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Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.

NOTTINGHAM TRENT UNIVERSITY

ISOLATION AND FUNCTIONAL CHARATERIZATION OF

CD4+CD25HIGHCD127LOW/- REGULATORY T CELLS FROM HUMAN

PERIPHERAL BLOOD

By

VIJAYKUMAR PATRA

Project report submitted in partial fulfilment of the MSc by Research Applied Biosciences

(Biotechnology), School of Science and Technology, Nottingham Trent University

Project Supervisor

Dr. Stéphanie Mcardle

The John van Geest Cancer research Center

Nottingham Trent University

2012-2013

DECLARATION OF OWNERSHIP

This submission is the result of my work. All help and advice, other than that received from

tutors, has been acknowledged and primary and secondary sources of information have been

properly attributed. Should this statement prove to be untrue, I recognize the right and duty of

the Board of Examiners to recommend what action should be taken in line with the University’s

regulations on assessment contained in the Handbook.

Signed.......................................................... Date.........................................


Table of Contents

Abstract

Acknowledgement

Figures

Tables

Abbreviations

Chapter 1: Introduction 1

1.1 Immune System 1

1.1.1 Innate and adaptive Immune system 1

1.1.1.1 Innate Immune system 1

1.1.1.2 Adaptive Immune system 2

1.1.2 T-Lymphocytes 2

1.1.2.1 CD4 T cells 3

1.1.2.2 CD8 T cells 4

1.1.3 Role of T cells 5

1.2. Regulatory T cells 7

1.2.1 History of Regulatory T cells (Tregs) 7

1.2.2 Natural Tregs (nTreg) and Adaptive Tregs (iTreg) 8

1.2.3 Properties of regulatory T cells 10

1.2.4 Key markers of regulatory T cells 10

1.2.4.1 CD25 10

1.2.4.2 CD127 11

1.2.4.3 FoxP3 11

1.2.5 Other potential markers 12

1.3. Prostate Cancer and Tregs 13

1.3.1 Source of Regulatory T cells in the Tumour 14

1.3.2 Mechanism of action of Foxp3+ Regulatory T cells 16


1.3.3 Targeting Regulatory T cells for Immunotherapy 17

1.4. Rationale of the Project 19

Chapter 2. Materials 20

2.1 General laboratory Materials 20

2.2 Electrical Equipment’s 20

2.3 Reagents for Culture Media 21

2.4 Materials for Flow Cytometer 21

2.4.1 Buffers 21

2.4.2 Antibodies 22

2.5 Materials for Proliferation & Suppression assay’s 22

2.6 Kits 22

Chapter 3. Methods 23

3.1 PBMC processing 24

3.2 Proliferation assay 25

3.3 CD4+ T cell isolation 26

3.4 Antibody compensation studies 27

3.5 CD4+ T cell Proliferation assay 27

3.6 CD4+CD25highCD127low/- (Tregs) & CD4+CD25-CD127+ (Tconv) cell sorting 28

3.7 Treg expansion 28

3.8 in vitro Treg suppression assay 29

Chapter 4. Results: 31

4.1 PBMC proliferation assay 31

4.2 Flow cytometer compensation studies 32

4.3 CD4+ Isolation 34

4.4 CD4+ Proliferation assay 37

4.5 Treg Expansion 39


Chapter 5. Conclusions and Future work 40

Chapter 6. Appendix 46

Chapter 7. Bibliography 48


Summary:

Purpose: Regulatory T cells are known to supress wide range of anti-tumour responses. It is

now evident that Tregs are one among the main barriers for cancer immunotherapy. Until

now there have been several methods described to isolate Tregs. It is also been documented

that the immunosuppressive functions of CD4+CD25High Tregs in cancer patients is

significantly higher than in healthy donors. This study has been designed to isolate

CD4+CD25highCD127low/- Tregs from human PBMCs (healthy donors) and functionally

characterize them based on their immunosuppression ability

Experimental Design: PBMCs were separated from whole blood, followed by enrichment of

CD4+T cells from PBMCs. Tregs (CD4+CD25highCD127low/- Tregs) and Tconv

(CD4+CD25lowCD127lhigh Tregs) were sorted from enriched CD4+T cells. The sorted Tregs

were expanded for a period of 20 days and used for further immunosuppression assays by

co-culturing Tregs with Tconv at varying concentrations.

Results: After separating PBMCs from whole blood, CD4+ T cell enrichment was done,

where the cells enriched showed above 95% purity. Proliferation assay was performed for

enriched CD4+T cells by stimulating them with anti-CD3 and anti-CD28 antibodies.

Maximum proliferation was observed at concentration of anti-CD3 at 0.5µg/mL and anti-

CD28 at 1 µg/mL. Tregs and Tconv were sorted from enriched CD4+ T cells, and thereafter

Tregs were expanded for a period of 20 days by CD3/CD28 coated micro-beads.

Conclusion: Tregs signify a small percentage in blood, hence there is need for reliable and

stable expansion protocols. The design for efficient and safer protocols for selective

expansion of Tregs is regarded fundamental for its future clinical applications. The

optimised protocol from this study will act as control for further functional assays which

will be performed for Tregs in benign and patients with metastatic prostate cancer.


Acknowledgement:

I am deeply grateful to my supervisor Dr.Stephanie McArdle, for her valuable

supervision, for constantly encouraging me and giving me an independence to plan

and work on the project. I would also like to thank Prof. Robert Rees and Prof.

Graham Pockley for giving me this project in first place and to allow me to work in

his wonderful group.

My sincere thanks to Steve, Amanda and Cathy for helping me in radioactive work

and also the support given during my lab work. My special thanks to Jay,

Shraddha, and Divya for their help during my lab work and also in writing my

thesis.

I am indebted to my parents for their love and faith they have in me. I would like to

thank all my friends, especially Meghana for being with me during my difficulties

and the friendship they offered.

My thanks to everyone who helped me knowingly and unknowingly for

completion of my Project.


List of figures:

Fig 1.1: CD4+ T cell differentiation chart.

Fig 1. 2: Thymus and peripheral generation of Foxp3+ regulatory T cells.

Fig 1.3: Movement of CD4+ CD25+ FoxP3+ Regulatory T cells towards Tumour

microenvironment under the influence of CCL22.

Fig 1.4: Differentiation of regulatory T cells in Tumour microenvironment via IL-10, TGF- β and

VEGF

Fig 1.5: conversion of CD4+CD25- T cells to CD4+CD25+ Regulatory T cells under the

influence of TGF- β.

Fig 1.6: Mechanism of action of Foxp3+ regulatory T cells

Fig 1.7: Targeting suppressive mechanisms including Tregs for therapeutic applications.

Fig 3.1: PBMC Processing using Leucosep™.

Fig 3.2: Proliferation plate setup

Fig 3.3: Plate diagram for suppression assay.

Fig 4.1: PBMC proliferation assay via anti-CD3 and anti-CD28 stimulation (n=3).

Fig 4.2 (a): Antibody compensation studies for flow cytometer (stained with CD4-PerCP Cy 5.5,

CD25 PE, and CD127 AF-647)

Fig 4.2 (b): Flow cytometer compensation studies

Fig 4.3 (a): Sample before CD4+ T cell isolation (stained with CD4 FITC and CD3 APC)

Fig 4.3 (b): Enriched CD4+ T cells (stained with CD4 FITC and CD3 APC)

Fig 4.3 (c): Sample before enrichment (stained with Treg sorting cocktail)

Fig 4.3 (d): Enriched CD4+ T cells (stained with Treg sorting cocktail)

Fig 4.4: CD4+ Proliferation assay with anti-CD3 and anti-CD28 antibody stimulation (n=3).


Fig 4.5: Regulatory T cell expansion.

Fig 6.1: Plate bound anti-CD3 stimulation and by using Dynabeads anti-CD3/anti-CD28 pre-

coated beads.

Fig 6.2: CD4+T cells Proliferation assay without IL-2.


List of Tables:

Table 1.1: Properties of Natural and Adaptive Regulatory T cells.

Table 1.2: Phenotypes of natural regulatory T cells.


