Role of T-REGULATORY CELLS IN CERTAIN AUTOIMMUNE DISORDERS AND GRAFT

REJECTION IN case of KIDNEY TRANSPLANTATION:

A FLOWCYTOMETRY APPROACH

ADissertation REPORT

SUBMITTED FOR

THE PARTIAL FULFILMENT OF THE Requirement OFTHE AWARD OF THE DEGREE OF

Master of Science in MEDICAL BIOTECHNOLOGY

Of Sardar Patel University

November 2011

Submitted byMr. Yash pandya

Msc. Medical biotechnology

Under the guidance ofDr.ARUNA VANIKAR, M.D., FICP

DR. H.L.TRIVEDI INSTITUTE OF TRANSPLANTATION SCIENCES

SUPERVISORSUPERVISORDR. DEVJANI CHAKRABORTYDR. DEVJANI CHAKRABORTY

ASST. PROF. IN BIOCHEMISTRYASST. PROF. IN BIOCHEMISTRYASHOK AND RITA PATEL INSTITUTE OF INTEGRATED STUDY ASHOK AND RITA PATEL INSTITUTE OF INTEGRATED STUDY AND RESEARCH IN BIOTECHNOLOGY & ALLIED SCIENCES AND RESEARCH IN BIOTECHNOLOGY & ALLIED SCIENCES (ARIBAS), (ARIBAS), NEW VALLABH VIDYANAGAR, GUJARAT.NEW VALLABH VIDYANAGAR, GUJARAT.

“Role of T-REGULATORY CELLS IN CERTAIN AUTOIMMUNE DISORDERS AND GRAFT REJECTION IN case of KIDNEY TRANSPLANTATION: A flowcytometry approach A Dissertation Report SUBMIITTED FOR THE PARTIAL FULFILMENT OF THE Requirement of THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN MEDICAL BIOTECHNOLOGY OF SARDAR PATEL UNIVERSITY NOVEMBER 2011 SUBMITTED BY MR. YASH PANDYA Msc. MEDICAL BIOTECHNOLOGY UNDER THE GUIDANCE OF DR. ARUNA VANIKAR M.D., FICP DR. H.L TRIVEDI INSTITUTE OF TRANSPLANTATION SCIENCES SUPERVISOR DR. DEVJANI CHAKRABORTY ASST. PROFESOR IN BIOCHEMISTRY ASHOK AND RITA PATEL INSTITUTE OF INTEGRATED STUDY AND RESEARCH IN BIOTECHNOLOGY & ALLIED SCIENCES (ARIBAS) NEW VALLBH VIDYANAGAR, GUJRAT

Charutar Vidyamandal’s

ASHOK & RITA PATEL INSTITUTE OF INTEGRATED STUDY & RESEARCH IN BIOTECHNOLOGY AND ALLIED SCIENCES (ARIBAS)

NOVEMBER 8, 2011CERTIFICATECERTIFICATE

This is to certify that the work presented in the project entitled ““ROLE OF T-regulatory CELL IN CERTAIN AUTOIMMUNE DISORDER AND GRAFT REJECTION IN case of KIDNEY TRANSPLANTATION :A FLOWCYTOMETRY APPROaCH” by Mr. YASH PANDYA of Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences (ARIBAS), Sardar Patel University, New Vallabh Vidyanagar comprises the result of independent and the original work carried out under my supervision for the partial fulfilment of the degree of M. Sc. INTEGRATED MEDICAL BIOTECHNOLOGY.I further certify that this work did not form a part of any other work published or unpublished.

DR. DEVJANI CHAKRABORTY DR. PRADIP PATEL ASST. PROF. IN BIOCHEMISTRY DIRECTORARIBAS ARIBAS

UNDERTAKINGUNDERTAKING

Date: 10.11.2011Date: 10.11.2011 I, Mr. YASH PANDYA, of Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology & Allied Sciences, New Vallabh Vidyanagar hereby undertake that the work presented in the dissertation project report entitled “ROLE OF T-regulatory CELL IN CERTAIN AUTOIMMUNE DISORDER AND GRAFT REJECTION IN case of KIDNEY TRANSPLANTATION :A FLOWCYTOMETRY APPROaCH’’’’ comprises the results of independent and original work carried out by me under the supervision of Dr. Devjani Chakraborty for the partial fulfillment of the award of the degree in M. Sc. INTEGRATED MEDICAL BIOTECHNOLOGY of Sardar Patel University, Vallabh Vidyanagar.I further declare that this work did not form a part of any other work published or unpublished.

