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Biology Theses Department of Biology

5-10-2017

Induction of arginase-1 in MDSC requires exposure to CD3/CD28 Induction of arginase-1 in MDSC requires exposure to CD3/CD28

activated T cells activated T cells

Ahmed Abdelbaky Abdelaal

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iv

Induction of arginase-1 in MDSC requires exposure to CD3/CD28 activated T cells

by

AHMED ABDELAAL

Under the Direction of Yuan LIU, PhD

ABSTRACT

Tumor-induced myeloid derived suppressive cells (MDSC) have been reported to inhibit T cell

responses. In our study MDSC isolated from tumor bearing mice showed potent inhibition of

T-cell proliferation. However, surprisingly we observed that freshly isolated MDSC from the bone

marrow of tumor bearing mice do not constitutively express arginase-1 until after exposure to T-

cell proliferation. The aim of this study is to determine the mechanism by which arginase-1 is

induced in MDSC following exposure to a proliferative T cell environment. We showed that

treatment of MDSC with culture supernatant isolated from T cells activated with CD3/CD28

antibodies successfully induced arginase-1 expression and this process is independent of IL-10

and IFNγ. This suggested that arginase-1 induction in MDSC can occur independently of cell-cell

contact. Interestingly IL-2, ConA or PMA activated T-cell supernatant as well as supernatant from

multiple cancer cell lines failed to induce arginase-1 in MDSC. We also showed that M-MDSC

v

expressed higher levels of arginase-1 than G-MDSC after co-culture with CD3/CD28 activated T

cells as well as its supernatant. In addition, other bone marrow cells have shown the potential to

express arginase-1 following exposure to the same conditions. For example, we observed that

healthy Ly6C+ monocytes but not mature granulocytes successfully expressed arginase-1.These

data demonstrated that T cells activated through stimulation of TCR but not other means of

activation induced arginase-1 enzyme expression.

INDEX WORDS: Myeloid derived suppressor cells, CD3, T cells, Arginase-1.

vi

Induction of arginase-1 in MDSC requires exposure to CD3/CD28 activated T cells

by

AHMED ABDELAAL

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

in the College of Arts and Sciences

Georgia State University

2016

vii

Copyright by AHMED MANSOUR ABDELBAKY ABDELAAL

2017

viii

Induction of arginase-1 in MDSC requires exposure to CD3/CD28 activated T cells

by

AHMED ABDELAAL

Committee Chair: Yuan Liu

Committee:

1- William Walthall

2- Deborah Baro

Electronic Version Approved:

Office of Graduate Studies

College of Arts and Sciences

Georgia State University

May 2017

ix

DEDICATION

This work is dedicated to my family. Mom, Dad, Eng. Mohammed, Drs. Islam and Eman

who have always been my strengths. I appreciate all of your patience and encouragement.

ACKNOWLEDGEMENTS

In the name of Allah, Most Gracious, Most Merciful .Praise be to God for his kindness

and thanks to him for the guidance.

I would like to express my deepest gratitude to my mentor Dr. Yuan Liu. I would like to

take the opportunity to thank her for her support, guidance and patience. Under your guidance, I

have learnt how to trust in my abilities to achieve my goals. Thank you for constantly inspiring

me to strive for excellence.

I would also like to thank my committee members, Drs. William Walthall and Deborah

Baro for their valuable guidance during my graduate career. Thank you for your constructive

feedback during the project. Thank you both for always supporting me, and helping me achieve

my goals, it is truly appreciated.

I would also like to express my gratitude to the ministry of higher education in Egypt and

USAID organization for providing financial support for my study for the past two years.

I would like also to extend my gratitude to the Department of biology at Georgia State

University for your scientific support.

Thank you to my lab members for helping me through all of my experiments. I sincerely

appreciate all of your help.

I would like to sincerely thank my professors in Egypt for all of their scientific and

emotional support. I appreciate all of your guidance during my undergraduate studies.

I would like to thank all of my friends Dr.Ahmed Abdelsattar, Dr.Ahmed Abdelbaki,

Dr.Moamen Almaz, Dr.Attia Mohammed, and Eng.M. El-sayed, Dr.M.Eldahshan, Mr. E.Elwan,

Mr.M.Abdelhaleem and Mr. A.Abdalla for all of their support in their own special ways.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .............................................................................................. i

LIST OF FIGURES .......................................................................................................... v

LIST OF ABBREVIATIONS ......................................................................................... vi

1 INTRODUCTION ...................................................................................................... 1

1.1.1 Myeloid Derived Suppressor cells................................................................... 1

1.1.2 History of MDSC ............................................................................................. 1

1.1.3 MDSC origin ................................................................................................... 1

1.1.4 MDSC phenotypic markers ............................................................................. 2

1.1.5 Characteristics of M-MDSC ........................................................................... 2

1.1.6 Characteristics of G-MDSC ............................................................................ 3

1.1.7 MDSC in different disease conditions ............................................................ 3

1.1.8 MDSC expansion and activation mechanisms .............................................. 5

1.1.9 Mechanisms of MDSC suppressive activity. .................................................. 6

1.2 Overview on T cells ........................................................................................... 11

1.2.1 T cell development and maturation ................................................................ 11

1.2.2 T cell activation .......................................................................................... 12

2 MATERIALS and METHODS ............................................................................... 14

2.1 Mouse tumor models ......................................................................................... 14

2.2 Isolation of bone marrow .................................................................................. 14

iv

2.3 Separation of leukocytes from bone marrow .................................................. 14

2.4 Isolation of splenocytes and T cells .................................................................. 15

2.5 T-cell proliferation assay .................................................................................. 16

2.6 Western blot (WB) analysis of arginase .......................................................... 17

2.7 Flow cytometry analysis.................................................................................... 17

2.8 Measurement of the cytokines ........................................................................... 17

3 Results ........................................................................................................................ 19

3.1 Bone marrow-derived MDSC from tumor bearing mice suppress T cell

proliferation… ......................................................................................................................... 19

3.2 Tumor bone marrow MDSC express arginase-1 upon exposure to TCR-

activated T cells ....................................................................................................................... 22

3.3 Arginase-1 expression by BM derived MDSC is independent of direct

contact with T cells. ................................................................................................................. 25

3.4 Arginase-1 induction in BM derived MDSC by activated T cells requires

ligation of TCR co-receptor (CD3). ....................................................................................... 26

3.5 Healthy Ly6Chigh monocytes but not Ly6G+ granulocytes express arginase-1

only after -the exposure to CD3/CD28 activated T cells. ..................................................... 29

4 Discussion .................................................................................................................. 31

REFERENCES ................................................................................................................ 33

v

LIST OF FIGURES

Figure 1-1MDSC origin. ............................................................................................................... 4

Figure 1-2MDSC mechanisms of suppression. ......................................................................... 10

Figure 2-1 T-cell proliferation assay………………………………………………………......16

Figure 3-1 Bone marrow-derived MDSC from tumor bearing mice suppress T cell

proliferation. ................................................................................................................................ 21

Figure 3-2 Bone marrow-derived MDSC express no arginase but until after exposure to

activated T cells. .......................................................................................................................... 24

Figure 3-3 Arginase-1 expression by BM derived MDSC is independent of direct contact

with T cells. .................................................................................................................................. 27

Figure 3-4 Arginase-1 induction in BM derived MDSC by activated T cells requires ligation

of TCR co-receptor (CD3). ......................................................................................................... 28

Figure 3-5 Healthy Ly6Chigh monocytes but not Ly6G+ granulocytes express arginase-1 only

after the exposure to CD3/CD28 activated T cells. .................................................................. 30

vi

LIST OF ABBREVIATIONS

BM-Bone Marrow

MDSC-Myeloid-derived suppressor cells

IMC-Immature myeloid cells

IL-Interleukin

VEGF-Vascular endothelial growth factor

MΦ-Macrophage

IFN-γ-Interferon gamma

ROS-Reactive oxygen species

RNS-Reactive nitrogen species

TCR-T cell receptor

MHC-Major histocompatibility complex

NF-κB-Nuclear factor kappa B

HSC-Hematopoietic stem cell

JAK-Janus kinase

STAT-Signal transducer and activator of transcription

Treg-Regulatory T cell

GM-CSF-Granulocyte/Macrophage colony-stimulating factor

G-CSF-Granulocyte colony-stimulating factor

CMP-Common myeloid progenitor

iNOS-Nitric oxide synthase

Arg-Arginase

CAT-2B Cationic amino acid transporter 2B

vii

G-MDSC-Granulocytic MDSC

M-MDSC-Monocytic MDSC

NO-Nitric oxide

ELISA-Enzyme-linked immunosorbent assay

HRP-Horseradish peroxidase

RBC-Red blood cell

FACS-Fluorescence activate cell sorting

CFSE-Carboxyfluorescein succinimidyl ester

CD-Cluster of differentiation

ConA - Concanavalin A

iNOS- Inducible nitric oxide synthase

COX2-Cyclooxygenase-2

SCF-Stem cell factor

CXCR- chemokine receptor

PD/PDL-Programmed death receptor/ligand

CTLA-Cytotoxic T-lymphocyte antigen

HVEM- herpes virus entry mediator

PKC –Protein kinase C

1

1 INTRODUCTION

1.1.1 Myeloid Derived Suppressor cells

Myeloid derived suppressor cells are a heterogeneous population of immature myeloid cells

that have been associated with a potent suppression of the immune system including both

innate and adaptive immunity[1, 2].

