (Liu et al., 2001). Together, these observations
suggest that the interaction of PX, PH and C-terminal with the catalytic region
determines the plasma membrane localization of PLD2.
Future studies can be aimed at assaying the catalytic activities of both
PLD1 and PLD2 mutant constructs and its effect of TCR signaling to pinpoint with
certainty the contribution of PLDs to the functioning of the immune system.
123
PLDc
1 933
PLDcPX PH C-term
Full length PLD2
C term
1 933
PX PHPH+PX+C
C
termC terminal only
Full length PLD2
PH+PX+C C terminal only
Merged
YFP
PI
124
Fig 32. Schematic representation of the mutant PLD2 constructs and
confocal microscopy of their sub-cellular localization. All the constructs are
tagged with YFP. Green indicates the localization of these constructs. Red is the
propidium iodide staining of the nucleus. These constructs were transfected in
jurkat T antigen cells. Live cells were isolated 24 hours later by ficoll gradient and
plated on lysine coated glass slides. Cells were then imaged using the confocal
microscope and the images were processed using LSM image examiner
software.
125
EFFECT OF PLD2 GENE DELETION ON T CELL RESPONSES
Delineating the isoform specific contribution of PLD1 and PLD2 would be
critical to address their role in regulatory T cell enrichment. In order to delineate
the potential role of PLD2 in regulating the balance between effector and
regulatory T cell subsets, we generated mice deficient in PLD2 specifically in T
cells. Mice carrying floxed PLD2 genes were crossed with CD4 CRE mice in
order to delete the gene in double positive (CD4+CD8+) and CD4 and CD8
single positive T lymphocytes. PLD2 gene was successfully deleted in CD4 T
cells as detected by the differential size of DNA segments (WT mice = 6 kb,
PLD2 +/- = 6 and 5.2kb, PLD2 -/- = 5.2Kb) on southern blots (Fig 33a).
At first, we characterized PLD2flox/floxCD4cre mice to determine the effect
of deletion of PLD2 on thymocyte differentiation and peripheral T cell activation.
The absence of PLD2 had minimal effect on T cell development as determined
by flow cytometric analysis of thymocytes from WT and PLD2 (+/- and -/-) mice
(Fig 33B). Also, CD4:CD8 ratios in the spleen were similar between PLD2 (+/-)
and PLD2 (-/-) mice (Fig 33 C). Based on our previous in vitro findings, we
expected increased frequency of regulatory T cells in PLD2 knock out mice.
Surprisingly, deletion of PLD2 did not alter the development of naturally occurring
CD4+ CD25+ Foxp3+ regulatory T cells (Fig 33D). These observations suggest
that deleting PLD2 at the double positive stage of T cell development does not
affect the development of CD4, CD8 and regulatory T cells.
126
WT +/- -/-
WT +/- - / -
CD
8
CD 4 CD 4
Foxp3
A)
B)
C)
D)
CD
8
CD
25
Fig 33. Phenotypic analysis of conditional PLD2 deficient mice. A) Southern
blot analysis of CD4 T cells from WT or PLD2 (+/-), PLD2 (-/-) mice. B) Flow
cytometric analysis of thymocytes from PLD2 deficient mice. X-axis corresponds
to CD4 T cells and y-axis corresponds to CD8 T cells. C and D) Flow cytometric
analysis of splenocytes from PLD2 deficient mice. Foxp3 versus CD25 is gated
on CD4+ T cells.
127
Next, we determined the functional consequences of TCR induced
signaling in PLD2 deficient T cells. Sorted CD4 T cells were stimulated with anti-
CD3 and irradiated APCs overnight. Expression of CD69 (very early activation
antigen) and CD25 (IL-2Rα) was determined by flow cytometry. Both WT and
PLD2 (-/-) mice up regulated activation markers to the same extent (Fig 34A).
Concomitant with activation status, both WT and PLD2 (-/-) mice had similar
proliferation kinetics 72 hrs after activation (Fig 34B). Although, our previous in
vitro experiments suggested that inhibition of PLD signaling using 1-
alcohol/adenosine blocks proliferation of effector CD4 T cells, proliferation of CD4
T cells from PLD2 knock out mice did not exhibit any significant differences. This
result indicates that the ability of PLD2 (-/-) CD4 T cells to initially become
activated is not affected by their lack of PLD2.