MR.YASH PANDYA

Ashok and Rita Patel Institute Integrated Study and Research in Biotechnology & Allied Sciences, New Vallabh Vidyanagar, Gujarat

P.O. Box NO. 61, New Vallabh Vidyanagar, Vitthal Udyognagar-388121, Dist. Anand, Gujarat,

India. Phone: (02692) 229189, 231894,645801, Fax: (02692) 229189, Website: www.aribas.edu.in

ACKNOWLEDGEMENT I believe that to achieve a goal, proper guidance of teachers, blessing of parents and the grace of GOD are the three basic requirements besides your hard work and proper planning.

I am thankful to my guide Dr. Aruna vanikar (M.D., Prof. & chief Department of Pathology Lab. Medicine & Transfusion Services in

I.K.D.R.C & I.T.S.) who guided me in all the practical aspects as well as research purposes of the topic. Madam Vanikar gave me her exceptional knowledge of the subject to understand the depth of immunology. Along with the basics, she also taught me the practical work by using flow-cytometry instrument and collection of blood as well as preparation of the samples by using markers explaining its importance. Her valuable guidance helped me achieve the goal.

The technical staff of the institute explained me the working of the machines like the flow cytometer and the fully automatic machine which measures the serum creatinine. Both these machines were attached with the computers to get the computerized data. I am also thankful to the technical staff of the institute for their help and support. My co-guide Dr. Devjani chakraborty explained to me various concepts of immunology in detail, which helped me, understand the subject in depth. Her valuable inputs made the thesis writing meaningful and simple. Her advice and guidance made a lot of difference. I thank from bottom of my heart to dr. pradip patel and Mr. brijesh jajal for their guidance and suggestions which helped me a lot. My hearty thanks to my guide, co-guide and all my faculty members.

10/11/2011 YASH J.PANDYA

CONTENT CHAPTERS PAGE NO.

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 27

3. Objective 33

4. Materials and method 34

5. Results 50

6. Conclusion 62

7. Discussion 63

8. References 72Abbreviation

ab : Antibody ADP : Adenosine bi phosphate APC : Antigen presenting cells ATP : Adenosine tri phosphate α : alph β : beta BM : Bone marrow CD : Cluster of differentiation CGN : Chronic globular nephritis CMV : Cytomegalovirus CNI : Alcineurin inhibitors DC : Dendritic cells FCS : Flow-cytomatry FITC : Fluorescein isothiocynate FOXP3 : Forkhead transcription factor

HSC : Hematopoietic stem cells Hb : Hemoglobin, IL : Interleukin MHC : Major Histocompatibility Complex MMF : Mycophenolate Mofetil nTreg : natural T regulatory cells O2 : Oxygen, PTC : Peritubular capillaries PE : Phycoerythrin TGF : T cell growth factor T-reg : T regulatory cells TX : Transplantation SCR : Serum creatinine

CHAPTER-1INTRODUCTION

Chapter 1: Introduction

1.1 BASIC IMMUNOLOGY:

The immune system evolved to protect multicellular organisms from the pathogen.

Protection by the immune system can be divided into two related activities –

RECOGNISATION and RESPONSE. Immune recognition is remarkable for its capacity

to distinguish foreign invaders from self component. The immune system is able to

discriminate between foreign molecules and the body’s own cells and molecules (self-

nonself discrimination). Typically, recognition of a pathogen by the immune system

triggers effector response that eliminates or neutralizes the invader. The multiple

components of immune system are exposure include a memory response characterized by

a more rapid and heightened immune reaction upon later attack. (Ref. - Book kuby et.al.)