Abbreviations:

TCR- T Cell receptor

nTreg- Natural Regulatory T Cell

iTreg- Induced Regulatory T Cell

NKT- Natural Killer T Cells

Th1- T Helper Cell 1



APC- Antigen Presenting Cell

MHC- Major Histocompatibility Complex

HIV- Human Immunodeficiency Virus

TB- Tuberculosis

Tregs- Regulatory T Cells

Tconv- Conventional T Cells

FoxP3- Forkhead Box P3

IL-2 –Interleukin 2

PCa- Prostate Cancer Antigen

TGF-β- Tumor Growth Factor- β

mRNA- Messenger Ribose Nucleic Acid

CCL-Chemokine Ligand

VEGF -Vascular Endothelial Growth Factor

FACS-Fluorescence Activated Cell Sorting

APC- Allophycocyanin

PE- Phycoerythrin

PerCP Cy - Peridinin Chlorophyll Protein combined with Cyanine dye

FITC- Fluorescein isothiocyanate

PBMC- Peripheral Blood Mononuclear Cells

PBS- Phosphate Buffer Saline


RT-Room Temperature

SEM-Standard Error of the Mean

JvGCRC- John van Geest Cancer Research Center


1

Chapter 1. Introduction:

1.1 Immune System:

The Immune system is a defence mechanism of the human body which

fights and protects against infection, pathogens and cancers. Our immune

systems has evolved to become highly versatile in defending against many

types of pathogenic infections and a wide range of cancers. What makes our

immune system a significant defence mechanism is the ability to produce

various cells and effector molecules capable of recognizing and eliminating

an unlimited range of foreign substances with high specificity.

1.1.1 Innate immunity and Adaptive immunity:

The immune system can be classified into two types:

1. Innate Immune system

2. Adaptive Immune system

1.1.1.1 Innate Immune system:

The innate immune system is a compilation of the defensive

mechanisms which the host uses to prevent or defend against the infection

or invading substance. Although this system functions in the absence of

adaptive immune system, it is also need for induction of the adaptive

immune system in numerous ways. The innate immune system is defined by

its quick response to invading agent, pathogen or effector cells, it is non-

specific in nature and is of a short duration. This system is however deficient

of immunological memory and there is no clonal expansion of immune cells

as observed in the case of the adaptive immune system.

The defence mechanisms which are in association with this system,

comprises of numerous barriers (skin), secretions which are accompanied by

serum


2

factors, cytokines and immunoglobulins. The cellular machinery includes cell

types such as Natural Killer cells, neutrophils, macrophages and dendritic

cells

(Janeway and Medzhitov, 2002; O’Gorman and Donnenberg, handbook of

immunology, 2nd edition).

1.1.1.2 Adaptive Immune system:

The Adaptive immune system, unlike the innate immune system, is

highly specific, flexible and also has an immunological memory, which gives

it the ability to respond rapidly to a second exposure to an antigen. The

ability of adaptive immune system to act specifically makes it very complex.

One significant mechanism which is common to both types of immune

systems is the ability to differentiate between self and non-self.

The primary elements of this system are T lymphocytes and B-lymphocytes.

Due to the presence of antigen specific receptors on their surfaces these T-

& B- cells show the distinctive ability to specifically target antigens. These

receptors are a result of the somatic reorganization of germ line gene

rudiments which form TCR genes and also the genes responsible for

immunoglobulin’s. The multiple interactions taking place between these two

systems results in co-amplifications of each of the respective response which

ultimately leads to the destruction and eliminating the pathogen.

1.1.2 T-Lymphocytes:

The specificity of the adaptive immunity is largely dependent on T or B

lymphocytes, where they recognize specific antigens and form antigen

specific antibodies. This process will demand the presence and function of T

helper lymphocytes. Immune response to harmful foreign pathogens is dealt


3

with cytotoxic T cells, which carry specific receptors which are capable of

recognizing the antigens present on the surface of infecting cells.

There is a significant interaction between T cells, B cells and T-T cells (CD4-

CD8 T cell interaction) in an adaptive immune response. The proliferation

and differentiation of naïve B cells and CD8+ T cells is stimulated by a

specific type of CD4+ T cell, which is the CD4+ T helper cell. The composite

interaction between the T cells, B cells and T-T cells along with the signals

involved in the proliferation and differentiation of both the cell types leads to

adaptive immune response. These CD4+ T cells are classified into a number

of cell types depending upon the migration arrays and its functional

capabilities. (O’Gorman and Donnenberg, handbook of immunology, pg 22).

1.1.2.1 CD4 T cells:

CD4 T cells are described to play a very important role in immune

defense. This is achieved by their capacity to promote antibody production

by stimulating B cells, inducing macrophages for development of improved

micobicidal activity, they also recruit innate immune cells to the infection

site and inflammation, and by producing various types of cytokines and

chemokine’s they license to kill infected cells by CD8+T cells and coordinate

the entire parade of the immune responses. (Jinfang Zhu and William E. Paul

2008).

CD4 T cell population are a divergent cell lineages of the T cells which are

already eminent from each other after they exit the thymus, like “natural

regulatory T cells (nTreg) and Natural killer T cells (NKT) or helper T cells

and quite a lot of cells represent different arrangements of differentiation of

naïve CD4 T cells (Sakaguchi S, 2004; Shevach EM, 2006; Bendelac A et al.,

2007).

Depending on the signals received by naïve CD4 T cells during their

interaction with an antigen, there can be various outcomes. The prominent


4

populations seen are “Th1, Th2, Th17 and induced regulatory T cells (iTreg)”

(Jinfang Zhu and William E. Paul 2008).

Fig 1.1: CD4+ T cell differentiation chart. The above chart displays the complex differentiation

of CD4 T cell upon stimulation via various antigens and the resulting different lineages have

been portrayed. Image adapted and modified from G G Brusselle et al., 2011.

1.1.2.2 CD8 T cells:

CD8 T cells are a subdivision of thymus derived T lymphocytes which

have clonally spread receptors, which are known to be responsible for cell

mediated lysis of the target cells or antigens (Cerottini et al., 1970; Golstein

et al., 1972; Cantor and Boyse, 1975;). These CD8 T lymphocytes are vital


5

intermediaries of adaptive immune response towards an antigen or an

infected cells. CD8+ T cells which have TCR specific for the antigens which

are derived, will undergo a selective clonal development when they

encounter with a microbe or antigen. This specificity of clonal selection is

compelled by interactions between the TCR and short peptides which have

been derived from pathogen and are being presented on surface of MHC

class I molecules. Antigen presenting cells (APC) have the capacity of

stimulating naïve T cells, which then proliferate and differentiate to respond

to an antigen, this is achieved by virtue of the expression of co-stimulatory

molecules by APC’s (Germain RN. 1994; Banchereau J, Steinman RM.

1998; Harty et al., 2000 ).

The activated CD8+ T cells can induce cytolysis of the cells infected by two

pathways.

A. granule exocytosis pathway.

B. up regulation of FasL (CD95L).

These pathways which have been activated in response to the TCR signals

will stimulate “caspase cascade” in the target cell which ultimately lead to

death by apoptosis. CD8+ T cells can also produce cytokines and

chemokine’s which function to recruit and/or activate the effector cells

(macrophages and neutrophils) (Harty et al., 2000).

1.1.3 Role of T cells:

The specific immunity of the immune system is solely dependent on

cytotoxic T or B lymphocytes recognizing the specific antigens and the

formation of the specific antibodies. This process of specific immunity will

require the presence and function of T helper lymphocytes. Response to

foreign molecules, which includes tumours, transplants or any virus infected

cell is given by cytotoxic T cells, as they carry receptors which recognize

intra-cellular antigens, present on their cell membrane.


6

Specific immunity is either acquired naturally or it can be induced via

vaccines. Vaccines play a vital role as a primary exposure, so that specific

antibodies are produced as soon as the antigen is encountered (Nossal

2003). 80-90% of those who are immunized with vaccine develop immunity

(Lambert et al. 2005).

The immune system is controlled by T helper and regulatory T cells. In case

of Human Immunodeficiency virus (HIV), it is seen that the T helper (CD4)

cells are destroyed by the virus and the patients succumb to some not so

harmful infections (Kanabus et al. 2008). Other infections which points

towards the importance for T helper (CD4) cell investigation are thrush in

oesophagus, Pneumocystis carinii, cytomegalovirus in retina and TB (Geha

and Rosen 2008). The potential of the immune system recognizing its body’s

own tissues and foreign molecules is attributed to the specificity of the

regulatory T cells. The immune response towards the body’s own tissues is

either down-regulated or completely switched off. Any failure in this

mechanism will lead to autoimmune diseases such as rheumatoid arthritis,

multiple sclerosis, type 1 diabetes mellitus (chapel et al., 2006).