In addition, we determined the cytokine secretion by CD4 T cells of PLD2
knock out mice. Sorted CD4 T cells were added to tissue culture plates coated
with anti-CD3 along with soluble anti-CD28 for 72 hrs. After 72 hrs of stimulation,
cells were rested for 48 hrs and then re-stimulated with PMA and ionomycin.
Intracellular cytokine staining on these cells indicate that in PLD2 (-/-) mice, the
CD4 T cells have 24% IL-2 positive cells compared to 5% (WT) and 12%
(PLD2+/-) mice (Fig 35). In contrast, the percentage of IL-4 secreting cells was
lower in PLD2 (-/-) mice (6%) compared to WT (18%) and PLD2 (+/-) mice
(12%). We did not observe any significant differences in the cytokine levels of IL-
17 and TNF-α between WT and PLD2 (-/-) mice. Thus, the data indicate that
128
A)
B)
αCD3
0.5
CD25
CD
69
Unstimulated μg/ml 0.2 μg/ml
PLD2 (+/+)
PLD2 (+/-)
PLD2 (-/-)
APC + CD3
0.E+00
1.E+02
2.E+02
3.E+02
4.E+02
5.E+02
6.E+02
0 0.1 0.3 0.5 1anti-CD3 concentration
O.D
APC + CD3 + IL2
0.E+00
5.E+05
1.E+06
2.E+06
2.E+06
3.E+06
0 0.1 0.3 0.5 1
ANTI-CD3 CONCENTRATION (ug/ml)
wtwt hetero d2hetero d2 homo d2homo d2
129
Fig 34. Characterization of CD4 T cell function in PLD2 deficient T cells. A)
Activation markers, CD69 (y-axis) and CD25 (x-axis) following stimulation. Cells
are gated on CD4 T cells after 24 hrs of stimulation with anti-CD3. B)
Proliferation analysis of PLD2 deficient CD4 T cells after 72 hrs of stimulation
with different concentrations of anti - CD3. WT, PLD2 (+/-) and PLD2 (-/-) mice
are littermates.
130
IL-2
IL-4
IL
-17
TNF-α
IFN- γ
PLD2 (-/-) PLD2 (+/-)PLD2 (+/+)
Fig 35. Cytokine profiles of PLD2 deficient CD4 T cells. A) CD4 T cells from
WT and PLD2 KO mice were stimulated with plate bound anti-CD3 and soluble
CD28 for 72 hrs, following which cells were rested for 2 days. Cells were re-
stimulated with PMA and ionomycin and intracellular FACS staining for IL-2,
IFN- γ, IL-4, IL-17 and TNF-α was performed.
131
PLD2 deficient CD4 T cells remain as IL-2 producers after three days of
stimulation and have impairment in differentiation into Th2 type T cells.
To test if PLD2 is necessary for effector T cell differentiation into different
subsets namely Th1, Th2 or Th17, CD4 T cells from WT and PLD2 knock out
mice were activated under Th1 (in the presence of IFN-γ, IL-12 and anti-IL-4),
Th2 (in the presence of IL-4 and anti-IFN-γ) or Th17 (in the presence of TGF-β
and IL-6) skewing conditions and FACS staining was performed to detect
intracellular cytokine expression. Under Th1 induction conditions, 78% of WT
CD4 T cells were IFN-γ positive and 11% were IL-2 positive while in PLD2 (-/-)
mice 21% were IL-2 positive and 66% were IFN-γ positive (Fig 36, top row)
indicating slight resistance to differentiate into Th1 phenotype. On the other
hand, under Th2 induction conditions 1.8% of WT CD4 T cells were IL-4 positive
in comparison to 0.8% of PLD2 (-/-) T cells (Fig 36, middle row). Under Th17
skewing conditions, ~8% of CD4 T cells in PLD2 (-/-) mice were IL-17 positive
compared to 6% of WT T cells (Fig 36, bottom row). Together, the data suggest
that PLD2 (-/-) mice have slight impairment in differentiation into Th1/Th2 cell
types and remain at IL-2 secreting stage. We can further assess effector T cell
differentiation in PLD2 (-/-) mice in vivo (eg: by using P25 peptide, a strong
inducer of Th1 response).