1.2 INNATE AND ADAPTIVE IMMUNE SYSTEM:The immune system must be pointed out that there are two systems of immunity:

Innate immunity and adaptive immunity, which collaborate to protect the body.

Innate immunity includes molecular and cellular mechanisms predeploye before an

infection and poised to prevent or eliminate it. Innate immunity is highly effective first

line of defense against infection. It distinguishes self and pathogens, but they are not

specialized to distinguish small differences in foreign molecules. Innate immunity are

present before the onset of infection and constitute a set of disease-resistance mechanism

that are not specific to a particular pathogen but include cellular and molecular

components that recognize classes of molecules particular to frequently encountered

pathogens.

Adaptive immunity develops in response to infection and adapts to recognized,

eliminate, and then remember the invading pathogen. Adaptive immunity is second line

of defense. The cells and molecules of the adaptive system possess slower temporal

dynamics; they possess a high degree of specificity and evoke a more potent response on

secondary exposure to the pathogen. The adaptive immune system frequently

incorporates cells and molecules of the innate system in its fight against harmful

pathogens.

ARIBAS 1

Chapter 1: Introduction

For example, complement (molecules of the innate system) may be activated by

antibodies (molecules of the adaptive system) thus providing a useful addition to the

adaptive system’s armamentaria.

1.3 CELLS IN IMMUNE SYSTEM:

Figure: 1 Hematopoiesis-Differentiation of Lymphoid cells and myeloid cells:

ARIBAS 2

Chapter 1: Introduction

1.4 Lymphocytes:Lymphocytes bearing antigen receptors are the central cells of adaptive immunity and are

responsible for its signature properties of diversity, specificity and memory.

Lymphocytes are types of white blood cells and play essential role in adaptive immunity,

presenting antigens, secreting cytokines, and engulfing and destroying microorganisms.

Lymphocytes constitute 20% to 40% of the body cells and 99% of the cells in the lymph.

There are approximately a trillion lymphocytes in the human body. Lymphocytes

circulate of continuously in the blood and lymph and are capable of migrating into the

tissue space and lymphoid organs, serving thereby as a bridge between parts of the

immune system.

The lymphocytes can be broadly subdivided into three major populations- Tcells, Bcells

and Natural killer cells (NK cell).

1.4.1 Natural killer cells:

The body contains a small population of large, granular lymphocytes called natural killer

cells that display cytotoxic activity against a wide range of tumor cells and against cells

infected with some but not all viruses. The extra-ordinary features of these cells which

constitute 5% to10% of lymphocytes in human peripheral blood, is their ability to tumor

or viruse infected cells lacking antigen specific receptor. It is innate immune system and

most do not have T- cell receptors or immunoglobulin incorporated in their plasma

membrane. Natural Killer cells are a subpopulation of circulating lymphocytes that lack

the conventional antigen receptors of T or B cells. These lymphocytes produce cytokines

such as interferon and IL-2.

70% to 80% of NK cells have the appearances of large granular lymphocytes (LGL).

These cells destroy target cells through an extracellular non-phagocytic process called

cytotoxic reaction. The target cells include tumor cells, some cells of the embryo, cells of

normal bone marrow, and microbial agents. NK cells will actively kill virally infected

target cells and certain tumor cells. (kuby et.al.2006)

ARIBAS 3

Chapter 1: Introduction

1.4.2 B- lymphocytes or B- cells:

Figure: 2 B- Lymphocytes

B- Lymphocytes mature in the bone marrow; on release, each express a unique antigen-

binding receptor on its membrane. In humans and mice bone marrow is site of B-cell

origin and development. Arising from lymphoid progenitors, immature B cells proliferate

and differentiate within the bone marrow and stromal cells within the bone marrow

interact directly with B cells and secrete various cytokines that are required for

development. B cells are the source of the about 90% of the immunoglobulin’s IgG and

IgA in plasma. In B cell maturation the progeny differentiate into effector cells called

plasma cells and into memory B cells.

B cells are lymphocytes that play a large role in the humoral immune response (as

opposed to the cell-mediated immune response, which is governed by T cells). The

principal functions of B cells are to make antibodies against antigens, and eventually

develop into memory B cells after activation by antigen interaction. B cells are an

essential component of the adaptive immune system.