Immunologic down regulation functions to put a stop to the immune

response. For quite some time down regulation mechanism was ignored by

immunologists (Beissert et al., 2006). During the past decade, there has

been great understanding of the underlying principles and mechanisms of

negative regulation. One of the early hypothesis which was suggested by

Gerhon and co-workers during 1972, was that a subset of T cell may show

negative regulation or supress the immune response, and these cells were

termed as “suppressor cells”(Gerhon et al., 1972). Since then, many


7

experiments have been conducted to study the presence of suppressor T

cells (Fisher and Kripke, 1978; Elmets et al., 1983).

2. Regulatory T cells:

The ‘Holy grail’ of immunology is tolerance, which can be classified into

central and peripheral tolerance. Central tolerance mainly deals with

immature lymphocytes differentiating in the primary lymphoid, whereas

peripheral tolerance deals with matured lymphocytes after they have moved

out of lymphoid organs and which are circulating in the periphery.

Regulatory T cells (Tregs) are believed to be accountable for sustaining

peripheral tolerance. These Tregs can be defined as “a T cell population,

which can functionally supress an immune response by impeding activities of

other immune cells” (Zou 2006).

2.1 History of Regulatory T cells (Tregs):

Gershon et al. During the early 1970’s described Tregs, by referring to them

as suppressive T cells (Gershon et al., 1972). Sehon and group, three years

later indicated negatively regulated immunity towards tumour and also

showed that Tregs encouraged the growth of tumour in mice (sehon et al.,

1975). Five years later, North et al, published experimental findings, in which

it was evident that CD4+ CD25+ T cells from mice with tumour, showed

inhibition for tumour rejection, which indicated the existence of suppressor T

cells.

Although there were pioneering studies made on regulatory T cells, there

was a wide uncertainty about the very existence of these population in

immunology and had left regulatory T cells and it suppressive capacity as a

grey area in immunology for many years.


8

In the year 1995, Sakaguchi and co-workers explained about naturally

occurring population of CD4+ CD25+ T cells which accounts for 5-10% of all

the T Helper (TH) cells present in mice. The CD4+ CD25+ T cells which were

Described in mice showed compelling evidence of regulatory functions

(Sakaguchi et al., 1995; Sakaguchi et al., 2001).

When CD4+ CD25- T cells were injected into mice, it induced colitis, the mice

used in the experiments was immunodeficient. Whereas when it was injected

with CD4+ CD25+ along with CD4+ CD25- T cells showed no bowel

inflammation. Also murine CD4+ CD25+ T cells down regulated the

proliferation of CD8+ T cells or CD4+ CD25- T cells in culture, which proved

that they had potent regulatory functions both in vivo and in vitro (Picciricco

and Shevach, 2001). A similar group of T cells, which were phenotypically

comparable with the above mentioned T cells in mice were identified in

human by the experiments conducted by Dieckmann et al. and Jonuleit et

al., 2001. These revolutionary studies established the very foundation of the

role of regulatory T cells in cancer immunology (Zou 2006).

Recent studies also show that Transcription factor fork head box P3 (FOXP3)

is one of the key intracellular marker and is involved in central

developmental and is a functional regulator for CD4+ CD25+ Regulatory T

cells ( Hori et al., 2003; Fotenot et al., 2003; khattri et al., 2003). Since

then, the concept of Regulatory T cells was revitalised and developed very

swiftly. In the past few years, many phenotypically diverse regulatory T cells

have been described. The classic regulatory T cells are the thymus derived

CD4+ CD25+ FOXP3+ T cells (Zou 2006).


9

2.2 Natural Tregs (nTreg) and Adaptive Tregs (iTreg):

It is well known that Foxp3 is responsible for the functional establishment of

regulatory T cell lineage. Previous studies have demonstrated and stated

that nTreg are generated in the thymus with the aid of MHC class II-

dependent T cell receptor (TCR) interfaces which results in high avidity

selection (M Lafaille and J Lafaille 2009). Although other selection

mechanisms may take place (van santen et al., 2004). Foxp3+ Tregs can also

be generated outside the thymus under various conditions and sub

immunogenic antigen presentation. The Foxp3+ iTregs cell lineage is derived

from naïve conventional CD4+ T cells.

Fig 1. 2: Thymus and peripheral generation of Foxp3+ regulatory

T cells. The above figure shows the generation of Tregs from the

thymus and maturation in the periphery. It also displays the generation

of nTreg and iTreg.


10

2.3 Properties of regulatory T cells:

2.4 Key markers of regulatory T cells:

There is an extensive need for dependable markers for identification and also

enriching viable Tregs. Tregs in humans as well as mice are heterogeneous

both phenotypically and functionally as well. It is well evident now, that

there exists a panel of co-stimulatory or co-inhibitory molecules, which are

expressed by Tregs (Chen and Oppenheim, 2011).

2.4.1 CD25:

CD25 is an IL-2 receptor. Regulatory T cells cannot make up IL-2 and

hence they are dependent on IL-2 secreting conventional T cells (Tconv).

Therefore, by expressing high level of CD25, they divert IL-2 from Tconv and

suppress the proliferation of conventional T cells. Co-expression of CD4 and

CD25 in human PBMC is also seen to be defined as functional regulatory T

Property Development Phenotype Other

Associated

Markers

Suppression Target

cells

CD28

Associati

on

In vivo

Role

In vitro

expansion

Natural

Tregs

(nTregs)

Thymus CD4+CD25+

CD127 low

CTLA-

4+GITR+

Foxp3+

Contact-,

Granzyme-B

dependent,

makes TGF

beta

APC &

Effector

T Cells

Thymic

developm

ent and

maintena

nce in

periphery

Suppressio

n of auto

reactive T

cells

TCR/CD28

stimulation

and IL-2

Induced

Tregs

(iTregs)-Tr1

Peripheral

(MALT)

CD4+ CD25- CD45RBlow

Foxp3-

IL-10

mediated

Effector

T Cells

Not

necessary

Mucosal

immunity&

inflammato

ry response

CD3, IL-10,

Retinoic

Acid

Induced

Tregs

(iTregs)-Tr2

Peripheral

(MALT)

CD4+CD25+

from CD25-

precursor

CD25low-

variable-

CD45RBlow

Foxp3+

TGF beta

mediated

? Not

necessary

Mucosal

immunity&

inflammato

ry response

CD3, TGF

beta

Table 1.1: Properties of Natural and Adaptive Regulatory T cells. The above table represents

properties of Natural and adaptive Tregs (Tr1 and Tr2).


11

cell. CD25 is expressed around 30% of total CD4+ T cells in Peripheral blood

(which also includes conventional T cells (Tconv)). Out of 30% of CD4+ cells,

only 1-2% of the cells also express CD25 (CD25high) and those are seen to

be enriched with stable suppressive functions and high expression of FoxP3+.

50% of the total FoxP3+ human regulatory T cells are expressed within

CD25high, also small population of CD25- expresses FoxP3. Thus, identifying

Tregs only based on CD25high will skip most of the CD4+ FoxP3+ regulatory T

cells. It is also seen that activated CD4+ Tconv, can also express high CD25

(Chen and Oppenheim, 2011).

2.4.2 CD127:

CD127 is an IL-7R alpha receptor. CD127low/- along with CD25high is

extensively used to characterize and define human regulatory T cells (Liu W

et al., 2006; Seddiki N et al., 2006).

It is seen that 40% of CD127low is FoxP3+ (Shevach EM, 2006), and

FoxP3lowCD45RO+ non-suppressive T cells present in normal humans are also

seen to express CD127low. CD127 is down regulated during activation of

Tconv, therefore the use of additional markers along with CD127low/- to

define regulatory T cells is needed. A Low levels of CD127 is not an inherent

characteristic of regulatory T cells, because it is well evident that Tregs

respond to IL-7 (Mazzucchelli R et al., 2008).

2.4.3 FoxP3:

FoxP3, at present is the most specific marker used to identify

functional regulatory T cells. FoxP3, which encodes Scurfin, is a member of

family of forkhead/winged helix transcriptional repressors.

FoxP3 has crucial roles in CD4+CD25+regulatory T cell biology. First, it

controls development of natural regulatory T cells (Hori S et al., 2003;

Fontenot JD et al., 2003). Second, it is evident that natural regulatory T cells


12

expressing FoxP3, have dominant self-tolerance (Hori S et al., 2003). Third,

FoxP3 expression associates with activation of T cells (Morgan ME, 2005).