In light of our previous findings, we next determined if ethanol/adenosine
inhibits effector T cell proliferation through inhibiting PLD2. To test this, we
treated WT, PLD2 (+/-) and PLD2 (-/-) mice with DMSO, A3 agonist or A3 agonist
plus 0.5% ethanol. We predicted that if A3 agonist inhibited PLD2 signaling, then
132
PLD2 (+/+) PLD2 (+/ -) PLD2 (- / -) IL
-2IL
-4IL
-17
TH1 induction
TH2 induction
TH 17 induction
IFN- γ
Fig 36. Differentiation of PLD2 CD4 T cells into Th1, Th2 and Th17 Effector
cells. Spleen CD4 T cells were stimulated with anti-CD3 in vitro and rested 5
days in the presence of Th1, Th2 or Th17 skewing conditions. T cells were re-
stimulated with PMA and Ionomycin and intracellular cytokine staining was
performed.
133
WT
PLD2 (+/-)
PLD2 (-/-)
DMSO AB-MECA
CTL
A4
Foxp3
Fig 37. Effect of A3 receptor agonist on PLD2 deficient CD4 T cells. CD4 T
cells isolated from WT and PLD2 KO mice spleen were stimulated with APC and
anti-CD3 either in presence of A3 agonist (AB-MECA) or carrier control (DMSO)
for 3 days. On day 4, cells were washed and placed in medium containing IL-2
for 4 days. On day 7, cells were harvested and stained for detection of Foxp3
expression and analyzed using flow cytometry.
134
WT
PLD2 (+/-)
PLD2 (-/-)
2% ethanol 1% ethanol0.5% ethanol
Foxp3
CTL
A4
Fig 38. Effect of ethanol on PLD2 deficient CD4 T cells. CD4 T cells isolated
from WT and PLD2 KO mice spleen were stimulated with APC and anti-CD3 in
the presence of varying concentrations of ethanol for 3 days. On day 4, cells
were washed and placed in medium containing IL-2 for 4 days. On day 7, cells
were harvested and stained for detection of Foxp3 expression and analyzed
using flow cytometry.
135
treatment of PLD2 (+/-) with A3 agonist and ethanol would lead to increased
frequency of Foxp3+ cells. However, we found no differences in the frequency of
Foxp3+ cells between WT and PLD2 knock out mice under these treatment
conditions (Fig 37 and 38). This suggests that A3 agonist/ethanol mediated
enrichment of Treg cells might involve additional signaling pathways other than
PLD2. Future experiments can be conducted to determine the level of PLD
activity in PLD2 (-/-) T cells. Based on the experiments of Treg enrichment using
AB-MECA/ethanol it is very likely that PLD1 compensates for the lack of PLD2 in
PLD2 (-/-) mice thus exhibiting no difference in PLDactivity levels.
Future studies in PLD2 (-/-) mice can be conducted to determine its role in
T cell signaling and function. Previous data from the lab indicated that PLD2 is
involved in proximal TCR signaling events. We can determine if PLD2 (-/-) mice
exhibit altered TCR signaling pathway compared to WT mice.
CHAPTER FOUR
DISCUSSION
Introduction
The response to pathogenic attack and maintenance of immune
homeostasis requires precise coordination between different cell types of helper
T cell subsets. Population balance between different types of cells is key to
sustaining a balanced immune system. Many studies have reported altered
balance between effector versus regulatory T cell subsets as the prime cause of
disease pathogenesis. For instance, increased frequency and suppressor
function of Treg cells have been reported and proposed as potential mechanism
of immune suppression in patients with tumor and HIV-infection (Strauss et al.,
2007a) (Strauss et al., 2007b) (Sempere et al., 2007). On the other hand,
reduced number/function of Treg cells and increased effector T cell function have
been reported in patients with allergic/autoimmune diseases (Dejaco et al., 2006)
(Bacchetta et al., 2006).