The abbreviation "B", in B cell, comes from the bursa of Fabricius in birds, where they

mature. In mammals, immature B cells are formed in the bone marrow, which is used as

AN ACRONYM for the cell. (kuby et.al. 2006)

ARIBAS 4

Chapter 1: Introduction

B cell types:

Plasma cells (also known as plasmocytes or effector B cells) are large B cells that have

been exposed to antigen and produce and secrete large amounts of antibodies, which

assist in the destruction of microbes by binding to them and making them easier targets

for phagocytes and activation of the complement system. They are sometimes referred to

as antibody factories. An electron micrograph of these cells reveals large amounts of

rough endoplasmic reticulum, responsible for synthesizing the antibody, in the cell

cytoplasm. These are short lived cells and undergo apoptosis when the inciting agent that

induced immune response is eliminated. This occurs because of cessation of continuous

exposure to various colony stimulating factors required for survival.

Memory B cells are formed from activated B cells that are specific to the antigen

encountered during the primary immune response. These cells live for a long time, and

respond quickly following a second exposure to the same antigen.

Figure: 3 Mechanism of B-Lymphocytes with antigen.

ARIBAS 5

Chapter 1: Introduction

1.4.3 T lymphocytes or T-cells:

T-lymphocytes also arise in the bone marrow but migrate to the thymus. Thymus is the

site of the T-cell development and maturation is a flat, bilobed organ situated above the

heart. The lymphocytes that mature in the thymus are called T-lymphocytes. These cells

are responsible for the cellular or cell-mediated immune response and help the B

lymphocytes. During its maturation within the thymus, the T cell comes to express on its

membrane a unique antigen-binding molecule called the T-CELL RECEPTOR. Unlike

membrane bound antibodies on B cells which can recognize antigen alone, T cell

receptors only recognize antigen that is bound to cell membrane protein called MAJOR

HISTOCOMPATIBLITY COMPLEX (MHC).

There are different types of T-cells.

T Helper Cells – These cells have CD4 cell receptors on their surface. Helper T

lymphocytes can be assigned to one of several subsets:

TH1 – are responsible for cell-mediated effector mechanism.

TH2 – play a greater role in the regulation of antibody production

TH0 – are an intermediate category.

TH1 and TH2 cells can promote development of cytotoxic cells and are believed to

develop from TH0 cells. TH1 cells interact most effectively with mononuclear phagocytes.

TH2 release cytokines that are required for B cell differentiation. TH cells express CD4

molecules on their cell surface, which enable the lymphocyte to bind to a MHC class II

molecule. The T cell receptor is unique in that it is only able to identify antigen when it is

associated with MHC molecule on the surface of the cell.

Cytotoxic-Tcells

TC are effector cells found in the peripheral blood that are capable of directly destroying

ARIBAS 6

Chapter 1: Introduction

virally infected target cells or tumor cells. Most TC are CD8 +. TC is the major effectors in

allograft organ rejection.

Cytotoxic T cells are primarily involved in the destruction of infected cells, notably by

viruses. Unlike TH cells, cytotoxic cells possess CD8cell surface markers, which bind to

antigenic peptides expressed on MHC class I molecules. The ratio is approximately 2:1 in

normal human peripheral blood.

Table: 1

Characteristics of Humoral-and Cell-Mediated Immunity:

  Humoral-Mediated Immunity Cell-Mediated ImmunityMechanism Antibody-mediated Cell-mediatedCell Type B Lymphocytes T Lymphocytes

 Mode of action  Antibodies in serumDirect cell-to-cell contact or soluble products secreted by

cells

 Purpose  Primary defense against bacterial infection

Defense against viral and fungal infections, intracellular

organisms, tumor antigens, and graft rejection

1.4.5 Mechanism of lymphocytes to encounter on antigen:

Resting B lymphocytes are able to react with free antigen directly when it binds to their

cell surface immunoglobins which act as receptors. T lymphocytes do not react with free

antigen and instead make use of APCs to phagocytes the antigen and then to express its

component proteins on the cell surface adjacent to special host proteins called major

histocompatibility complex (MHC) class II molecules.