1.1.2 History of MDSC

Myeloid derived suppressor cells (MDSC) were first observed associated with tumor evasion in

the early 1900s[3]. This observation was explained as an extreme deprivation of neutrophils

(neutropenia) and an extramedullary hematopoiesis, which was also associated with abnormal

myeloid cells differentiation [3] . Null cells , Veto or natural suppressor cells were used to describe

this population of cells as they do not have any distinct cell surface markers that distinguish them

from other cells as T cells, B cells, NK cells or macrophages [3]. A few years later, these cells

were reported to inhibit T- cell proliferation and activity [4].In 2007, the name “ Myeloid derived

suppressor cells” was formally used to describe this population of suppressor cells[5].Recently,

many reports describe the biology and the significance of this population of cells in different

disease conditions[6].

1.1.3 MDSC origin

Different factors including granulocyte macrophage colony-stimulating factor (GM-CSF),

macrophage colony-stimulating factor (M-CSF), stem-cell factor (SCF) control myelopoiesis.

During normal myelopoiesis, Hematopoietic stem cells (HSCs) form common myeloid progenitor

2

(CMP) cells which differentiate into immature myeloid cells (IMCs)[2] .Lastly IMCs differentiate

into macrophages, granulocytes and dendritic cells. Under different disease conditions such as

cancer, trauma and inflammation, immature myeloid cells (IMCs) lose their ability to form mature

cells and instead give rise to myeloid derived suppressor cells (MDSC)[2] (Fig. 1-1).

1.1.4 MDSC phenotypic markers

One of the most challenging issues in the field of MDSC, is the lack of specific surface marker to

distinguish suppressive granulocytes from non-suppressive granulocytes [7]. In mice, CD11b and

Gr-1 protein, which include two isoforms (LY6C and LY6G) still the most useful markers to

describe MDSC. However, these markers are not MDSC specific [7] . MDSC involve two

populations of myeloid leukocytes: Granulocytic MDSC (G-MDSC) , which can be described as

CD11b+, Ly6Clow, Ly6G+cells, and monocytic MDSC (M-MDSC), which can be described as CD11b+,

Ly6Chigh, Ly6G-[8]. Both M-MDSC and G-MDSC have been reported to inhibit T cell responses

including T-cell proliferation and functions [2]. In addition to the difference in phenotype, M-

MDSC and G-MDSC are also different in the mechanisms of their immunosuppressive function[9].

1.1.5 Characteristics of M-MDSC

There are 2 subpopulations of MDSCs, the monocytic MDSC, and the granulocytic MDSC.As

previously mentioned M-MDSC and G-MDSC have different surface markers as well as different

immunosuppressive mechanisms. Monocytic MDSC immunosuppressive function is believed to

be generated primarily by the upregulation of both arginase-1(Arg-1) enzyme and inducible nitric

oxide synthase (iNOS) as well as by suppressive cytokines[10].Arginase-1 enzyme acts by

depleting L-arginine from the environment by degrading it into L-ornithine and urea. Inducible

3

nitric oxide synthase (iNOS) breaks down L-arginine into nitric oxide (NO) which induces T cell

apoptosis[10].

1.1.6 Characteristics of G-MDSC

Granulocytic-MDSC, which is characterized by high expression of the neutrophilic marker Ly6G+,

shares some common characters with the mature neutrophils including the formation of

intracellular granules and the expression of NADPH oxidase, the enzyme responsible for the

production of reactive oxygen species (ROS)[2]. ROS works in combination with NO to form

peroxynitrite radicals, that inhibit cytotoxic T cell responses through nitration of the T-cell

receptor making T-cells unresponsive to antigen stimulation[10, 11].In addition, it has been

reported that G-MDSC produces TGF-β, which enhances tumor invasion, metastasis and

suppresses the immune response[10, 12].

1.1.7 MDSC in different disease conditions

As previously discussed, MDSCs were first discovered in patients with cancer or in tumor-bearing

mice[2]. It also has been reported that there is a marked expansion of myeloid derived suppressor

cells in patients with different cancers [2, 13]. However, MDSCs are also found in other diseases

such as trauma, acute and chronic inflammation, transplantation and sepsis[2]. Expansion of

MDSCs is also associated with different autoimmune diseases such as inflammatory bowel

disease (IBD), which suggests that MDSCs are observed under different pathological conditions[2,

14].

4

Figure 1-1MDSC origin.

Under healthy conditions, hematopoietic stem cells (HS) give rise to common myeloid progenitor cells (CMP), then into immature myeloid cells (IMC) that finally differentiate into mature granulocytes, macrophages or dendritic cells. In the case of tumor, trauma or infections, IMC are forced by the diseases environment to stay in the immature state and become myeloid derived suppressor cells (MDSC).

5

1.1.8 MDSC expansion and activation mechanisms

Many studies have shown that many factors are able to regulate MDSCs expansion or activation.

An interesting review by Dmitry I. Gabrilovich et al. showed that these factors can be classified

into two main groups: 1) those that control MDSCs expansion, and 2) those that control MDSCs

activation. The first group are produced mainly by tumor cells, and work by preventing the

formation of mature myeloid cells. The second are produced by tumor stromal and activated T

cells [2].

1.1.8.1 Factors that control MDSC expansion

Factors reported to control MDSC expansion include vascular endothelial growth factor (VEGF)[2,

15], cyclooxygenase-2 (COX2)[2, 16], granulocyte/macrophage colony-stimulating factor GM-

CSF[2, 17] and IL-6 [2, 18] , stem-cell factor (SCF)[19] and prostaglandins[2].Interestingly, most of

these factors activate the JAK-STAT3 signaling pathway.Stat-3 phosphorylation is also

upregulated in MDSCs from tumor bearing mice [2, 20]. It has been reported that selective STAT3

inhibitors successfully enhance T- cell responses by reducing MDSCs expansion[2, 20]. A recent

study from our laboratory showed that, CXCR2-expressing granulocytic MDSC (G-MDSC) but not

monocytic MDSC (M-MDSC) isolated from tumor bearing mice expand during tumor progression.

This suggests that G-MDSC are the most important immunosuppressive cells regulated during

tumor growth.

6

1.1.8.2 Factors that control MDSC activation

In addition to the factors that promote MDSCs expansion, MDSCs require factors to induce their

activation. These factors include IL-4, IL-13, Transforming growth factor-β (TGFβ) and

IFNγ[2].Treatment of freshly isolated MDSCs from the spleen of tumor bearing mice or cloned

MDSCs cell lines with IL-4 upregulate arginase-1 expression[21].

It has been demonstrated that IL-13 and IL-4 treatment upregulate arginase-1 expression in bone

marrow derived macrophages (BMDM)[22].Importantly, IL-13 and IL-4 binds to (IL-4Rα) and this

pathway involves STAT-6 activation[2]. However, in a different study using a breast tumor model

, IL-4Rα deficient mice contain MDSCs after surgery[23].

Another study has attributed the upregulation of Arg-1 and iNOS in MDSCs isolated from

spleen of tumor bearing mice to STAT-1 signaling pathway which is mediated by IFNγ[24].

1.1.9 Mechanisms of MDSC suppressive activity.

MDSCs use different mechanisms to suppress both adaptive and innate immunity components

including depletion of L-arginine, ROS and RNS production or induction of Treg. Many studies

have shown that MDSCs suppressive function require direct contact with T cells[2].

1.1.9.1 Depletion of L-arginine

L-arginine plays an important role in T-cell proliferation, activation, metabolism and functions

[25, 26]. L-arginine can act as a substrate for many enzymes including arginase- I, arginase- II, and

nitric oxide synthase [25].

It is now well established that arginase-1 activity promotes tumor growth and escape from

immune-surveillance through depletion of L-arginine [27]. Arginase-1 activity has been detected

7

in the tumor microenvironment of skin, breast and prostate cancer and is considered as a marker

of tumor progression in patients with different tumor models[27].

