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.........................................
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
Chapter 5. Conclusions and Future work 40
Chapter 6. Appendix 46
Chapter 7. Bibliography 48
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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).
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
List of Tables:
Table 1.1: Properties of Natural and Adaptive Regulatory T cells.
Table 1.2: Phenotypes of natural regulatory T cells.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- 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
Th2- T Helper Cell 2
Th17- T Helper Cell 17
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
RT-Room Temperature
SEM-Standard Error of the Mean
JvGCRC- John van Geest Cancer Research Center
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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).
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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).
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- 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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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)
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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)
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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).
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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).
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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).
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory 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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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.
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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
Isolation and Functional Characterization of CD3+CD4+CD25High CD127low/- Regulatory T cells.
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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