Hence, ideal treatment for conditions of excessive immune suppression
would be to enhance the frequency/ function of effector T cells and vice–versa for
autoimmune diseases. Therapeutic potential of this strategy has spurred the
136
137
interests of researchers leading to the discovery of many mechanisms for
specific enrichment of either Treg cells or effector T cells.
Previously, our lab reported the differential requirement for PLD signaling
in Treg cells versus effector T cells. As a consequence, inhibition of PLD
signaling led to preferential enrichment of Treg cells by inhibiting effector T cell
proliferation. Interestingly, other reports suggest increased PLD expression and
signaling in autoimmune myocarditis and chronic inflammation (Ahn et al., 2004).
For my dissertation work, I focused on identification of mechanisms regulating
PLD signaling in T cells. The data demonstrated that various exogenous
(ethanol, bacterial toxins) and endogenous (adenosine) factors alter the immune
balance through regulating PLD signaling. PLD2 deficient mice showed that the
effect of these molecules on the Teff: Treg balance is not dependent on PLD2
alone. Further the structure that controls the sub cellular localization of PLD1 and
PLD2 was determined using mutant construct analysis.
IMMUNE BALANCE: EFFECT OF EXOGENOUS FACTORS
The human body is exposed to myriads of pathogens, pollutants, and
environmental and bacterial toxins in day-to-day life. The immune system has
evolved in many ways to combat these intruders and to maintain peace with self
through mechanisms like plasticity of helper T cell subsets. But some pathogens
and various exogenous molecules selectively manipulate the immune balance to
their advantage by shifting the equilibrium between these activities one way or
138
the other. For instance, environmental toxin like dioxin has been shown to cause
immune suppression by increasing Treg frequency (Quintana et al., 2008). Our
data demonstrate that by inhibiting PLD signaling, exogenous factors like ethanol
and C. difficile toxin tip the balance towards immune suppression.
Our results suggest that these external factors by means of inhibiting PLD
signaling, block the up regulation of activation induced CD25 expression on
naïve CD4 T cells. IL-2 is an important T cell growth factor and CD25 is required
for the proper function/signaling of IL-2. However, Treg cells constitutively
express CD25 and thus expand in the presence of exogenous IL-2. The
functional significance of ethanol/C. difficile toxin mediated inhibition of PLD
signaling is suggested by the decreased proliferation of antigen specific effector
T cells and increased frequency of antigen specific Treg cells in vivo.
There is extensive evidence that ethanol consumption leads to immune
suppression. Recently, both acute and chronic alcohol intake have been shown
to result in specific defects in innate and adaptive immunity (Nelson and Kolls,
2002). It is suggested that ethanol results in loss of splenic and circulating T and
B cells through apoptosis (Shao et al., 1995) (Sibley and Jerrells, 2000). Our
experiments using ethanol provide evidence to suggest that this loss of T cells
might be due to lack of PLD signaling and its effect on naïve T cells. In addition
to mediating its effect through CD4 T cells, alcohol has been shown to modulate
the functioning of other immune cells like macrophages, neutrophils, dendritic
cells with antigen presenting capabilities (Szabo et al., 1993) (Nelson and Kolls,
139
2002). The lack of activation induced marker expression on CD4 T cells might
also be mediated through lack of antigen presentation through these APCs.
Thus, in our experimental set up we cannot rule out the effect of ethanol on these
APCs and the consequent reduction in TCR activation and PLD signaling.
In contrast to mice injected with ethanol intraperitoneally, mice fed on
alcohol diet showed increased frequency of Treg cells only locally (mesenteric
lymph nodes). This result can be explained based on previous studies
suggesting that oral administration of ethanol results in increased availability and
metabolism of ethanol in the gut resulting in less entry of ethanol into the blood
stream while intraperitoneal injections result in rapid appearance of ethanol in the
blood stream (Livy et al., 2003). Thus oral exposure of alcohol reflects the
localized effect of ethanol on T cells while intraperitoneal injection reflects
systemic effect of ethanol on T cells. This phenomenon of local immune
suppression could in principle be exploited for treatment of gut associated
inflammatory diseases like IBD and colitis.