In a study using 3LL murine lung carcinoma model, it has been reported that mature myeloid cells

isolated from the tumor microenvironment; successfully inhibit T-cell proliferation and

expression of signaling molecules in an arginase-1 dependent manner[28]. However, arginase-1

expression and suppressive activity is not restricted to mature myeloid cells.

Using arginase-1 and iNOS, MDSCs break down L-arginine into L-ornithine and urea [2,

25].Arginase-1 expression in MDSC is associated with inhibition of T-cell responses including

down regulation of CD3ζ expression [2, 29]while NO inhibits T cell responses through the

induction of T-cell apoptosis or inhibition of MHC class II expression [2, 30, 31]. Interestingly,

many reports showed that inhibition of arginase-1 was able to rescue T-cell proliferation [32-34]

[33]and is also to inhibit tumor growth which suggests that maintaining L-arginine levels in T-cell

environment is very important in preventing the immunosuppression by MDSCs[32] .

Importantly, Murine MDSCs deplete the extracellular L-arginine levels by increasing the uptake

of L-arginine through the cationic amino acid transporter 2B (CAT-2B) while human MDSCs do

not uptake L-arginine but release arginase-1 enzyme within granules into the microenvironment.

This induces L-arginine depletion which inhibits T-cell proliferation [28, 35, 36].

1.1.9.2 ROS

In the tumor microenvironment, reactive oxygen species (ROS) are produced by MDSCs,

neutrophils, eosinophils, regulatory T-cells, macrophages and tumor cells. ROS are associated

with the inhibition of T-cell responses and the enhancement of tumor growth [37, 38]. One of

the main characteristics of MDSCs in cancer patients and tumor-bearing mice is elevated ROS[2,

8

39, 40].Inhibition of ROS production by MDSCs in these cancer patients and tumor-bearing mice

completely rescues T-cell functions[2, 39, 40].It has been shown that, integrins on the MDSCs

surface play a role in ROS production since blocking integrins (CD11b, CD18, and CD29) by specific

antibodies completely abolishes the suppressive function of MDSCs [2, 40].

ROS inhibit T-cell responses through different mechanisms such as blocking the interaction

between T-cell receptor (TCR) and the major histocompatibility complex (MHC)[2, 11, 41] ,

inhibiting the differentiation of MDSCs into macrophages so MDSCs will stay in the immature

state [2, 42] or depleting cystine and cysteine which causes inhibition of T-cell activation and

function[37, 43].In addition, it has been reported that MDSCs can induce T-cell apoptosis via ROS

dependent mechanisms[37, 44, 45].However, low levels of ROS can induce T-cell activation and

proliferation[37, 46, 47].

1.1.9.3 Reactive nitrogen species (RNS)

RNS are considered as an important immune-suppressive instrument used by MDSCs to suppress

T cell responses. For example peroxynitrite (ONOO-) induces cysteine, tryptophan, methionine

and tyrosine nitration and nitrosylation[2, 48]. It has been shown that peroxynitrite makes T-cell

unresponsive to different stimuli through the nitration of TCR and CD8 molecules[11].Another

study showed that peroxynitrite induces T-cell apoptosis through impairment of tyrosine

phosphorylation[49].

9

1.1.9.4 Other mechanisms

In addition to previous mechanisms by which MDSCs inhibit T-cell responses, several studies

demonstrate that MDSCs can also induce regulatory T-cell (Treg ) development which is shown

to be independent of NO but requires the presence of TGF-β and IL-10 [2, 50] .Furthermore,

arginase-1 is reported to enhance Treg expansion in lymphoma bearing mice[51].

10

Figure 1-2MDSC mechanisms of suppression.

11

1.2 Overview on T cells

1.2.1 T cell development and maturation

T cell development takes place in the thymus where the bone marrow hematopoietic stem cells

(HSCs) generate lymphoid progenitor cells which give rise to thymocytes[52]. Two characteristics

are required for the bone marrow cells to generate T cell lineage progenitors. First, the ability to

move to the thymus[52, 53]. Second, they must have a potential to generate T cells in the

thymus[52, 54]. Based on the expression of either CD4 or CD8 surface marker, T cells undergo

different selection steps in the thymus[55].The earliest thymocytes are double negative which

mean that no expression of either CD4 or CD8 surface markers (CD4– CD8–)[55]. These double

negative cells can be further classified based on the expression of CD117, CD44 or CD25[55].Then

they become double positive (CD4+CD8+) through their development and end as single positive

thymocytes (CD4+CD8− or CD4−CD8+) which leave to the peripheral organs [55].

T cell fate is determined by the strength of the interaction between the major histocompatibility

complex (MHC) and the T cell receptor[56]. Double positive thymocytes that express T cell

receptor and have intermediate interaction with the major histocompatibility complex of the

cortical epithelial cells, undergo positive selection process to form a mature single positive naïve

T cell. If the T cell receptor of the double positive thymocytes failed to interact with the major

histocompatibility complex, the thymocytes will die by neglect. The thymocytes will be deleted

by negative selection if the interaction is too strong. Each day, about 50 million double positive

thymocytes (CD4+CD8+) are generated in the mouse thymus[57].More than 90 % of these cells

have T cell receptor that lack positive selection ability thus these cells undergo death by neglect

as they failed to interact with the major histocompatibility complex[56].

12

1.2.2 T cell activation

T cell activation requires two signals. The first signal is driven after the interaction between the

T cell receptor (TCR) and the major histocompatibility complex (MHC) on the surface of antigen

presenting cell. The second signal is a costimulatory signal between co-stimulatory molecules on

the surface of T cells and costimulatory receptor on the surface of the antigen presenting cell[58].

For example, the costimulatory molecule CD28 binds to CD80 or CD86 receptors on the surface

of the antigen presenting cell[58]. In addition to the costimulatory molecules , there are co-

inhibitory molecules as CTLA-4 and PD-1 which lead to T cell exhaustion after the binding with

CD80 , CD86 or PDL-1 receptors[58].

The TCR consists of α and β-chain that is linked with CD3 subunits[59]. The binding between TCR

and (MHC) complex lead to initiation of downstream signaling pathways that cause T cell

proliferation, differentiation and cytokines secretion[58, 59]. Although the first signal through

TCR is enough for T cell activation, the costimulatory signals are very important to sustain the

proliferation of naïve T cells through the activation of NF-κB signaling pathway[59, 60].The CD28

role in enhancing T cell proliferation was confirmed using CD28 deficient T cells [61].In addition

to the antigen (Ag)-dependent activation of T cells through the binding of TCR to the MHC

complex ,T cells activation can occur independently of the binding between TCR and MHC

complex in an Ag-independent way. Different substances as anti-CD3 monoclonal antibody, PMA

plus ionomycin or concanavalinA (ConA) are known to activate T cells[62]. Anti-CD3 monoclonal

antibody activates T cells by stimulating the signaling down stream of TCR, while PMA plus

Ionomycin induce protein kinase C (PKC) activation [62].ConcanavalinA (ConA) binds to the TCR

and other cell surface glycoproteins[62, 63] and its role in T cell activation is shown to be

13

regulated by herpes virus entry mediator (HVEM)[62], which promotes T cell activation ,

proliferation and cytokine production by co-operating with CD3/TCR signaling[64].

14

2 MATERIALS and METHODS

2.1 Mouse tumor models

C57BL/6J mice (6-8 week, 20-22 g, Jackson Laboratory) were subcutaneously (s.c.) engrafted

(allograft) with B16 melanoma with 2 × 105 B16 (B16F10) cells injected in 300 µl sterile PBS. B16

cells (ATCC) were cultured in DMEM supplemented with 10% FBS. After engraftment, tumor

developed in 2-3 weeks.

2.2 Isolation of bone marrow

The femur and tibia of euthanized mice were removed from each mouse and the surrounding

tissue was removed. The bone ends were removed from the diaphysis with scissors surgically,

so the bone marrow rich medullary cavity was exposed. A syringe was filled with 4mls ice-cold

PBS. The PBS washed out all the bone marrow into a 15ml BD Falcon conical collection tube as

it pass through the medullary cavity. The cell suspension was centrifuged at 1000RPM, 4°C for

10 minutes. The pellets were resuspended in sodium bicarbonate, ammonium chloride, EDTA

lysis buffer to lyse any red blood cells from the bone marrow. After lysis of the RBCs, the cells

were washed in PBS and centrifuged again.

2.3 Separation of leukocytes from bone marrow

Bone marrow leukocytes were collected by flushing the femur and tibia of previously

euthanized mice using iced-cold PBS followed by lysis of RBCs using RBCs lysis buffer.