It is important to note the limitations of FoxP3. Firstly, FoxP3 is an

intracellular nuclear protein, which limits its use for isolating regulatory T

cells. Second, its exact role in regulatory T cell biology is controversial,

especially in humans (Huanfa Yi et al., 2006).

2.5 Other potential markers:

There are a wide range of potential markers for regulatory T cells.

Human Mouse

Cell surface

receptors

CD39,CD27, CD45ROhigh,

CD58, CD83, CD95high, HLA-

DRlow, a4β1, a4β7, CCR4,

CCR8, granzyme A

CD11b/CD18high,

CD103(aE), CCR2, CCR6,

CCR7, CXCR3, LAG-3,

Nrp1, PD-1, TLR4, TLR7,

galectin-1, Ly6A/E high,

TGFβR1, Granzyme B,

CD122high, CD132, CD28,

CD38high, CD44high, CD62L,

CD54, CD71, CD127, GITR,

TLR5, TLR8, TGF-β, CD25

intermediate-high, CD30,

CD45RBlow, CD5high, CD69,

CD150low, TNFRII, OX40, 4-

IBB, TRANCER

Intracellular

molecules

CTLA4+, FoxP3+

Table 1.2: Phenotypes of natural regulatory T cells. Adapted and modified from

`Huanfa Yi et al., 2006.


13

1.3. Prostate Cancer and Tregs:

One of the most common cancer known to man is prostate cancer. Cancers

are commonly named after the part where they have formed. In this case,

prostate is a minor gland which is found only in men. And the cancer that

affects this gland is named as prostate cancer. It is commonly found to affect

men aged over 50, and rarely observed in younger men. In the UK, each

year there are around 37,000 men who are diagnosed with prostate cancer.

Stages of Prostate Cancer:

1. Early prostate cancer (localized): At this stage the cancer is seen to be

found only in prostate and will not be spread to surrounding tissues.

2. Locally advanced prostate cancer: the cancer is observed to have spread

to the tissues surrounding the prostate gland.

3. Advanced prostate cancer (metastatic): at this stage the cancer advances

to spread beyond the prostate gland.

Early stages of prostate cancer can be cured surgically, more advanced

stages may result in cancer becoming metastatic, which makes it a deadly

disease. Recent research indicates that prostate cancer is one of the

promising subject for immunotherapy (Flamminger et al., 2012). More

knowledge about immunological processes at the microenvironment level

can be obtained by studying the tumour infiltrating lymphocytes (Yu P and

Fu YX, 2006).

A vast range of anti-tumour immune responses are suppressed by regulatory

T cells (Tregs). It has been observed that a high number of Tregs exist in

peripheral blood and also in the microenvironment of different tumour bodies

(Beyer et al., 2006). The presence of high number of Tregs in the cancer


14

environment may result in antagonistic effect (Wilke et al., 2010). But

depending on the type of tumour, Tregs may be disadvantageous or

beneficial to the patient’s life.

The Presence of Tregs in prostate cancers has previously been described by

several investigators. It has been reported that the number of Tregs in the

peripheral blood of early stage prostate cancer is seen to be higher than that

of healthy donors. It has also been reported that these numbers of Tregs are

present more in the surrounding areas around the prostate cancer gland

than in benign prostate gland (Miller et al., 2006; Ebelt et al., 2009;

Flamminger et al., 2012).

Prostate cancer are seen to have the presence of prostate-infiltrating

lymphocytes (PIC). CD4+ CD25+ regulatory T cells, regulate immunogenic

tolerance to self-antigens (Sakaguchi S et al., 1995; Sakaguchi S et al.,

2001; Valdman A et al., 2010). Damage to anti-tumour immunity is because

of the increased population of Tregs. Tregs tend to stop the immunogenic

responses to tumour by antagonizing the CD8- and CD4- positive cells (Chen

ML et al., 2005; Nishikawa H et al., 2005) by cell to cell contact and by

producing cytokines IL-10 or TGF-β (Curiel TJ 2007). FoxP3, the transcription

factor is believed to play an important role in CD4+ CD25+ regulatory T-Cell

function and this is also a very key marker for Tregs (Hori S et al., 2003).

1.3.1 Source of Regulatory T cells in the Tumour:

Functional natural regulatory T cells (nTreg) and induced regulatory T cells

(iTreg) are found in tumour microenvironment. Tregs cells differentiate in

thymus and tumour associated Tregs tend to express FoxP3 and mRNA. It

may be possible that these Tregs cells move to tumour site for instance,

from thymus, bone marrow etc. to tumour microenvironment which is


15

influenced by CC-chemokine ligand 22 (CCL22) produced in tumour

microenvironment.

Regulatory T cells can also be induced and differentiated in the periphery of

tumour microenvironment via factors such as vascular endothelial growth

factor (VEGF), transforming growth factor- β (TGF-β) and IL-10.

Fig 1.3: Movement of CD4+ CD25+ FoxP3+

Regulatory T cells towards tumour

microenvironment under the influence of

CCL22. Image adapted and modified from Zou,

2006

Fig 1.4: Differentiation of regulatory T cells in

tumour microenvironment via IL-10, TGF- β and

VEGF. Image adapted and modified from Zou, 2006.


16

Regulatory T cells can also be present in tumour microenvironment by

conversion from CD4+CD25- T cells via TGF- β which is present abundantly

in tumour microenvironment.

1.3.2 Mechanism of action of Foxp3+ Regulatory T cells:

Many studies have suggested that Treg cells facilitate suppression by

inhibition of the induction of IL-2 mRNA in responder Foxp3- T cells (Ethan

M. Shevach, 2009). Treg cells are seen to express high affinity IL-2R-CD25,

CD122, CD132 and IL-2.

Treg cells can discharge suppressor cytokines which can directly impede the

function of responder T cells and also myeloid cells. Tregs are known to

express CD25High, the IL-2 receptor and carry the capability to compete

with effector T cells for the IL-2, which results in cytokine mediated deficit of

effector cells and Bim mediated apoptosis. Activated Foxp3+Tregs can

function as cytotoxic cells and can kill the effector cells directly like CD8+

cytotoxic cells. These activated Foxp3+Tregs can express known (E.g.

galectin-1) or any other unknown proteins on their surface which can

interact with receptors on the effector T cells, resulting in cell cycle arrest.

Fig 1.5: conversion of CD4+CD25- T cells to CD4+CD25+

Regulatory T cells under the influence of TGF- β. Image adapted

and modified from Zou, 2006.


17

All these contrivances can be used by Foxp3+Tregs to impede the function of

APC or other cells of immune system (Shevach, 2009).

1.3.3 Targeting Regulatory T cells for Immunotherapy:

The current clinical effectiveness of current methods involved in tumour

immunotherapy and vaccination is not adequate. One of the important

reason is that the complex suppressive mechanisms pre-dominate in

patients suffering with advanced stages of cancer (Dunn G P et al., 2004;

Zou W, 2005). The concepts involving reversing immunosuppression in

Fig 1.6: Mechanism of action of Foxp3+ regulatory T cells. 1. Foxp3+Tregs secreting

suppressive cytokines, which cause Apoptosis, Cell cycle arrest, Co-stimulation Blockade and

Contact dependent inhibition. 2. Foxp3+Tregs releasing various Granzyme, which result in

apoptosis. 3. Foxp3+Tregs, process of metabolic disruption. 4. Dendritic cells being targeted

by Foxp3+Tregs by other mechanisms. Image adapted from Ethan and Todd, Nature Reviews,

Regulatory T cells, 2011.


18

cancer have an advantage in therapeutic line. New studies have emerged

targeting the suppressive molecules and regulatory T cells, pointing towards

ways to successful application in tumour immunotherapy (Fig 1.7).

It is clear that tumours have outstanding ability to escape then tumour

immunity by means of various mechanisms, and thus they defeat the

traditional tumour immunotherapy. It is important to keep in mind that Treg

targeting may be an attractive way in treating human tumours. It is

therefore probable that multiple therapeutic intervention is required to attain

an effective, stable and reliable clinical efficacy (Zou 2006).

Fig 1.7: Targeting suppressive mechanisms including Tregs for therapeutic

applications. Patients with cancer are subjected to traditional therapy after the clinical

diagnosis. Conventional immunotherapy involves supplementing the immune system and to

provide necessary immune elements. Novel immunotherapeutic methods include targeting

the suppressive mechanism of Tregs, suppressive molecules and dysfunctional APCs.