Our studies with C. difficile toxin demonstrate a new facet of immune
evasion strategy through PLD signaling. C. difficile toxins have long been known
to induce reorganization of actin filaments due to modification of Rho proteins
(Ottlinger and Lin, 1988). Since PLD is an effector molecule of Rho, C. difficile
was also reported to inhibit PLD signaling. However, functional relevance for the
lack of PLD signaling was not previously established. On the basis of our
experimental results, we propose that by inhibiting PLD signaling in naïve T cells
140
through toxin B, C. difficile eliminates effector T cell functions. The consequent
increase in the frequency of Treg cells aids the pathogen’s success in promoting
illness and furthering its own survival. Although we report the effect of Toxin B on
altering the immune balance, we have not tested the effect of blocking Toxin B
using anti-Toxin B antibodies. Based on our results, we predict that blocking of
Toxin B would mitigate the effects of C. difficile on the immune system and
ameliorate the disease progression by promoting effector and memory T cell
expansion.
IMMUNE BALANCE: EFFECT OF ENDOGENOUS FACTORS
One of the drawbacks of a powerful immune response is uncontrolled immune
response against pathogens and associated collateral damage to self. The
restraint of uncontrolled immune response is mediated through negative feed
back mechanisms. Adenosine (Ado) is an endogenous molecule well known for
its role in mediating the negative feed back mechanisms of immune responses.
Previous studies have reported inhibition of inflammation following activation of
adenosine receptors. Adenosine receptors are expressed by all immune cells
and A2a receptor signaling on macrophages, dendritic cells and T cells have
been shown to inhibit effector function (Zarek et al., 2008). Based on many
studies, A2a receptors are considered the dominant receptor dictating
lymphocyte responses in T cells.
141
Here we demonstrate the role of A3 receptor in inhibiting effector T cell
responses. The data indicate that stimulation of T cells in the presence of A3
receptor agonist inhibits TCR induced activation of PLD signaling. This inhibition
of PLD signaling abrogates CD25 expression, effector T cell proliferation, and
cytokine secretion. Although, signaling through A3 receptor in other immune cells
results in low cAMP concentrations, we found that signaling through A3 receptor
in CD4 T cells results in elevated cAMP levels and increased PKA activity.
Previous studies on A3 receptors report both pro and anti-inflammatory
effects depending on the system investigated (mast cells, macrophages) and
species examined (rat, humans) etc (Gessi et al., 2008). However, the specific
effect of activating A3 receptors on CD4 T cells has never been studied. In the
heart, A3 receptors mediate cardioprotection through modulating PLD signaling
(Mozzicato et al., 2004). This led us to determine the effect of A3 receptor
signaling on CD4 T cells. We found that A3 receptor signaling blocked TCR
induced PLD activation. At present, we do not know the exact mechanism of A3
receptor signaling leading to PLD signal inhibition. Inhibition of PLD by A2a
receptors is mediated by interference with the translocation of small GTPases,
Arf and Rho (Thibault et al., 2000). In future, we can test if A3 receptor inhibits
PLD through translocation of small GTPases as well. One recent study also
demonstrated inhibition of PLD1 activity directly by Gβγ subunits (Preininger et
al., 2006).
142
Since adenosine receptors are coupled to G-protein, signaling is thought
to occur either through the inhibition or stimulation of adenylyl cyclase resulting in
a decrease or increase of intracellular cAMP concentration respectively. The
classical signaling pathway associated with A3 receptor suggests Gi mediated
inhibition of adenylyl cyclase with a concomitant decrease in cAMP
concentrations. We experimentally determined the effect of A3 receptor
activation on cAMP concentrations. Contrary to previous reports, we found
increased cAMP levels in A3 receptor agonist treated CD4 Tcells. This increased
cAMP levels also resulted in increased PKA activity. The effect of A3 receptor
activation on inhibition of effector T cell proliferation and concomitant increase in
Treg frequency was indeed reproducible by the cell permeable analog of cAMP,
db-cAMP. Future experiments can be done to determine if the effect of A3
receptor is solely mediated by cAMP and PKA by co-culturing CD4 T cells with
A3R agonist and PKA inhibitor H-89.