Enrichment and separation of the myeloid cells from the collected leukocytes were performed

using percoll dentistry gradient as previously described. In brief, Percoll gradient was prepared

using Percoll (Sigma) by mixing of 9 mls of Percoll with 1 ml of 10 × HBSS followed by filtration

and dilution by 1 × HBSS to prepare different Percoll solutions with different densities: 70%

15

(1.097 g/mL)g/mL), 60% (1.083 g/mL), 50% (1.071 g/mL), 40% (1.058 g/mL) . Five-layer

discontinuous density gradients were prepared by adding one layer (2 ml/layer) upon one

another starting with the layer with the highest density at the bottom in a 15 ml tube using a

syringe fitted with a 22G needle. About 7 x107 bone marrow leukocytes were suspended in 1

mL HBSS and then placed on top of the gradients using a syringe with 22 G needle, followed

by centrifugation at 600 g x g for 40 min in a swinging bucket rotor. After the centrifugation,

the cells were separated and formed bands at the interface between each 2 layers. Cells in band

III were collected, washed and analyzed by FACS. PE-conjugated anti-Ly6G antibody and PE-

conjugated magnetic microbeads were used to isolate granulocytes (LY6G+) by positive

selection, while the rest without binding to the beads contained about 90 % of

Ly6Chigh monocytic leukocytes.

2.4 Isolation of splenocytes and T cells

To isolate cells from tumor tissues or the spleen, tumor tissues or spleen were grounded and were

digested with collagenase D (400 U/ml) at 37οC (45 min) in HBSS with agitation following by

filtration through nylon mesh with the pore size of 70 µm diameter and collection in ice-cold

RPMI 1640 Spleen tissues were grinded in HBSS. The pellets were resuspended in red blood

cell lysis buffer to lyse any red blood cells from the spleen. After lysis of the RBCs, the cells were

washed in HBSS and centrifuged again followed by collection in ice-cold RPMI 1640 with 10%

FBS. To purify CD4+ and CD8+ T cells, PE-conjugated anti-CD4+ antibody and PE-conjugated

anti-CD8+ antibody, PE-conjugated magnetic microbeads (Miltenyi Biotec.) were used for

positive selection and the finally purified CD4+ and CD8+ T-cells were confirmed to have a

purity of > 95% by using of fluorescence microscopy.

16

2.5 T-cell proliferation assay

Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to label splenocytes (6 × 105)

following their isolation from mice or purified T cells (6 × 105) after further purification as

mentioned above, followed by washing. Labeled splenocytes (6 × 105) or purified T cells were

seeded in 96-well pre-coated with anti-CD3 (1 µg/ml, clone 145-2C11, Biolegend). RPMI1640

with 10% FBS and soluble anti-CD28 (0.5 µg/ml, clone 37.51, Biolegend) was added followed by

the incubation of cells at 37°C, 5% CO2 for 4 days. For the co-culture experiment, MDSC isolated

from the bone marrow were added to splenocytes at the ratio of 1:3 or to purified T cells at the

ratio of 1:1.After 4 days, Cells were collected and labelled with fluorescent anti-CD8 and CD4

antibodies (Biolegend) and analyzed by FACS to evaluate T-cell proliferation.

Figure 2 1T cell proliferation assay.

Splenocytes labelled with CFSE, activated by CD3 and CD28 antibodies, after each cell division, the intracellular CFSE will become more diluted.

17

2.6 Western blot (WB) analysis of arginase

Freshly isolated bone marrow derived M-MDSC or G-MDSC that were separated by

Percoll, BM Ly6G+ granulocytes and Ly6C+ monocytes after co-culture with splenocytes or

treated with CD3/CD28 activated T cell supernatant were lysed in a buffer containing 100

mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, protease inhibitors (Sigma, Product

Code: P8340) and 3 mM PMSF following by centrifugation at (10,000 rpm, 10 min), cell

lysates were separated by 10 % SDS-PAGE after adding of 5x loading buffer , followed by

transferring onto nitrocellulose for 2 hrs , constant current at 0.3 A then blocking with

5% non-fat milk. The membrane was incubated with anti-arginase 1 (mAb H-52, Santa

Cruz Biotechology), secondary antibody and ECL. Β-actin in cell lysates was detected by

using anti- β-actin (Santa Cruz Biotechology).

2.7 Flow cytometry analysis

FACS analysis was performed to the leukocytes following their separation by percoll. After lysis

of red blood cells, leukocytes were resuspended in PBS following by labelling with fluorescence-

conjugated antibodies (Ly6G (1A8), Ly6C (HK1.4), Gr-1 (RB6-8C5) and CD11b (M1/70) (all from

Biolegend or R&D systems) and analyzed using BD LSR Fortessa (BD Biosciences). Data analysis

was performed using FlowJo software.

2.8 Measurement of the cytokines

Splenocytes isolated from the spleen of healthy mice were seeded to 96 well plate at the density

of (6 × 105) per well that had been coated or not with anti-CD3e antibodies (BioLegend).Cells

were resuspended in complete RPMI (10% FBS, 0.1% β-mercaptoethanol and CD28, 0.5 µg/ml,

18

clone 37.51, Biolegend). For non-activated T cells, cells were cultured in complete RPMI (10 %

FBS) without CD3 or CD28 antibodies. In case of Concanavalin A (ConA), PMA+Ionomycin and IL2

activated T cells, T cells were cultured in RPMI (10 % FBS) + (ConA) at the final concentration of

(0.5 μg/ ml), PMA (30ng/ml)/Ionomycin (80ng/ml) or IL-2 (1000 IU/ml) .Cell supernatant was

collected after 1 or 4 days. Cytokines concentration was measured using standard sandwich ELISA

technique. In brief, antibodies against IL-6, IL-10, IFNγ and GM-CSF were used to coat a 96-well

flat-bottom ELISA plate. Following washing, both activated and non-activated T cells media were

incubated in the wells, each sample in duplicate. Following by the incubation of Biotin labelled

antibodies and HRP-conjugated streptavidin antibodies .wells were washed with 0.005% Tween-

20 PBS following the incubation of each antibody. Color change was detected at wavelength

450nm following the addition of HRP substrate, o-phenylenediamine dihydrochloride (OPD,

Sigma) to each well. All of the antibodies were purchased from BD Biosciences and BioLegend.

Data analysis was performed using SoftMax Pro microplate data software.

19

3 RESULTS

Although aginase-1 is believed to be an important enzyme used by myeloid derived suppressor

cells to inhibit T cell responses, the mechanisms that control arginase-1 expression has been

subjected to major debate and research. One study using 3LL tumor model cells showed that

arginase-1 expression in tumor infiltrating MDSC is independent of T cell cytokines but requires

tumor released factors[65]. A second study using C26 colon carcinoma tumor model attributed

arginase-1 expression in blood MDSC to IFNᵧ signaling [66]while a third study proved that IFNᵧ

does not control MDSC suppressive function and targeting of IFNᵧ as a potential therapy will not

limit MDSC number[67]. The variability of these results suggests that, MDSC formed under

different tumor conditions or isolated from different tissues, might be controlled differently.

Understanding this mechanism will help to rescue of T cell responses as well as to understand

the interplay between MDSC and T cells. In this study we hypothesized that arginase-1 expression

in bone marrow derived MDSC requires the exposure to T cells that activated through ligation of

CD3 and is independent of tumor released factors.

3.1 Bone marrow-derived MDSC from tumor bearing mice suppressed T cell proliferation

We had previously shown that bone marrow-derived Ly6C+ monocytes, both in bone marrow and

the peripheral blood of C57BL6 mice, are natural M-MDSC, and display potent inhibition in CD3

and CD28 ligation-induced T cell proliferation. In addition, tumor conditions induced an extensive

expansion in bone marrow of low-density granulocytes (Ly6G+), which displayed characteristics

of G-MDSC and as well potently inhibited T-cell proliferation. Different from M-MDSC, which are

present constitutively, G-MDSC appear only under pathogenic conditions such as tumor. Using

Percoll density gradients, M-MDSC and G-MDSC (both in band III) can be separated from mature

20

granulocytes, especially mature neutrophils (PMN), which displayed high cell density (band IV)

and non-myeloid cells in bone marrow (band I-II) (Fig. 3.1A).

Because mice MDSCs express both CD11b and Gr-1 protein, flow cytometry analysis using both

CD11b and Gr-1 antibodies was used to identify cell types from bone marrow of both B16 bearing

mice and control mice (healthy) following the separation by percoll. Flow cytometry analysis

results showed that Both band III and IV from B16 bearing mice contain CD11b+Gr-1+ leukocytes

and then further gating using Ly6G and Ly6C antibodies in fraction III show that band III contains

immature myeloid cells that have the same phenotype of M-MDSC or G-MDSC while fraction IV

contains about 98 % mature granulocytes (Fig. 3.1A).