Combinatorial therapy can be done to obtain an effective, reliable and stable clinical

effectiveness. Image adapted from Zou, 2006.


19

4. Rationale of the Project:

Recently, a wide range of regulatory T cell subsets have been identified

based on their surface markers such as CD4+ Treg cells, CD8+ Treg cells,

veto CD8+ cells, γδ T cells, Natural killer cells (NK 1.1+ CD4- CD8-) and many

others (Mills KH et al., 2004). Collective evidence also points towards the

fact that natural regulatory T cells (CD4+CD25+ Treg cells) are actively

involved in down regulation of autoimmune responses and to maintain

immune homeostasis (Akbari O et al., 2003; Almeida AR et al., 2005).

Absence of clear molecular characterisation of the regulatory T cells has

hindered researchers to define their biological processes and their role in the

immune system. This study will enable us to isolate a distinct population of

Tregs cells (CD4+CD25high CD127low/- Tregs) and functionally characterize

them based on their immune suppressive ability.

Tregs only account for about 0.5-3% in peripheral blood, hence there is a

need for reliable and stable expansion protocols. The design for efficient and

safer protocols for selective expansion of Tregs is regarded fundamental for

its future clinical applications.


20

Chapter 2. Materials:

2.1 General laboratory Materials:

Material Company

5mL, 10mL and 25mL pipettes Sarsted, UK

20mL universals Sarsted, UK

6 well plate Sarsted, UK

96 well round bottom plate Sarsted, UK

Centrifuge Tubes (15mL and 50mL) Sarsted, UK

FACS Tubes Elkay, UK

1.2mL and 0.5 mL eppendorf tubes Sarsted, UK

Pipette tips (<1mL) Sarsted, UK

Haemocytometer Weber

0.5-10µL tips Sartorius, UK

20-100µL tips Sartorius, UK

200-1000µL tips Sartorius, UK

PRESEPT AST, J&J

10mL Syringe BD Plastipak

40µL Cell strainer BD Falcon

LS & MS MACS columns Miltenyi Biotec

Ultima gold Packard

2.2 Electrical Equipment’s:

Equipment Manufacturer

MoFlo™ XDP (FACS) Beckman Coulter, Inc.

Gallios Flow Cytometer Beckman Coulter, Inc.

Cell Harvester, Filter mate Harvester Packard

Micro plate -Scintillation counter

Refrigerated Centrifuge Sanyo

-800 C Freezer Ultima II, reveo


21

Class II safety Cabinets Walker

370 C Incubator Forma Scientific

96 well plate Harvester Packard

Light Microscope Nikon

Nikon Microscope camera Nikon

Micro centrifuge, Microcentraur MSE

2.3 Reagents for Culture Media:

Reagent Company

1640 RPMI Lonza

DPBS PAA & Lonza

C.T.L

Supplements added to Culture Media

HEPES Buffer Lonza

Foetal Calf Serum PerBio (Thermal Fisher)

PenStrip Biowittaker

Cell culture reagents:

DMSO Sigma

Trypan Blue Sigma

IL-2 [105 U/mL] R&D Systems

2.4 Materials for Flow Cytometer:

2.4.1 Buffers:

FACS Buffer

1 X PBS (10 tablets/L)

0.1% BSA (1g/L)

0.02% Sodium Azide (0.2g/L)


22

2.4.2 Antibodies:

Antibody Company

APC anti-Human CD3 BioLegend

(#300311, 300312)

PE anti-Human CD25 BioLegend

(#302605, 302606)

PerCP CY 5.5 anti-Human CD4 BD Biosciences

(#51-9006621)

Alexa Flour 647 Anti-Human CD127 BD Biosciences

(#51-9006622)

APC-e Fluor 780 anti-Human CD127 eBioscience

(#47-1278)

FITC anti-Human CD4 eBioscience

(#11-0049)

2.5 Materials for Proliferation & Suppression assay’s:

Anti-Human CD3 BD Pharmingen

(#555726)

Anti-Human CD28 BD Pharmingen

(#550368)

[3H]-Thymidine Amersham

2.6 Kits:

Human Regulatory T cell Sorting Kit BD Pharmingen

(#560753)

CD4+ Cell isolation kit (Human) Miltenyi Biotec

(#130-096-533)

CD4+ Cell isolation kit (Human) II Miltenyi Biotec

(#130-091-155)

Treg Expansion Kit (Human) Miltenyi Biotec

(#130-095-353)


23

Chapter 3. Methods:

Overview of the experimental design followed in this study:

PBMC Processing

(Leucosep, Greiner Bio-one)

Anti-CD3 and Anti-CD28 stimulation and proliferation studies

([3H]-thymidine assay)

Enrichment of CD4+ T cells

(CD4+ isolation Kit Human, Miltenyi Biotec)

Antibody compensation studies for flow cytometer analysis

(Beckman Coulter Gallios™ Flow Cytometer)

CD4+CD25+ CD127low/- (Tregs) & CD4+CD25-CD127+ (Tconv) cell

sorting

(Beckman Coulter MoFlo™ XDP)

Treg expansion

(Treg Expansion Kit, Human, Miltenyi Biotech)

Suppression assays

([3H]-thymidine assay)

The above flow chart represents overview of the experimental design

followed in this project.


24

3.1 PBMC processing:

Peripheral Blood mononuclear cells (PBMC) are separated from Human whole

blood by using Leucosep™ (greiner bio-one).

Preparation of Leucosep Tubes:

The Leucosep separation medium is kept at room temperature. 15mL of

Leucosep is added to the Leucosep tube. The tubes were centrifuged for 30

seconds at 1000xg and room temperature (RT). The separation medium falls

below the porous barrier after centrifugation. The tubes will be ready for

sample separation.

Procedure:

The anti-coagulated blood sample (diluted with salt solution (PBS) in 1:2

ratio) was poured to the prepared Leucosep tube carefully. It was

centrifuged for 10 min at 1000xg and at room temperature or at 15 min at

800xg at RT, the brakes were kept off during this step. After centrifugation,

layers were formed within the tube as shown in figure 8. The enriched cell

fraction (b) was harvested very carefully using a Pasteur pipette. The

enriched cell fraction (PBMC) was washed with 10mL of PBS and centrifuged

for 10 min at 300xg. The washing step was repeated twice and the cell pellet

was resuspended in growth media.

Fig 3.1: PBMC Processing using Leucosep™. Various steps involved in PBMC processing are

shown above. The sample is being poured into the tube carefully, and is subjected to centrifugation.

There are different layers formed after centrifugation [a) Plasma, b) enriched fraction (PBMC), c)

separation medium, d)porous barrier, e)separation medium, f)pellet]. The PBMC is harvested using a

Pasteur pipette. Figure adapted from Leucosep™ Instruction manual. www.gbo/bioscience


25

3.2 Proliferation assay:

Proliferation assay was performed in a 96 well round bottom plate. The

PBMCs were thawed from frozen state with CTL Thaw. The cells were

counted and the volume was adjusted so that 100µL/ well gives 1x105 cells.

Anti-CD3 and anti-CD28 were taken at different concentrations ranging from

0.5µg/mL- 4µg/mL. The plate was set up as shown in fig 3.2, with different

titrations of antibodies. The final volume in each well was 200µL. The plate

was kept in 37oC incubator for 3 days. 20µL media containing [3H]-

thymidine was added to all the wells 18 hours prior to cell harvesting.

Fig 3.2: Proliferation plate setup. The Proliferation assay was done in 96 well round bottom

plate in the arrangement as shown above with different concentrations of anti-CD3 and anti-

CD28 antibodies for stimulation.


26

3.3 CD4+ T cell isolation:

CD4+ T cells from human PBMCs was isolated using Miltenyi Biotec’s CD4+ T

cell isolation kit (#130-096-533). This kit isolates human CD4+ T cells using

negative selection.

Buffer preparation: Prepared solution containing PBS (pH 7.2), 0.5% BSA

(bovine serum albumin) and 2mM EDTA. This will be the MACS buffer to be

used for isolating CD4+ T cells.

Magnetic labelling: Thawed PBMCs were counted. The cell suspension was

centrifuged at 350xg for 10 min, and the supernatant was pipetted off very

carefully. The cell pellet was resuspended in 40µL of MACS buffer per 107

total cells. 10µL of Biotin-antibody cocktail was added per 107 total cells. It

was mixed well and incubated for 10 minutes at 4-8oC. 30µL of buffer was

added per 107 total cells followed by 20 µL of anti-biotin micro beads per 107

total cells. It was mixed well again and incubated for 15 minutes at 4-8oC.