ARIBAS 7

Chapter 1: Introduction

There are two major types of MHC molecules:

1. Class I MHC molecules, expressed by nearly all nucleated cells of vertebrate

species, and

2. Class II MHC molecules, which are expressed by only a few cell types that are

specialized for antigen presentation.

When T cell recognizes antigen combined with an MHC molecule on a cell, under

appropriate circumstances the T cell proliferates and differentiates into various effectors

T cell and memory T cells.

Antigen presenting cells which express MHC class II molecules include dendritic cells

and macrophages. This “afferent” phase must occur in order for the T cell to recognise

the antigen. The “efferent” phase occurs when activated lymphocytes enter the tissue and

meet antigen again. This results in multiplication and secretion of cytokines or

immunoglobins in order to destroy the antigen.

There are large numbers of lymphocytes produced daily in the primary lymphoid

organs, thymus and bone marrow. Some of these cells migrate via the circulation into the

secondary lymphoid tissues- spleen, lymph nodes and mucosa-associated lymphoid

tissues. The average human adult has about 2 x 1012 lymphoid cells and the lymphoid

tissue as a whole represents about 2% of total body weight. The lymphocyte is the “key

player” in immune response. The majority of circulating lymphocytes in the peripheral

blood (60 to 80%) are T cells and these become differentiated in the thymus. Lymphoid

cells account for approximately 20%-40% of the leukocytes in the adult circulation.

Many mature lymphoid cells are long-lived, and persist as memory cells for many years.

Lymphocytes represent the only immunologically specific cellular components of the

immune system. They recognize foreign antigens, destroy some cells, and produce

antibodies as plasma cells.

The primary lymphoid organs are the thymus and the bone marrow. The thymus

exercises control over the entire immune system. The development of diversity occurs

ARIBAS 8

Chapter 1: Introduction

mainly in these primary lymphoid organs. Progenitor cells that migrate to the thymus

divide and differentiate under the influence of the humoral factor. The thymus also

regulates immune function by secretion of multiple soluble hormones. The thymus

gradually loses up to 95% of its mass during the first 50 years of life. This may account

for the increased susceptibility of older adults to infections, autoimmune disease, and

neo-plasms. The bone marrow is the source of the progenitor cells. These cells can

differentiate into lymphocytes and other hematopoietic cells (granulocytes, erythrocytes,

and megakaryocytic populations).

The secondary lymphoid tissues include lymph nodes, spleen and blood. Mature

lymphocytes and accessory cells antigen-presenting cells (APC) are found throughout the

body, although the relative percentages of T and B cells are different in different

locations. Proliferation of the T and B lymphocytes in the secondary and peripheral

lymphoid tissues is dependent on antigenic stimulation.

Blood is the most frequently tested lymphoid organ. Proteins that appear on cell surfaces

can be used as markers to differentiate T cells and B cells. Proteins can also be used to

distinguish the developmental stages of the two types of cells according to when these

proteins appear. A number of laboratories have developed monoclonal antibodies, and

each used its own nomenclature for the sets of antigens found. In an attempt to relate

research findings and standardize the nomenclature scientist came up with the “clusters of

differentiation” (CD) term. As each antigen, or CD, was found it was assigned a number.

The name cluster of differentiation came about because the exact nature of the proteins

identified by the various antibodies was not known. These antigens are most important in

characterizing T and B lymphocytes. (Ref-Yuan Zhai et.al. 2001)

ARIBAS 9

Chapter 1: Introduction

FIGURE: 5 MECHANISM OF T-LYMPHOCYTE CELLS

ARIBAS 10

Chapter 1: Introduction

1.5. Regulatory T cell

Regulatory T cells (Treg, sometimes known as suppressor T cells) are a specialized

subpopulation of T cells that act to suppress activation of the immune system and thereby

maintain immune system homeostasis and tolerance to self-antigens. The existence of a

dedicated population of suppressive T cells was the subject of significant controversy

among immunologists for many years. However, recent advances in the molecular

characterization of this cell population have firmly established their existence and their

critical role in the vertebrate immune system. Interest in regulatory T cells has been

heightened by evidence from experimental mouse models demonstrating that the

immunosuppressive potential of these cells can be harnessed therapeutically to treat

autoimmune diseases and facilitate transplantation tolerance or specifically eliminated to

potentiate cancer immunotherapy. (Ref.-2 Wikipedia, from internet)