Tumor-induced myeloid derived suppressive cells have been strongly correlated with the

inhibition of T cell proliferation in different tumor models[2, 68].To confirm that fraction III

formed in mice engrafted with B16 melanoma (s. c.) are immunosuppressive, fraction III

separated cells were co-cultured with total mice splenocytes from healthy C57BL6 mice ,

labelled with CFSE and activated by incubating with CD3 and CD28 antibodies. As expected, CD4

and CD8 T-cell proliferation was potently inhibited while fraction IV displayed no inhibition

(Fig.3.1B). In addition, the co-culture of either purified Ly6G+ granulocytic cells or Ly6Chigh

monocytic cells from fraction III with CD3/CD28 activated T cells showed inhibition of T-cell

proliferation. Same fraction III from tumor bearing mice, inhibited Con.A, PMA or IL-2 induced T-

cell proliferation (Fig.3.1D, E). These results were also repeated using different tumor models as

B16 melanoma, MC38 colon carcinoma and EL4 lymphoma (Fig.3.1C).These results suggest that

these cells resemble MDSC phenotypically and functionally.

21

Figure 3-1 Bone marrow-derived MDSC from tumor bearing mice suppress T cell proliferation. A) BM collected from tumor-bearing mice were added to percoll density gradients in 15 ml tube.

After centrifugation the cells separated into four fractions (I, II, III and IV) and MDSC present in

fraction III followed by FACS analyses for cells in fraction III and IV using LY6Chigh and LY6G+

antibodies. C) Testing of MDSC suppressive activity. Cells from fraction III or IV were co-

cultured with splenocytes that activated by anti CD3/CD28 antibodies. After 4 days cells were

stained for CD4 and CD8 T cells and cell proliferation was determined by FACS by the evaluation

of CFSE signal. MDSC were added to splenocytes at the ratio of 1:3. Ctr is only T cells without

any leukocytes. D, E) ConA, IL-2 and PMA activated T cells inhibited by Cells from Fraction III.

Cells from Fraction III were co-cultured with T cells in RPMI (10 % FBS+ 0.2ug/mL ConA, PMA

20ng/mL / Ionomycin 60ng/mL or IL-2 500 IU/mL followed by FACS analysis of T cell

proliferation. Percentage of Ly6Chigh monocytes and Ly6G+ granulocytes in fractions III and IV

were calculated as a percent of total BM cells. Data are presented as mean ± SEM (n=3). ***, p

≤ 0.001.

22

3.2 Tumor bone marrow MDSC express arginase-1 upon exposure to TCR-activated T

cells

Since arginase-1 is believed to be a major feature of MDSC suppressive activity, we tested

arginase-1 expression in MDSC isolated by percoll using western blotting analysis. Although

MDSC isolated from tumor bearing mice, profoundly inhibited T-cell proliferation,

surprisingly, no arginase-1 expression was detected in MDSC following their isolation from

the bone marrow. Interestingly, the incubation of MDSC from tumor bearing mice with

CD3/CD28 activated T cells induced arginase-1 expression. As shown in figure 3.2A, the

incubation of MDSC with CD3/CD28 activated splenocytes but not non-activated splenocytes,

induced arginase-1 expression.

Since splenocytes consists of different cell types such as T cells, B cells, macrophages and

dendritic cells, we purified T-cells using CD4 and CD8 positive selection that affinity isolated

T cells (> 97% purity). Incubation of MDSC with only CD3/CD28 activated T cells induced

arginase-1 expression while the incubation with CD3/CD28 activated splenocytes (depleted T

cells) failed to induce arginase-1 expression). As shown in Fig.3. 2B, MDSC co-cultured with

purified T cells in the presence of CD3/CD28 ligation were induced to express arginase-1.

Depletion of T cells from splenocytes in the co-culture, or removal of antibodies for TCR

ligation, failed to induce arginase-1 expression in MDSC.

We next tested weather Ly6Chigh monocytes (M-MDSC) and Ly6G+ granulocytes (G-MDSC)

have the same potential to express arginase-1. Our results show that, both M-MDSC and G-

MDSC isolated from the bone marrow of tumor bearing mice tend to express arginase-1 only

after the exposure to CD3/CD28 activated T cells. As shown in Figure 2C, the co-culture of

either M-MDSC or G-MDSC with CD3/CD28 activated T cells successfully induced arginase-

23

1 expression that was not detected following the isolation from tumor-bearing mice. Consistent

with that, no arginase-1 expression was detected in the mature granulocytes from tumor

bearing mice before or after the exposure to CD3/CD28 activated T cells (Fig.3. 2C). Arginase-

1 expression was detected after (18 – 24 hrs.) but not shorter time (3 or 6 hrs.) (Fig.3. 2D).

24

Figure 3-2 Bone marrow-derived MDSC express no arginase but until after exposure

to activated T cells.

A) Western blotting to detect arginase-1 expression in MDSC before (-) or after (+) co-culture with activated or non-activated splenocytes after incubating for overnight (18 hrs.). B) Arginase-1 expression in MDSC requires activated T cells. Western blotting of arginase-1 expression before or after co-culture with activated splenocytes (SP), splenocytes without T cells or purified T cells. C) Fraction III Ly6G+ granulocytes and Ly6Chigh monocytes, and fraction IV mature PMN isolated from B16 melanoma-bearing mice were tested for arginase expression before (-) and after (+) addition into the CD3/CD28 activated T cells for 18 h. D) Time course for arginase-1 expression by MDSC after co-culture with CD3/CD28 activated T cells.

25

3.3 Arginase-1 expression by bone marrow derived MDSC is independent of direct contact

with T cells.

We next investigated whether the induction of arginase-1 expression in bone marrow derived

MDSC that primarily triggered by CD3/CD28 activated T cells requires direct contact with T cells.

We found that arginase-1 expression is induced in bone marrow derived MDSC by a mechanism

that does not necessarily require contact between MDSC and CD3/CD28 activated T cells. As

shown in figure (3.3.A), the culture of MDSC isolated from bone marrow of tumor-bearing mice

in culture supernatant collected from CD3/CD28 activated total splenocytes or purified CD4 and

CD8 T cells successfully induced arginase-1 expression while the incubation in culture supernatant

collected from non-activated T cells, B16 tumor cells failed to induce arginase-1 expression.

Furthermore, incubation of MDSC in culture supernatant isolated from either CD3/CD28 activated

CD4 or CD8 T cells induced arginase-1 expression (Fig. 3.3.B).In addition, both M-MDSC and

G-MDSC expressed arginase-1 only after being treated with CD3/CD28 activated T cells

supernatant which is similar to the result that we got from the co-culture system (Fig.3.3C). This

result suggested that, bone marrow derived MDSC from tumor bearing mice do not constitutively

express arginase-1 but instead arginase-1 expression induced once MDSC was exposed to

activated T cell environment.

26

3.4 Arginase-1 induction in BM derived MDSC by activated T cells requires ligation of

TCR co-receptor (CD3).

Since different activators such as Concanavalin A (ConA), PMA plus Ionomycin and IL-2 have

the ability to induce T-cell proliferation, we sought to determine whether T-cells activated by

ConA, PMA plus Ionomycin or IL-2 induce arginase-1 expression in MDSC. Although ConA,

PMA plus Ionomycin or IL-2 successfully activated T cell proliferation, interestingly, only T cells

activated via ligation of CD3 induced arginase-1 expression. As shown in figure (3.4A,B),

incubation of MDSC with culture supernatant isolated from T cells that had been activated by IL-

2, ConA or PMA plus Ionomycin failed to induce arginase-1 expression but only MDSC that

incubated with cell culture supernatant isolated from CD3/CD28 activated cells successfully

induced arginase-1.This result suggested that T cells activation through ligation of CD3 but not

other ways of activation can induce arginase-1 enzyme expression in myeloid derived suppressor

cells.

27

Figure 3-3 Arginase-1 expression by BM derived MDSC is independent of direct contact with T cells.

A) Arginase-1 expression in MDSC (FIII) isolated from tumor bearing mice after the treatment using 50% CD3/CD28 activated splenocytes, T cells or B16 cells supernatant. B) Induction of arginase-1 expression by either CD3/CD28 CD4 or CD8 activated T cells supernatant. C) Arginase-1 expression detection in fraction III Ly6G+ granulocytes and Ly6Chigh monocytes. Fraction III Ly6G+ granulocytes or Ly6Chigh were tested for arginase-1 expression after overnight treatment with supernatant collected from T cells after overnight activation with CD3/CD28 antibodies.B-actin was used as a loading control.