The cells were washed by adding buffer 10-20x labelling volumes and

centrifuged for 10 min at 300xg. The supernatant was removed completely.

Cells were resuspended in 500µL of buffer for up to 108cells. The cells were

then ready for magnetic separation.

Magnetic separation: appropriate column was placed in magnetic field of

suitable MACS separator (MS column: upto 107 cells; LS column: upto 108

cells). The column was prepared by rinsing with appropriate amount of

buffer (MS: 500µL; LS: 3mL). The cell suspension was applied onto the

column. The cells were allowed to pass through the column and the effluent

fraction was collected which is the enriched CD4+ T cells. The column was

washed with appropriate amount of buffer (MS: 3x 500µL; LS 3x 3mL)

thrice. The entire effluent was collected along with enriched CD4+ T cells

fraction.


27

3.4 antibody compensation studies:

Compensation was done for the panels used in the project to avoid false

signals occurring due to spectral overlap of the fluorescent dyes when used

in multi-colour staining panels. First, 6 FACS tubes were labelled and

prepared as follows:

Tube1: unstained compensation beads

Tube2: PE compensation beads-20µL

Tube3: PerCP-Cy 5.5 compensation beads-20 µL

Tube4: Alexa Fluor® 647 compensation beads-20 µL

Tube5: cell only (PBMCs)-2x105 cells

Tube6: cells+ antibody cocktail- 2x105 cells+ Treg sorting kit.

After preparing the tubes, they were incubated at 18-22oC for 30 minutes,

keeping them protected from light. After the incubation wash buffer was

added to fill the tube and centrifuged at 250xg for 15 minutes. The

supernatant was discarded and the pellet was resuspended in 300µL of

Isoton. It was then subjected to flow cytometer to compensate and to set up

the protocols.

3.5 CD4+ T cell Proliferation assay:

Enriched CD4+ T cells were incubated at 37oC for overnight. The cells were

counted before setting up the proliferation assay. The plate setup is similar

to the Method followed for PBMCs proliferation assay (3.2). Anti-CD3 and

anti-CD28 antibodies were used at different concentrations. 1x105 cells per

well were added in a 96 well round bottom plate. The final total volume in

each well was adjusted to 200µL. The plate was incubated at 37oC incubator

for 3 days. 20µL media containing [3H]-thymidine was added to each well 18

hours prior to cell harvest.

Note: 50 U/mL IL-2 was added to the cells to get maximum

proliferation.


28

3.6 CD4+CD25highCD127low/- (Tregs) & CD4+CD25-CD127+ (Tconv) cell

sorting:

The cell sample to be sorted, PBMCs or enriched CD4+ T cells were counted.

The cells were centrifuged at 350xg for 10 minutes and the supernatant was

discarded carefully. The cells were resuspended in 200-500µL (depending

upon the number of cells) of PBS+ 1% human serum. The antibody cocktail

(Human regulatory T cell sorting cocktail) was added (240µL for 120x106

cells; 100µL for 50x106 cells). Tube was then incubated at 18-22oC for 30

minutes, protected from light. After incubation, enough wash buffer (1xPBS

+ 1% human AB serum) was added to fill the tube and is centrifuged at

250xg for 15 minutes. The supernatant was aspirated out completely and

the sample is resuspended in wash buffer about 100-500µL.

The reception tubes were filled with PBS +1% human AB serum+ 1%

antibiotics (PenStrip). The sorting was done using Beckman Coulter’s

MoFlo™ XDP (FACS). After the sorting, the cells were centrifuged at 250xg

for 15-20 minutes. It was then resuspended in growth medium.

3.7 Treg expansion:

The sorted Tregs (CD4+CD25highCD127low/-) were expanded using Treg

expansion kit (Miltenyi Biotec).

CD3/CD28 MACSiBead Particle Preparation: resuspend 200µL of CD3/CD28

MACSiBead Particles, and transferred it to a suitable tube. 300-600µL of

culture medium was added and was centrifuged at 300xg for 5 minutes. The

supernatant was removed completely. The pellet (CD3/CD28 MACSiBead

Particles) was resuspended in 200µL of culture medium (concentration of

2x107 beads/mL).


29

Cell preparation and expansion: The isolated Tregs (CD4+CD25highCD127low/-)

were counted. 1x105 cell per well are needed. The Cells were centrifuged at

350xg for 10 minutes and the supernatant was completely removed. The

cells were resuspended at a concentration of 1x106 cells/mL of medium

which included 500 U/mL IL-2. 100µL was pipetted in a well of 96 well round

bottom plate (day 0). 20µL of prepared CD3/CD28 MACSiBead Particles were

added to each well. On day 1, 100µL of media was added which contained

500 U/mL of IL-2. On day 3-5, depending upon the media usage, the cells

were split or 100 µL was aspirated and 100 µL of media including 500 U/mL

of IL-2 was added. The cells were undisturbed and kept in 37oC incubator.

Removal of MACSiBead particles: After 14 days of culture, the cells were

harvested and transferred to tube and washed with buffer. The cells were

resuspended in buffer at density of 2x107 cells per 1mL and vortexed

properly. It was then placed in a magnetic field, for 2-4 minutes. Keeping the

tube in magnetic field, supernatant was collected which will contain Tregs

and placed in a new tube. The cells was washed in buffer at 300xg for 10

minutes, and the pellet was resuspended in growth media and was ready to

be used as required.

3.8 in vitro Treg suppression assay:

This experiment was setup in 96 well round bottom plate in a total volume of

200µl. Fig 3.3, shows the plate layout. The Tregs and Tconv were sorted

from PBMC. Tregs and Tconv were counted and adjusted in T cell media

(growth media) to 5x104/mL and 1x105/mL respectively (Tregs: Tconv 1:2).

Growth media was added to wells 1-11. 100µl of Tregs were added to well

12. They were mixed properly and 50µl was titrated into well 11 which gave

us two fold dilution. This step was repeated in the successive well, 50µl

every time until well 6 with no Tregs (to determine the maximum

proliferation of Tconv). 50µl of Tconv were added to all the wells. 0.5µg/mL

of anti-CD3 and 1 µg/mL of anti-CD28 was added to all the wells, which was


30

adjusted with the media. The plate was incubated at 37OC with 5% CO2 for 3

days. The plate was pulsed with 20µL media containing [3H]-thymidine, 18

hours prior to cell harvest. The cells were harvested and counts per minute

(CPM) was determined.

Fig 3.3: Plate diagram for suppression assay. (a) 96 well plate setup for suppression

assay. (b) Procedure for titration of Tregs. Image adapted from Collison and Vignalli,

2011


31

Chapter 4. Results:

4.1 PBMC proliferation assay:

PBMC proliferation assay was done to verify that there was clonal

proliferation occurring when stimulated by anti-CD3 and anti-CD28

antibodies. PBMC proliferation assay was also a major pre-requisite for

assessing the functional capacity of CD4+T cells and for proliferation. The

maximum proliferation of PBMCs were observed at the concentrations of

0.5µg/mL of anti-CD3 and 4 µg/mL of anti-CD28 antibodies.

Co

ntr

ol

0.5

: 1

0.5

: 2

0.5

: 3

0.5

: 4

1:

11:

2

1:

3

1:

4

2:

1

2:

2

2:

3

2:

4

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

a n t i-C D 3 : a n t i-C D 2 8 µ g /m l

CP

M

Fig 4.1: PBMC proliferation assay via anti-CD3 and anti-CD28

stimulation (n=3). PBMCs at the concentration of 1x105 cells/well were

stimulated with anti-CD3 and anti-CD28 at different concentrations, without

IL-2 in a 96 well round bottom plate for 3 days at 370C. 20µL media

containing [3H]-thymidine was pulsed to all the wells, 17 hours prior to

quantification. All the experiments were done in triplicate wells. Maximum

proliferation was observed at concentrations of 0.5 µg/mL of anti-CD3 and 4

µg/mL of anti-CD28. The error bars represent Standard error of the mean

(SEM).


32

4.2 Flow cytometer compensation studies:

One important consideration when carrying out using multi-colour

fluorescence studies is spectral overlap. During experiments with these

multiple colours, there is a chance of the emission profiles will overlap with

each other, which will make the fluorescent emission measurement very

difficult. Hence, to avoid this, fluorescence compensation is applied during

the data analysis. Required adjustments are done to minimise the spectral

overlap. The below results are obtained after the compensation was

performed with compensation beads, and various populations are observed

clearly.