A3 receptor is coupled to trimeric GTP binding proteins, Gαi and/or Gαo
and Gβγ dimers. The αi subunits directly inhibit adenylyl cyclase. However βγ
dimers from Gi/o are known to activate adenylyl cyclase (AC) isozymes II and IV
(Yao et al., 2002). We speculate that the increase in cAMP levels caused by A3R
activation is mediated through βγ dimers. We can test if this is indeed the case by
inhibiting βγ signaling. One way to inhibit βγ signaling is through over expression
of the carboxyl terminal of βARK1 retrovirally in CD4 T cells. Previous evidence
143
suggests that expression of carboxyl terminus of βARK1 binds free βγ dimers and
thus inhibits βγ signaling (Yao et al., 2002) (Koch et al., 1994).
Depending on the cell type studied, cAMP has been reported to inhibit (Le
Stunff et al., 2000; Thibault et al., 2002) or activate (Mamoon et al., 1999) PLD
activity through PKA signaling. In our experiments, we detected increased
cAMP levels in CD4 T cells treated with A3R agonist 5 mins following T cell
stimulation and observed a decrease in the PLD activity 15-30 mins after TCR
stimulation. Based on these results, it is very likely that increased cAMP levels
caused by A3 receptor signaling results in inhibition of PLD signaling. The
experiment using inhibitor of PKA (H-89) and/or constitutively active form of PKA
could also be used to determine PLD activity following PKA signaling.
Based on our various observations, we propose the following model
depicting A3 receptor signaling in CD4 T cells (Fig 39). Two different pathways
can mediate the effect of A3 receptor signaling in T cells. A central role for Gi/o
βγ is proposed. A3 receptor activation can lead to increased cAMP levels and
PKA signaling. PKA has been demonstrated to inhibit PLD activation. Another
possibility is βγ dimers can directly inhibit PLD activity (Preininger et al., 2006).
The ability of A3 receptor agonist to promote regulatory T cell enrichment
in vivo suggests that it could be used for treatment of autoimmune diseases.
Studies in mice and rats demonstrate that the anti-inflammatory effects of
methotrexate are in part mediated via A3 receptors (Montesinos et al., 2003).
144
Perhaps, methotrexate acting through A3 receptor on CD4 T cells blocks PLD
signaling and leads to Treg enrichment.
Interestingly, it has been demonstrated that A3 receptor is over expressed
in cancer tissues in comparison to normal tissues (Gessi et al., 2004a). It should
be noted that the concentration of adenosine in solid tumor reaches as high as
100μM due to high levels of hypoxia (Hasko and Cronstein, 2004). Thus, it is
highly likely that T cells activated by MHC class II positive APCs in these tissues
are selectively blocked via A3 receptor signaling. Moreover, since A3 receptor
agonists inhibit IFN-γ secretion, A3 receptor antagonists could be effective in
tumor immunotherapy.
145
A3 R
adenosine
α i/o - βγ
AC II and IV
cAMP
PKA
PLD
adenosine
PA PC
Fig 39. Schematic representation of A3 receptor signaling in CD4 T cells. A
central role for Gi/o βγ subunits is proposed. A3 receptor activation leads to
increased cAMP levels and activates PKA. PKA has been demonstrated to inhibit
PLD activation. Another possibility is βγ dimers directly inhibit PLD activity
(Preininger et al., 2006).
146
AB-MECA AND ETHANOL: POTENTIAL USE AS A TOLEROGENIC
ADJUVANT
Our data show that concurrent presence of PLD suppressive factors such
as adenosine and ethanol impose substantial suppression of immune response.
We observed that 0.5% ethanol had no effect of inhibition of effector T cell
proliferation or expansion of Treg cells. However, combination of 0.5% ethanol
with A3 receptor agonist led to substantial decrease in effector T cell
proliferation. This suggests that there exists a threshold for PLD activity in T cells
and the combination of ethanol and AB-MECA pushes PLD activity well below
this threshold. Under these conditions, we see a sizable effect of PLD inhibition
on effector T cell proliferation and subsequent enrichment of Treg cells.