28

Figure 3-4 Arginase-1 induction in BM derived MDSC by activated T cells requires ligation of TCR co-receptor (CD3).

A) T cell proliferation after incubating of T cells with PMA (30ng/ml)/Ionomycin (80 ng/ml), ConA (0.5 μg/ ml) or IL-2 (1000 IU/ml). B) Arginase-1 expression in F III (MDSC) after treatment with supernatant collected from activated T cells showed in (A).

29

3.5 Healthy Ly6Chigh monocytes but not Ly6G+ granulocytes express arginase-1 upon the

exposure to CD3/CD28 activated T cells.

We had shown that Ly6Chigh monocytes but not Ly6G+ granulocytes isolated from the bone

marrow of healthy C57BL6 mice, inhibited T cell proliferation in a similar manner to their

counterpart isolated from tumor bearing mice (fig. 3-5.A).We next sought to determine whether

Ly6Chigh monocytes isolated from bone marrow of healthy mice express arginase-1 after being

exposed to CD3/CD28 activated T cell or its supernatant. As shown in figure 3-5B, Ly6Chigh

monocytes but not Ly6G+ granulocytes from healthy mice expressed arginase-1 after co-culture

with CD3/CD28 activated T cells. Furthermore, treatment with supernatant isolated from T cells

activated by CD3/CD28 antibodies, induced arginase-1 expression while no arginase-1 expression

was detected in mature granulocytes before or after treatment with similar supernatant. This

suggested that not only MDSC that formed under tumor or disease conditions have the potential

to express arginase-1 but also Ly6Chigh monocytes can express arginase-1 after being exposed to

CD3/CD28 activated T cells and can be considered as natural MDSCs.

30

Figure 3-5 Healthy Ly6Chigh monocytes but not Ly6G+ granulocytes express arginase-1 only after the exposure to CD3/CD28 activated T cells. A) Evaluation of LY6Chigh suppressive activity by inhibiting T cell proliferation. Splenocytes were

labelled with CFSE and incubated in plates immobilized with anti-CD3 and in the presence of

soluble anti-CD28 antibodies, in presence of myeloid cells isolated from healthy mice or without

any myeloid cells (ctl.) for 4 days before FACS analyses. The ratio of splenocytes: myeloid cells

was 3:1. B) Arginase-1 detection in LY6Chigh or LY6G+ cells from band III isolated by percoll from

BM of healthy mice before and after co-culture with CD3/CD28 activated T cells. Data are

presented as mean ± SEM (n=3). ***, p ≤ 0.001.

CD4 CD8

31

4 DISCUSSION

MDSC are one of the suppressive cell types found in the tumor microenvironment associated

with suppression of T cell responses and enhancing tumor progression[69]. Blocking MDSC

activity had been shown to have a negative effect on tumor progression[70]. Suppression of T

cell responses by MDSC had been shown to be associated with arginase-1 enzyme[71].

However, We found that arginase-1 enzyme is not constitutively expressed in bone marrow

derived MDSC isolated from tumor-bearing mice, instead required to be induced .In our study

we showed that T cells activated via the ligation of CD3 induced arginase-1 expression in BM

derived MDSC. Although many reports had shown that MDSC inhibition of T-cell

proliferation require direct contact between MDSC and T cells [21, 72], our results showed

that direct contact between MDSC and activated T cells is not required for arginase-1 enzyme

induction in bone marrow derived MDSC. Supernatant collected from CD3/CD28 activated T

cells, induced arginase-1 expression suggesting the presence of diffusible factors that are

released after T cell activation leading to the induction of arginase-1 expression in bone

marrow derived MDSC. The culture of bone marrow derived MDSC with resting T cells failed

to induce arginase-1 enzyme expression. This result is consistent with previous results that

showed that arginase-1 suppressive activity required the presence of activated T cells[72]. We

also showed that both monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC)

expressed arginase-1 after being treated with CD3/CD28 activated T cell supernatant.

Even though tumor released factors had been shown to induce arginase -1 expression in tumor

infiltrating myeloid suppressor cells [65], we did not see any arginase-1 expression in bone

marrow derived MDSC after treatment with supernatant collected from B16 cells. But instead,

our data demonstrated that soluble factors released from CD3/CD28 activated T cells induced

32

arginase-1 in bone marrow derived MDSC in vitro. Thus, we hypothesized that bone marrow

derived MDSC express no arginase-1, but once they enter the tumor microenvironment and

encounter activated T cells, MDSC will express arginase-1 as a result of CD3 activated T cell

released factors.

Interestingly, our data showed that arginase-1 expression required CD3/CD28 activated T cells

but not other ways of activation. In our study, the co-culture of MDSC with ConA, IL-2 or

PMA activated T cells potently inhibited T-cell proliferation. However, we failed to detect

arginase-1 expression in MDSC after the co-culture with IL-2, ConA or PMA activated T cells.

This result suggested the presence of a different mechanism of suppression rather than

arginase-1 enzyme activity which is believed to be an NO-dependent mechanism[72]. This was

confirmed by the use of an iNOS inhibitor (l-NMMA) which profoundly rescued T-cell

proliferation[72]. Furthermore, neither IL-2, ConA nor PMA activated T cells culture

supernatant induced arginase-1 expression in bone marrow derived MDSC suggesting a

difference in soluble factors released from activated T cells using different activators.

We and others had shown that LY6Chigh monocytes isolated from healthy mice suppressed T-

cell proliferation[73]. Our results demonstrated that in a similar manner to their counterparts

from tumor-bearing mice, LY6Chigh monocytes from healthy mice expressed arginase-1 only

after the exposure to CD3/CD28 activated T cells. Indeed, this observation might provide a

mechanism to treat autoimmune diseases.

33

REFERENCES

1. Ostrand-Rosenberg, S., P. Sinha, D.W. Beury, and V.K. Clements. Cross-talk between

myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells enhances

tumor-induced immune suppression. in Seminars in cancer biology. 2012. Elsevier.

2. Gabrilovich, D.I. and S. Nagaraj, Myeloid-derived suppressor cells as regulators of the

immune system. Nature Reviews Immunology, 2009. 9(3): p. 162-174.

3. Talmadge, J.E. and D.I. Gabrilovich, History of myeloid-derived suppressor cells. Nature

Reviews Cancer, 2013. 13(10): p. 739-752.

4. Bennett, J.A., V.S. Rao, and M.S. Mitchell, Systemic bacillus Calmette-Guerin (BCG)

activates natural suppressor cells. Proceedings of the National Academy of Sciences,

1978. 75(10): p. 5142-5144.

5. Gabrilovich, D.I., V. Bronte, S.-H. Chen, M.P. Colombo, A. Ochoa, S. Ostrand-

Rosenberg, and H. Schreiber, The terminology issue for myeloid-derived suppressor cells.

Cancer research, 2007. 67(1): p. 425-425.

6. Marvel, D. and D.I. Gabrilovich, Myeloid-derived suppressor cells in the tumor

microenvironment: expect the unexpected. The Journal of clinical investigation, 2015.

125(9): p. 3356-3364.

7. Damuzzo, V., L. Pinton, G. Desantis, S. Solito, I. Marigo, V. Bronte, and S.

Mandruzzato, Complexity and challenges in defining myeloid‐derived suppressor cells.

Cytometry Part B: Clinical Cytometry, 2015. 88(2): p. 77-91.

8. Bronte, V., S. Brandau, S.-H. Chen, M.P. Colombo, A.B. Frey, T.F. Greten, S.

Mandruzzato, P.J. Murray, A. Ochoa, and S. Ostrand-Rosenberg, Recommendations for

myeloid-derived suppressor cell nomenclature and characterization standards. Nature

communications, 2016. 7.

9. Youn, J.I. and D.I. Gabrilovich, The biology of myeloid‐derived suppressor cells: the

blessing and the curse of morphological and functional heterogeneity. European journal

of immunology, 2010. 40(11): p. 2969-2975.

10. Qu, P., L.-z. Wang, and P.C. Lin, Expansion and functions of myeloid-derived suppressor

cells in the tumor microenvironment. Cancer letters, 2015.

11. Nagaraj, S., K. Gupta, V. Pisarev, L. Kinarsky, S. Sherman, L. Kang, D.L. Herber, J.

Schneck, and D.I. Gabrilovich, Altered recognition of antigen is a mechanism of CD8+ T

cell tolerance in cancer. Nature medicine, 2007. 13(7): p. 828-835.