Fig 4.2 (a): Antibody compensation studies for flow cytometer.

PBMCs were stained with Human regulatory T cells cocktail (CD4-PerCP Cy

5.5, CD25 PE, and CD127 AF-647) and inherent overlap of emission spectra


33

from each antibody (PerCP Cy 5.5, PE, AF 647) was measured and

compensated successfully.

Fig 4.2 (b): Flow cytometer compensation studies. PBMCs stained with

CD4-FITC, CD25-PE and CD127 AF-780. The above figure represents the

compensated analysis and no spectral overlap is seen when used with

multiple flurochromes.


34

4.3 CD4+ Isolation:

The current study in this project revolves around CD4+ Tregs. The important

studies included CD4+ T cell proliferation, Functional and phonotypical

studies on CD4+ T cells. It was thus important to isolate CD4+ T cells from

PBMCs. This was achieved by using CD4+ T cells isolation kit (human), in

order to verify that the isolated cells are CD4+ T cells, flow cytometer

analysis was performed for cells before isolation (PBMCs) and enriched CD4+

T cells. The cells were stained with two different panels as shown in below

results. The percentage of enrichment was observed and calculated.

Fig 4.3 (a): Sample before CD4+ T cell isolation. Flow cytometer

analysis was done by staining the cells with CD4 FITC and CD3 APC. A

histogram was plotted and two distinct populations can be observed.

Analysis was done using Kaluza 1.2 flow analysis software.


35

Fig 4.3 (b): Enriched CD4+ T cells. CD4+ T cell enrichment was done as

per the protocol using Miltenyi Biotec’s CD4+ T cell isolation kit (#130-096-

533). The above histogram shows the purity of the enriched CD4+ T cells

(stained with CD3 APC and CD4 FITC) that were rested for 2-3 hours at 370C

after isolation.

Fig 4.3 (c): Sample before enrichment.

The sample was stained with Human Treg

sorting cocktail (CD4 PerCP Cy 5.5, CD25 PE,

and CD127 AF 647). The histogram shows

two distinct populations.


36

Fig 4.3 (d): Enriched CD4+ T cells. The above histogram shows the

purity of the enriched CD4+ T cells (stained with CD4 PerCP Cy 5.5, CD25

PE, and CD127 AF 647) that were rested for 2-3 hours at 370C after

isolation.


37

4.4 CD4+ Proliferation assay:

After enriching CD4+T cells from PBMCs, proliferation assay were performed

to observe the clonal proliferation of CD4+ T cells by stimulating with

different concentrations of anti-CD3 and anti-CD28 antibodies. In further

studies, our plan was to assess the immunosuppressive capacity of CD4+

Tregs, it was therefore very important to observe the maximum proliferation

of CD4+ T cells when stimulated by various concentrations of anti-CD3 and

anti-CD28 antibodies. The maximum proliferation of CD4+T cells was seen at

concentrations of anti-CD3 0.5µg/mL and anti-CD28 1µg/mL.

Co

ntr

ol

0.5

: 0.5

0.5

: 1

0.5

: 2

1:

0.5

1:

1

1:

2

0

1 0 0 0

2 0 0 0

3 0 0 0

CP

M

a n t i-C D 3 :a n ti-C D 2 8 µ g /m L

*

Fig 4.4: CD4+ Proliferation assay with anti-CD3 and anti-CD28

antibody stimulation (n=3). Enriched CD4+ T cells at the concentration of

1x105 cells/well were stimulated with anti-CD3 and anti-CD28 at different

concentrations, with 50 U/mL of IL-2 in a 96 well round bottom plate for 3

days at 370C. 20µL media containing [3H]-thymidine was pulsed to all the


38

wells, 17 hours prior to quantification using scintillation counter. All the

experiments were done in triplicate wells. Maximum proliferation was

observed at concentrations of 0.5 µg/mL of anti-CD3 and 1 µg/mL of anti-

CD28. Unpaired t test was performed and was observed that concentration

of 0.5 µg/mL of anti-CD3 and 1 µg/mL of anti-CD28 was statistically

significant. The error bars represent Standard error of the mean (SEM).


39

4.5 Treg Expansion:

The percentage of Tregs in PBMCs was very low (less than 1%), hence we

enriched CD4+T cells, which either had CD4+CD25highCD127low/- Tregs or

CD4+CD25lowCD127high Tconv. We sorted Tregs and Tconv and then expanded

the Tregs using the kit which provided us with high number of Tregs which

were then used for immunosuppression assay. The below figure shows the

beads attached to Tregs which initiated the selective clonal expansion.

Fig 4.5: Regulatory T cell expansion. Treg expansion was done using

Miltenyi Biotech’s Human Treg expansion kit. The above image was captured

20x/0.25 magnification on Day 8, clearly showing micro beads attached to

Treg cell surface.

Micro-beads

CD4+ CD25High CD127 low/-

Regulatory T cell


40

Chapter 5. Conclusions and Future work:

It is now well evident that CD4+ Tregs can be classified into natural Tregs

and adaptive Tregs based on their differentiation and migration patterns (van

santen et al.., 2004; M Lafaille and J Lafaille 2009). Human natural CD4+

express higher levels of CD25 and FoxP3 (Hori S et al., 2003; Chen and

Oppenheim, 2011). The reason to CD127low/- as a marker is because CD127

low/- corresponds to high expression of FoxP3 (W Liu et al., 2006; JvGCRC)

and also to justify our aim to see the immunosuppressive ability of Tregs

(CD4+CD25highCD127low/- Tregs). Previous studies (J Yokokawa et al., 2008)

have studied the functionality of only CD4+CD25high FoxP3+ Regulatory T

cells in PBMCs of prostate cancer patients and found that there was no

significant difference in number of Tregs in patients with PCa than those

present in health donors. However, the suppressive functionality of the Tregs

in PCa was considerably greater than that of healthy donors.

This project aims at optimizing the method to isolate Tregs

(CD4+CD25highCD127low/- Tregs) from Human PBMCs (Healthy donors) and

functionally characterize them based upon their immunosuppressive ability.

Peripheral blood mononuclear cells (PBMC) was separated from Human

whole blood by using Leucosep™ (greiner bio-one) by the means of density

gradient centrifugation. During centrifugation the lymphocytes and PBMCs

were separated from other cells on the basis of their buoyant density and

they were enriched in an interphase formed above the separation medium.

After reviving the PBMCs from frozen state, we observed a high amount of

RBCs, which affected the purity of CD4+ T cell isolation. RBC lysis was done

to remove the RBCs present in PBMC.


41

PBMC proliferation results indicates that the, the maximum proliferation is

observed at the concentrations of anti-CD3 at 0.5 µg/mL and anti-CD28 at 4

µg/mL which were added as soluble agents. In our experiments, soluble

anti-CD3 and anti-CD28 showed the maximum proliferation and with

reproducible results. Anti-CD3 plate bound proliferation as per the protocol

mentioned by Shevach E et al., 2004 was also simultaneously performed in

this study Although the proliferation was as significant as the soluble anti-

CD3, the time duration (overnight) to prepare the plate, and the chances of

contamination and lack of reproducible results were reasons the method was

not chosen for further proliferation assays. Proliferation assays were also

performed with Dynabeads® Human T-activator CD3/CD28, as per the

protocol given by the company. The proliferation results were not as

significant as soluble anti-CD3/anti-CD28.

CD4+ T cell enrichment was done using Miltenyi Biotec’s CD4+ T cell isolation

kit. Due to the presence of RBCs in the PBMCs. The RBC cell lysis buffer was

used to devoid the PBMCs of the RBCs, this increased the efficacy of isolation

and also the purity. To get the best results, buffers and reagents were kept

on ice and the experiment was performed as fast as possible avoiding delay

in any of the steps. Passing the cell sample through 40 micron cell strainer

to get single suspension gave us a good enrichment. Incubating enriched

cells 3-4 hours at 37oC, showed more than 97% purity in the flow cytometer

analysis. We also tried to change the amount of antibody and micro bead

(CD4+ T cell biotin-antibody and anti-biotin micro beads) added during the

isolation step, and noticed that changing the concentration of these did not

show any significant outcome.

The results of flow cytometer showed that we achieved more than 95%

purity in enriching CD4+ T cells.