A very high concentration of ethanol leads to cell death due to the toxic
effects of by products of ethanol (Lieber, 1997). However, we can avoid these
toxic effects by using a combination of low doses of ethanol and AB-MECA.
Induction/expansion of antigen-specific Treg cells is of great therapeutic
interest for the treatment of autoimmunity, allergy and organ transplantation. Our
studies using AB-MECA/ethanol show expansion of antigen specific Treg cells in
vivo. Therefore, AB-MECA alone or combination of AB-MECA and low dose
ethanol may be used in immunotherapy of various diseases.
A recent study proposed the use of immunosuppressants like
dexamethasone (DEX) as tolerogenic adjuvants (Kang et al., 2008). They
demonstrated that antigenic immunization combined with DEX led to block of
147
dendritic cell (DC) maturation and preferential expansion of antigen specific
Foxp3+ cells. Based on the similar effect observed with AB-MECA /ethanol, we
propose that agents inhibiting PLD signaling can be used as tolerogenic
adjuvants. However, we do not know the effect of AB-MECA and ethanol on the
status of DC maturation. Future experiments can be designed to evaluate the
effect of PLD signaling in antigen presenting cells like DC.
PLD LOCALIZATION: INTERACTION BETWEEN MULTIPLE STRUCTURES
PLD1 and PLD2 are 50% identical and display similar domain structures.
However, they localize to different regions in the cell. It is suggested that
localization determines intermolecular interaction with downstream target
molecules thus serving different functions in signal transduction. A previous
report shows that deletion of the loop region in PLD1 led to an increase in its
basal activity levels but the localization of the loop deletion mutant was not
determined. We over expressed loop deletion mutant in the Jurkat T cell line and
found that it had plasma membrane localization similar to PLD2. Since PLD2 is
characterized by high basal activity it is very likely that the increased activity of
the loop deletion mutant is in part due to its plasma membrane localization and
potential modification by membrane attached enzymes (eg: phosphorylation).
A puzzling observation was that the isolated loop domain had plasma
membrane localization as well. Since deletion of loop from PLD1 abrogates its
distribution from vesicular structures we hypothesize that the coordinated
148
interaction of loop within PLD1 and with unknown structure in the cell sequesters
it to vesicular structures. One way to test this hypothesis is to insert the isolated
loop domain into full length PLD2. If the interaction of loop with the other domains
is the factor governing its vesicular localization, then PLD2 will localize similar to
PLD1.
A previous report suggests that PH, PX and palmitoylation sites on the N-
terminal region of PLD1 serve as potential membrane binding determinants
(Sugars et al., 2002). In agreement with previous studies, in a deletion mutant
encompassing all 3 potential sites (N-terminal deletion mutant), the membrane
binding determinants redistributed to the cytoplasm. We also tested the cellular
distribution of the isolated ‘PH+PX’ domain of PLD1. The localization of this
mutant did not resemble that of full length PLD1. Rather it was found to have a
diffuse localization in both cytoplasm and nucleus similar to vector construct.
Based on this result, we suggest that ‘PH+PX’ domain cannot be studied in
isolation. Other studies suggest that an intact C-terminus of PLD1 is required for
its function (Liu et al., 2001). We made a deletion mutant containing the C-
terminus fused with YFP. Interestingly, this mutant localized only in the nucleus
of the cell. Taken together, we propose that a single domain does not determine
the localization of PLD1. Rather interactions between different domains of PLD1
and with other cellular components regulate localization of PLD1 in sub cellular
structures. The presence of C-terminal region alone determines its nuclear
localization while interaction of the C-terminal region with catalytic domains and
149
the loop region redistributes PLD1 to the cytoplasm. Further, the interaction of N-
terminal domains (PX+PH) with the rest of PLD1 determines its localization in
membranous vesicles like golgi.
We undertook experiments to determine the domains necessary for the
plasma membrane localization of PLD2. As proposed for PLD1, similar
interaction between domains determines its plasma membrane localization.