12. Yang, L., J. Huang, X. Ren, A.E. Gorska, A. Chytil, M. Aakre, D.P. Carbone, L.M.

Matrisian, A. Richmond, and P.C. Lin, Abrogation of TGFβ signaling in mammary

carcinomas recruits Gr-1+ CD11b+ myeloid cells that promote metastasis. Cancer cell,

2008. 13(1): p. 23-35.

13. Almand, B., J.I. Clark, E. Nikitina, J. van Beynen, N.R. English, S.C. Knight, D.P.

Carbone, and D.I. Gabrilovich, Increased production of immature myeloid cells in cancer

patients: a mechanism of immunosuppression in cancer. The Journal of Immunology,

2001. 166(1): p. 678-689.

14. Haile, L.A., R. Von Wasielewski, J. Gamrekelashvili, C. Krüger, O. Bachmann, A.M.

Westendorf, J. Buer, R. Liblau, M.P. Manns, and F. Korangy, Myeloid-derived

34

suppressor cells in inflammatory bowel disease: a new immunoregulatory pathway.

Gastroenterology, 2008. 135(3): p. 871-881. e5.

15. Gabrilovich, D., T. Ishida, T. Oyama, S. Ran, V. Kravtsov, S. Nadaf, and D.P. Carbone,

Vascular endothelial growth factor inhibits the development of dendritic cells and

dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood,

1998. 92(11): p. 4150-4166.

16. Li, Z.-L., S.-B. Ye, L.-Y. OuYang, H. Zhang, Y.-S. Chen, J. He, Q.-Y. Chen, C.-N. Qian,

X.-S. Zhang, and J. Cui, COX-2 promotes metastasis in nasopharyngeal carcinoma by

mediating interactions between cancer cells and myeloid-derived suppressor cells.

Oncoimmunology, 2015. 4(11): p. e1044712.

17. Serafini, P., R. Carbley, K.A. Noonan, G. Tan, V. Bronte, and I. Borrello, High-dose

granulocyte-macrophage colony-stimulating factor-producing vaccines impair the

immune response through the recruitment of myeloid suppressor cells. Cancer research,

2004. 64(17): p. 6337-6343.

18. Bunt, S.K., L. Yang, P. Sinha, V.K. Clements, J. Leips, and S. Ostrand-Rosenberg,

Reduced inflammation in the tumor microenvironment delays the accumulation of

myeloid-derived suppressor cells and limits tumor progression. Cancer research, 2007.

67(20): p. 10019-10026.

19. Pan, P.-Y., G.X. Wang, B. Yin, J. Ozao, T. Ku, C.M. Divino, and S.-H. Chen, Reversion

of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor

cell development by blockade of stem-cell factor function. Blood, 2008. 111(1): p. 219-

228.

20. Nefedova, Y., S. Nagaraj, A. Rosenbauer, C. Muro-Cacho, S.M. Sebti, and D.I.

Gabrilovich, Regulation of dendritic cell differentiation and antitumor immune response

in cancer by pharmacologic-selective inhibition of the janus-activated kinase 2/signal

transducers and activators of transcription 3 pathway. Cancer research, 2005. 65(20): p.

9525-9535.

21. Bronte, V., P. Serafini, C. De Santo, I. Marigo, V. Tosello, A. Mazzoni, D.M. Segal, C.

Staib, M. Lowel, and G. Sutter, IL-4-induced arginase 1 suppresses alloreactive T cells

in tumor-bearing mice. The Journal of Immunology, 2003. 170(1): p. 270-278.

22. Rutschman, R., R. Lang, M. Hesse, J.N. Ihle, T.A. Wynn, and P.J. Murray, Cutting edge:

Stat6-dependent substrate depletion regulates nitric oxide production. The Journal of

Immunology, 2001. 166(4): p. 2173-2177.

23. Sinha, P., V.K. Clements, and S. Ostrand-Rosenberg, Interleukin-13–regulated M2

macrophages in combination with myeloid suppressor cells block immune surveillance

against metastasis. Cancer research, 2005. 65(24): p. 11743-11751.

24. Kusmartsev, S. and D.I. Gabrilovich, STAT1 signaling regulates tumor-associated

macrophage-mediated T cell deletion. The Journal of Immunology, 2005. 174(8): p.

4880-4891.

25. Bronte, V. and P. Zanovello, Regulation of immune responses by L-arginine metabolism.

Nature Reviews Immunology, 2005. 5(8): p. 641-654.

26. Popovic, P.J., H.J. Zeh, and J.B. Ochoa, Arginine and immunity. The Journal of nutrition,

2007. 137(6): p. 1681S-1686S.

27. Serafini, P., I. Borrello, and V. Bronte. Myeloid suppressor cells in cancer: recruitment,

phenotype, properties, and mechanisms of immune suppression. in Seminars in cancer

biology. 2006. Elsevier.

35

28. Rodriguez, P.C., D.G. Quiceno, J. Zabaleta, B. Ortiz, A.H. Zea, M.B. Piazuelo, A.

Delgado, P. Correa, J. Brayer, and E.M. Sotomayor, Arginase I production in the tumor

microenvironment by mature myeloid cells inhibits T-cell receptor expression and

antigen-specific T-cell responses. Cancer research, 2004. 64(16): p. 5839-5849.

29. Rodriguez, P.C., A.H. Zea, K.S. Culotta, J. Zabaleta, J.B. Ochoa, and A.C. Ochoa,

Regulation of t cell receptor cd3ζ chain expression byl-arginine. Journal of Biological

Chemistry, 2002. 277(24): p. 21123-21129.

30. Harari, O. and J.K. Liao, Inhibition of MHC II gene transcription by nitric oxide and

antioxidants. Current pharmaceutical design, 2004. 10(8): p. 893-898.

31. Rivoltini, L., M. Carrabba, V. Huber, C. Castelli, L. Novellino, P. Dalerba, R. Mortarini,

G. Arancia, A. Anichini, and S. Fais, Immunity to cancer: attack and escape in T

lymphocyte–tumor cell interaction. Immunological reviews, 2002. 188(1): p. 97-113.

32. Ochoa, A.C., A.H. Zea, C. Hernandez, and P.C. Rodriguez, Arginase, prostaglandins,

and myeloid-derived suppressor cells in renal cell carcinoma. Clinical Cancer Research,

2007. 13(2): p. 721s-726s.

33. Munder, M., H. Schneider, C. Luckner, T. Giese, C.-D. Langhans, J.M. Fuentes, P.

Kropf, I. Mueller, A. Kolb, and M. Modolell, Suppression of T-cell functions by human

granulocyte arginase. Blood, 2006. 108(5): p. 1627-1634.

34. Highfill, S.L., P.C. Rodriguez, Q. Zhou, C.A. Goetz, B.H. Koehn, R. Veenstra, P.A.

Taylor, A. Panoskaltsis-Mortari, J.S. Serody, and D.H. Munn, Bone marrow myeloid-

derived suppressor cells (MDSCs) inhibit graft-versus-host disease (GVHD) via an

arginase-1–dependent mechanism that is up-regulated by interleukin-13. Blood, 2010.

116(25): p. 5738-5747.

35. Rodriguez, P.C., M.S. Ernstoff, C. Hernandez, M. Atkins, J. Zabaleta, R. Sierra, and A.C.

Ochoa, Arginase I–producing myeloid-derived suppressor cells in renal cell carcinoma

are a subpopulation of activated granulocytes. Cancer research, 2009. 69(4): p. 1553-

1560.

36. Munder, M., F. Mollinedo, J. Calafat, J. Canchado, C. Gil-Lamaignere, J.M. Fuentes, C.

Luckner, G. Doschko, G. Soler, and K. Eichmann, Arginase I is constitutively expressed

in human granulocytes and participates in fungicidal activity. Blood, 2005. 105(6): p.

2549-2556.

37. Chen, X., M. Song, B. Zhang, and Y. Zhang, Reactive oxygen species regulate T cell

immune response in the tumor microenvironment. Oxidative Medicine and Cellular

Longevity, 2016. 2016.

38. Holmdahl, R., O. Sareila, L.M. Olsson, L. Bäckdahl, and K. Wing, Ncf1 polymorphism

reveals oxidative regulation of autoimmune chronic inflammation. Immunological

reviews, 2016. 269(1): p. 228-247.

39. Schmielau, J. and O.J. Finn, Activated granulocytes and granulocyte-derived hydrogen

peroxide are the underlying mechanism of suppression of t-cell function in advanced

cancer patients. Cancer research, 2001. 61(12): p. 4756-4760.

40. Kusmartsev, S., Y. Nefedova, D. Yoder, and D.I. Gabrilovich, Antigen-specific inhibition

of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive

oxygen species. The Journal of Immunology, 2004. 172(2): p. 989-999.