42

Proliferation assay was performed for the enriched CD4+ T cells as per the

plate setup as shown in Fig 3.2. 1x105 cells were added to each well, with 50

U/mL of IL-2. Various concentration of anti-CD3 and anti-CD28 was added

and incubated for 3 days at 370C and 20µL media containing [3H]-thymidine

was pulsed 17 hours prior to harvesting with liquid scintillation counter.

Statistical analysis (t test) was done to the results obtained and found that

0.5 µg/mL of anti-CD3 and 1µg/mL of anti-CD28 showed maximum

proliferation and was also statistically significant. Alternatively proliferation

assay was performed without adding IL-2, which showed less proliferation.

Regulatory T cells (CD4+CD25highCD127low/- Tregs) and conventional T cells

(CD4+CD25lowCD127high Tconv) were sorted using Beckman Coulter’s MoFlo™

XDP. PBMCs or enriched CD4+ T cells were stained with Human Regulatory T

cell sorting kit (Miltenyi Biotec) by following the staining protocol provided in

the kit. Total percentage of Tregs in PBMCs was around 0.7-1% and was 1.3-

3% in enriched CD4+ T cells. Therefore, we enriched CD4+T cells first and

then sorted for Tregs. As the number of Tregs obtained after sorting is still

low to perform enough suppression assays, there was need to expand them

in order to get sufficient cells (Tregs) for further studies. Expansion of Tregs

was done as per the protocol listed in the Treg Expansion kit (Miltenyi

Biotec), an important observation was that expansion protocol requires

1x105 cell per well. Scaling down the CD3/CD28 micro beads provided with

the kit will not work efficiently if there are less than 1x105 cell per well. The

plate was incubated undisturbed at 370C for 14 days, and the cells were split

and media was changed when needed. One of the study conducted by

Hoffmann P et al., 2006, indicates that Treg cells maintain FoxP3 expression

after isolation with Human regulatory T cell sorting kit.


43

In this study, we were able to optimise the protocol of isolating Tregs from

PBMCs, where we first separated PBMCs from whole blood, performed

proliferation assays to obtain concentrations of anti-CD3 and anti-CD28

which showed maximum proliferation. Later CD4+ T cells were enriched from

PBMCs and analysed by flow cytometer for the purity of isolation.

Proliferation assay was done with enriched CD4+ T cells to obtain the

concentration anti-CD3 and anti-CD28 which showed maximum proliferation.

Later, Tregs (CD4+CD25highCD127low/- Tregs) were sorted from enriched

CD4+T cells and expanded for over 20 days. After expansion, there were

enough number of cells to perform suppression assay by co-culturing Tregs

(CD4+CD25highCD127low/- Tregs) with Tconv (CD4+CD25lowCD127high Tconv) in

varying ratios was performed.


44

5.1 Future Work:

Now, that the isolation protocol is optimized, future work include performing

suppression assay by co-culturing Tregs (CD4+CD25highCD127low/- Tregs) with

Tconv (CD4+CD25lowCD127high Tconv) in varying ratios, as it was done only

once. Now, one glitch is that, the number of Tconv cells are low, to overcome

this problem, CD3+T cells can be sorted and immunosuppression assays can

be done by co-culturing with Tregs, where they are seen as Tconv cells.

Alternatively, Tconv cells can be sorted initially after the PBMC processing,

and frozen to be used later, that way, when Tregs are completely expanded,

we can do the immunosuppression assay straight away by reviving the

frozen Tconv cells (Tregs take 15-30 days to expand).

It is now well documented that patients with metastatic prostate cancer

have high levels of Tregs (CD4+CD25high Tregs) in the Peripheral blood (J

Yokokawa et al., 2008). Research on various cancer have also shown

increased levels of Tregs (CD4+CD25high) in the peripheral blood (Woo EY et

al., 2001; Liyanage UK et al., 2002; Sasada T et al., 2003; Curiel TJ et al.,

2004; Schaefer C et al., 2005).

By optimizing the method for isolating Tregs (CD4+CD25highCD127low/- Tregs)

from healthy donor PBMCs, it will provide a strong base to isolate Tregs

(CD4+CD25highCD127low/- Tregs) from PBMCs of prostate cancer (PCa)

patients and functionally characterize them and compare the results of

suppressive functions of Tregs in healthy donors, progressive PCa, localized

PCs and metastatic PCa as described by J Yokokawa et al., 2008, where the

authors have described about functionality of only CD4+CD25high FoxP3+

Tregs.


45

Even when most of human CD4+CD25highCD127low/- Tregs are FoxP3+, there

is still a need for more specific marker (surface marker) by which we can

characterize human Tregs by phenotypic and functional analysis. This project

also provides an excellent pathway for in depth phenotypic analysis of Tregs

in Human PBMCs.

Also, at present there is limited understanding on the increase of Tregs in

peripheral blood. Previously it was described that the increase of Tregs in

peripheral blood of PCa was due to active proliferation rather than migration

from other sites such as bone marrow or secondary lymphoid organs (Wolf D

et al., 2006). However, it is now seen that there is an wide array of antigens

and factors such as TGF-β, chemokine’s etc. that are secreted by tumour

microenvironment which results in increased levels of Tregs in peripheral

blood which can be detected due to the “convergence of lymphatics and

blood vessels” (Knutson KL et al., 2007). Isolating Tregs from human PBMCs

can be used to further study the cause of the increased Tregs in PCa

patient’s peripheral blood.

There are also many suppressive mechanism of Tregs described, cytokine

profiling can simply be done by collecting the supernatant from all the wells

during the immunosuppression assays is performed, and this supernatant

can be analysed using flow cytometer to study the cytokine secretion to

provide an insight into the mechanism of suppression of Tconv by Tregs.

Finally, by studying the functional activity of Tregs (CD4+CD25highCD127low/-

Tregs) in healthy donors and patients with PCa can provide basis for novel

immunotherapeutic approaches, which can target Tregs. Analysing the

functions of Tregs and also the level of expression can be used as an

excellent diagnostic method and also to observe PCa patients before and

after vaccinations.


46

Chapter 6.Appendix:

1. Alternate strategies such as plate bound anti-CD3 stimulation and also

using Dynabeads was performed with PBMCs. It was observed that the

proliferation results were very low and were not as significant as with using

soluble anti-CD3, as there were lack of reproducibility and also the time

taken to prepare the plates. The below results show the proliferation of

PBMCs by plate bound anti-CD3 and also using Dynabeads at a bead to cell

ration of 1:1.

c e lls a

lon

e

be a d

s 1:1

Be a d

s 1:1

+ IL

2

pla

te b

ou

nd

an

t i-C

D3 0

.5µ

g/m

L

pla

te b

ou

nd

an

t i-C

D3 1

µg

/mL

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

CP

M

Fig 6.1: Plate bound anti-CD3 stimulation and by using Dynabeads

anti-CD3/anti-CD28 pre-coated beads. Although the proliferation is seen

to maximum with concentration of anti-CD3 of 0.5µg/mL, it is still very less

significant than using soluble anti-CD3.


47

2. Proliferation studies were also done with CD4+ T cells without using IL-2,

as initially while performing the PBMC proliferation which did not use IL-2

showed good proliferation. But, when IL-2 was not added to CD4+ T cells,

the CPM count was seen to be very low. Although there was difference in the

proliferation with various concentrations of anti-CD3/anti-CD28, it was still

not as significant as proliferation obtained by adding IL-2.

Cells a

lon

e

0.5

: 0.5

0.5

: 1

0.5

: 2

0.5

: 3

1:

0.5

1:

1

1:

2

1:

3

0

1 0 0

2 0 0

3 0 0

4 0 0

a n t i-C D 3 : a n t i-C D 2 8 µ g / m l

CP

M

Fig 6.2: CD4+T cells Proliferation assay without IL-2. Enriched CD4+ T

cells at the concentration of 1x105 cells/well were stimulated with anti-CD3

and anti-CD28 at different concentrations, without any IL-2 in a 96 well

round bottom plate for 3 days at 370C. 20µL media containing [3H]-

thymidine was pulsed to all the wells, 17 hours prior to quantification using

scintillation counter. All the experiments were done in triplicate wells.

Although the maximum proliferation was observed at concentrations of 0.5

µg/mL of anti-CD3 and 1 µg/mL of anti-CD28 which was similar to that of


48

proliferation assay done by adding IL-2, but the CPM count is significantly

lower than done by adding IL-2.

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