Isolated C-terminus mutant of PLD2 had nuclear localization while the mutant
with the presence of PH+PX along with the C-terminus redistributed to the
cytoplasm. Further, the interaction of the region containing the catalytic domains
with the rest of PLD2 results in its plasma membrane localization. Our studies
provide evidence for the role of different domains of PLD1 and PLD2 in
determining their respective localizations under basal condition. Future
experiments can be conducted to determine the cellular binding target of PLD
proteins particularly the loop and N-terminal half. It is possible that various
modifications of typical residues mediate the hierarchy of localization signals.
This can be addressed using a site directed mutagenesis approach. Also, we
have not examined the functional effect of these mutations. Functional studies in
future will provide a better understanding of the role of localization in determining
specific functions.
PLD2 AND T CELL FUNCTIONING
As a result of the critical role of PLD in development, PLD null mutants are
embryonic lethal (LaLonde et al., 2006). In PLD2flox/flox CD4 cre mice, PLD2 is
150
deleted relatively late (double positive stage). Our data demonstrate that PLD2
is dispensable for T cell development after the double positive stage of
thymocyte differentiation. Our data does not address the role of PLD2 in earlier
aspects of T cell development.
Also, deficiency of PLD2 did not affect CD4 T cell activation and
proliferation. On the other hand, we did observe differences in cytokine secretion
profiles of PLD2 deficient T cells. PLD2 deficient T cell cultures had more IL-2
positive cells than wild type T cells and showed resistance to differentiate in to
Th1 and Th2 lineage in vitro. This effect on effector T cell differentiation might be
caused by blockade of transcription factors like T-bet and GATA3. These
possibilities can be investigated in the future.
Surprisingly, PLD2 deletion had very little effect, if any, on the expansion
of regulatory T cells. One possible explanation is that PLD1 compensates for the
lack of PLD2. We can test for this possibility by inducibly deleting PLD1 alone in
PLD2 CD4 cre knock out mice. Another possibility is that PLD2 deficient T cells
are inherently different due to their thymocyte development and differentiation in
the absence of PLD2 signals. The other exciting possibility is perhaps that PLD2
is more important under conditions of physiological stress. This possibility can be
addressed by inducing autoimmunity or by observing the immunological
response to pathogens in PLD2 deficient mice in vivo. Nonetheless, these
experiments in PLD2 knock out mice have brought to light previously unknown
questions and answers.
151
CONCLUDING REMARKS
PLD signaling in T cells was discovered in 1991. However, until now the
specific role of PA and PLD in T cell functioning has not been clear. Our lab
group was the first to highlight the differential requirement of PLD signaling in
effector versus regulatory T cells. This led us to investigate the role of PLD
signaling in T cells. My dissertation work has focused on identifying the
regulation of PLD signaling in T cells. I found that physiological molecules like
adenosine maintain immune homeostasis by modulating PLD signaling. In
addition, I discovered that pathogens/ exogenous molecules could alter the host
immune response by virtue of their PLD inhibiting properties. Further, I
determined the structure that controls the sub-cellular localization of PLD
isoforms and their specific role in T cell function. These studies highlight the
complex array of functions mediated by PLD in T cells. In the process of studying
PLD, we have also discovered the role of adenosine A3 receptor signaling in T
cells. This study highlights the role of crosstalk between different signaling
pathways in human health and disease.
152
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VITA
The author, Uma Chandrasekaran was born in Chennai, India on March
13, 1983 to Mr. and Mrs.Chandrasekaran. She received a Bachelor of
Engineering in Electronics & Instrumentation and Masters in Biological Sciences
from Birla Institute of Technology & Sciences (BITS-Pilani), India in May 2005.
In August of 2005, Uma joined the graduate program in Biomedical
Sciences at Medical College of Georgia (Augusta, GA). Shortly, thereafter she
joined the laboratory of Dr.Makio Iwashima. Uma moved with Dr. Iwashima to
Loyola University, Chicago in Aug 2007. In Dr.Iwashima’s laboratory, Uma
studied mechanisms of regulation of Phospholipase D (PLD) signaling in T cells.
After completing her Ph.D., Uma will begin a post-doctoral position in Dr.
Alessandra Pernis’s laboratory at Weill Medical College of Cornell University
(New York, NY) where she will study the role of IL-17 producing T cells in the
pathogenesis of autoimmune disorders like Rheumatoid Arthritis and Lupus.