41. Liu, Y., J. Wei, G. Guo, and J. Zhou, Norepinephrine-induced myeloid-derived

suppressor cells block T-cell responses via generation of reactive oxygen species.

Immunopharmacology and immunotoxicology, 2015. 37(4): p. 359-365.

36

42. Kusmartsev, S. and D.I. Gabrilovich, Inhibition of myeloid cell differentiation in cancer:

the role of reactive oxygen species. Journal of leukocyte biology, 2003. 74(2): p. 186-

196.

43. Srivastava, M.K., P. Sinha, V.K. Clements, P. Rodriguez, and S. Ostrand-Rosenberg,

Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and

cysteine. Cancer research, 2010. 70(1): p. 68-77.

44. Aguiló, J.I., A. Anel, E. Catalán, A. Sebastián, R. Acín-Pérez, J. Naval, R. Wallich, M.M.

Simon, and J. Pardo, Granzyme B of cytotoxic T cells induces extramitochondrial

reactive oxygen species production via caspase-dependent NADPH oxidase activation.

Immunology and cell biology, 2010. 88(5): p. 545-554.

45. Hildeman, D.A., T. Mitchell, T.K. Teague, P. Henson, B.J. Day, J. Kappler, and P.C.

Marrack, Reactive oxygen species regulate activation-induced T cell apoptosis.

Immunity, 1999. 10(6): p. 735-744.

46. Devadas, S., L. Zaritskaya, S.G. Rhee, L. Oberley, and M.S. Williams, Discrete

generation of superoxide and hydrogen peroxide by T cell receptor stimulation. Journal

of Experimental Medicine, 2002. 195(1): p. 59-70.

47. Jackson, S.H., S. Devadas, J. Kwon, L.A. Pinto, and M.S. Williams, T cells express a

phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation. Nature

immunology, 2004. 5(8): p. 818-827.

48. Vickers, S.M., L.A. MacMillan-Crow, M. Green, C. Ellis, and J.A. Thompson,

Association of increased immunostaining for inducible nitric oxide synthase and

nitrotyrosine with fibroblast growth factor transformation in pancreatic cancer. Archives

of surgery, 1999. 134(3): p. 245-251.

49. Brito, C., M. Naviliat, A.C. Tiscornia, F. Vuillier, G. Gualco, G. Dighiero, R. Radi, and

A.M. Cayota, Peroxynitrite inhibits T lymphocyte activation and proliferation by

promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic

death. The Journal of Immunology, 1999. 162(6): p. 3356-3366.

50. Huang, B., P.-Y. Pan, Q. Li, A.I. Sato, D.E. Levy, J. Bromberg, C.M. Divino, and S.-H.

Chen, Gr-1+ CD115+ immature myeloid suppressor cells mediate the development of

tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host. Cancer

research, 2006. 66(2): p. 1123-1131.

51. Serafini, P., S. Mgebroff, K. Noonan, and I. Borrello, Myeloid-derived suppressor cells

promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells. Cancer

research, 2008. 68(13): p. 5439-5449.

52. Schwarz, B.A. and A. Bhandoola, Trafficking from the bone marrow to the thymus: a

prerequisite for thymopoiesis. Immunological reviews, 2006. 209(1): p. 47-57.

53. Petrie, H.T., Cell migration and the control of post-natal T-cell lymphopoiesis in the

thymus. Nature Reviews Immunology, 2003. 3(11): p. 859-866.

54. Shortman, K. and L. Wu, Early T lymphocyte progenitors. Annual review of

immunology, 1996. 14(1): p. 29-47.

55. Zúñiga-Pflücker, J.C., T-cell development made simple. Nature Reviews Immunology,

2004. 4(1): p. 67-72.

56. Klein, L., B. Kyewski, P.M. Allen, and K.A. Hogquist, Positive and negative selection of

the T cell repertoire: what thymocytes see (and don't see). Nature Reviews Immunology,

2014. 14(6): p. 377-391.

37

57. Kyewski, B. and L. Klein, A central role for central tolerance. Annu. Rev. Immunol.,

2006. 24: p. 571-606.

58. Bonnefoy-Berard, N., V. Besnard, P. Morel, Z. Hmama, B. Verrier, B. Mandrand, C.

Vincent, and J. Revillard, Second signal for T lymphocyte activation: multiple targets for

pharmacological modulation. Developments in biological standardization, 1991. 77: p.

41-48.

59. Malissen, B., C. Gregoire, M. Malissen, and R. Roncagalli, Integrative biology of T cell

activation. Nature immunology, 2014. 15(9): p. 790-797.

60. Pagán, A.J., M. Pepper, H.H. Chu, J.M. Green, and M.K. Jenkins, CD28 promotes CD4+

T cell clonal expansion during infection independently of its YMNM and PYAP motifs.

The Journal of immunology, 2012. 189(6): p. 2909-2917.

61. Khoruts, A., A. Mondino, K.A. Pape, S.L. Reiner, and M.K. Jenkins, A natural

immunological adjuvant enhances T cell clonal expansion through a CD28-dependent,

interleukin (IL)-2–independent mechanism. Journal of Experimental Medicine, 1998.

187(2): p. 225-236.

62. Ando, Y., C. Yasuoka, T. Mishima, T. Ikematsu, T. Uede, T. Matsunaga, and M. Inobe,

Concanavalin A-mediated T cell proliferation is regulated by herpes virus entry mediator

costimulatory molecule. In Vitro Cellular & Developmental Biology-Animal, 2014.

50(4): p. 313-320.

63. Weiss, A., R. Shields, M. Newton, B. Manger, and J. Imboden, Ligand-receptor

interactions required for commitment to the activation of the interleukin 2 gene. The

Journal of Immunology, 1987. 138(7): p. 2169-2176.

64. Chen, L. and D.B. Flies, Molecular mechanisms of T cell co-stimulation and co-

inhibition. Nature Reviews Immunology, 2013. 13(4): p. 227-242.

65. Rodriguez, P.C., C.P. Hernandez, D. Quiceno, S.M. Dubinett, J. Zabaleta, J.B. Ochoa, J.

Gilbert, and A.C. Ochoa, Arginase I in myeloid suppressor cells is induced by COX-2 in

lung carcinoma. Journal of Experimental Medicine, 2005. 202(7): p. 931-939.

66. Gallina, G., L. Dolcetti, P. Serafini, C. De Santo, I. Marigo, M.P. Colombo, G. Basso, F.

Brombacher, I. Borrello, and P. Zanovello, Tumors induce a subset of inflammatory

monocytes with immunosuppressive activity on CD8+ T cells. The Journal of clinical

investigation, 2006. 116(10): p. 2777-2790.

67. Sinha, P., K.H. Parker, L. Horn, and S. Ostrand‐Rosenberg, Tumor‐induced myeloid‐derived suppressor cell function is independent of IFN‐γ and IL‐4Rα. European journal of

immunology, 2012. 42(8): p. 2052-2059.

68. Beury, D.W., K.H. Parker, M. Nyandjo, P. Sinha, K.A. Carter, and S. Ostrand-

Rosenberg, Cross-talk among myeloid-derived suppressor cells, macrophages, and tumor

cells impacts the inflammatory milieu of solid tumors. Journal of leukocyte biology,

2014. 96(6): p. 1109-1118.

69. Ostrand-Rosenberg, S., Myeloid-derived suppressor cells: more mechanisms for

inhibiting antitumor immunity. Cancer Immunology, Immunotherapy, 2010. 59(10): p.

1593-1600.

70. Highfill, S.L., Y. Cui, A.J. Giles, J.P. Smith, H. Zhang, E. Morse, R.N. Kaplan, and C.L.

Mackall, Disruption of CXCR2-mediated MDSC tumor trafficking enhances anti-PD1

efficacy. Science translational medicine, 2014. 6(237): p. 237ra67-237ra67.

38

71. Zea, A.H., P.C. Rodriguez, M.B. Atkins, C. Hernandez, S. Signoretti, J. Zabaleta, D.

McDermott, D. Quiceno, A. Youmans, and A. O'Neill, Arginase-producing myeloid

suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer

research, 2005. 65(8): p. 3044-3048.

72. Mazzoni, A., V. Bronte, A. Visintin, J.H. Spitzer, E. Apolloni, P. Serafini, P. Zanovello,

and D.M. Segal, Myeloid suppressor lines inhibit T cell responses by an NO-dependent

mechanism. The Journal of Immunology, 2002. 168(2): p. 689-695.

73. Slaney, C.Y., A. Toker, A. La Flamme, B.T. Bäckström, and J.L. Harper, Naive blood

monocytes suppress T-cell function. A possible mechanism for protection from

autoimmunity. Immunology and cell biology, 2011. 89(1): p. 7-13.