1.5.1 T regulatory cell populations:

T regulatory cells are a component of the immune system that suppresses immune

responses of other cells. This is an important "self-check" built into the immune system

to prevent excessive reactions. Regulatory T cells come in many forms, including those

that express the CD8 transmembrane glycoprotein (CD8+ T cells); those that express

CD4, CD25, and Foxp3 (CD4+CD25+ regulatory T cells, or "Tregs"); and other T cell

types that have suppressive function. These cells are involved in shutting down immune

responses after they have successfully tackled invading organisms, and also in regulating

immune responses that may potentially attack one's own tissues (autoimmunity).

CD4+Foxp3+ regulatory T cells have been referred to as "naturally-occurring" regulatory

T cells to distinguish them from "suppressor" T cell populations that are generated in

vitro. The regulatory T cell field is further complicated by reports of additional

suppressive T cell populations, including Tr1, Th3, CD8+CD28-, and Qa-1 restricted T

cells. The contribution, however, of these populations to self-tolerance and immune

homeostasis is less well defined. The lack of a clear defining marker for regulatory T

cells presents a serious challenge to researchers. (Ref.-3 A.Sanschez-fueyo et.al)

ARIBAS 11

Chapter 1: Introduction

1.5.2 Treg cells

Several different Treg cell populations have been described in the past decade. These

include cross-regulatory CD4+ Th1 and Th2 cells, IL-10 producing CD4+ Tr1 cells, and

TGFβ-producing CD4+ Tr2/Th3 cells. Both Tr1 and Tr2/Th3 Treg cells can acquire

CD25 expression. CD8+ Tr1 and Tr2 cells have also been described. All of these Treg

cell types are induced in the course of an immune response and mediate their suppressive

activity via the production of inhibitory cytokines. In contrast, naturally occurring CD4+

T regulatory (Trn or nTreg cells) constitutively display CD25 and mediate their

suppressive effect through an antigen-nonspecific mechanism that involves cell contact

and does not necessarily require IL-10 or TGFβ. Finally, CD4+CD25- T cells that are

activated in the presence of IL-2 and TGFβ express CD25 and develop an inhibitory

phenotype that is indistinguishable from Trn cells. These inhibitory T cells are termed

peripherally induced Treg (Tri) cells. Unlike Th cells that proliferate in response to

antigenic stimulation of the T cell receptor, Treg cells are usually unresponsive to

antigenic stimulation, at least in tissue culture. However, Treg cells do proliferate when

stimulated through the T cell receptor in the presence of IL-2 (and sometimes IL-15).

Treg cells act as feedback regulators of Th cells, inhibiting both Th1 and Th2 cells in an

antigen-nonspecific manner.

1.5.3 Naturally occurring Treg cells

Over the past decade considerable attention has been focused on naturally occurring

CD4+CD25+ Treg (nTreg or Trn) cells. Trn cells develop in the thymus and make up

5-10% of the peripheral naïve CD4+ T lymphocyte pool in normal mice and humans.

Thymectomy of neonatal mice results in an absence of Trn cells and a propensity to

ARIBAS 12

Chapter 1: Introduction

develop T cell-mediated autoimmune disease. Trn cell development in the thymus

appears to involve CD28 costimulation since CD28-/- non-obese diabetic mice

develop diabetes more rapidly than their wildtype littermates. IL-2 is also essential

for Trn cell development/maintenance since mice that lack IL-2 or the α chain or β

chain of the IL-2 receptor have few or no Trn cells and die prematurely from

autoimmune lympho-proliferation. T cell receptor stimulation and IL-2 are required

to induce suppressor activity by freshly isolated CD4+CD25+ Trn cells. Suppression

of both CD4+ and CD8+ T cell responses is antigen-nonspecific and involves the

suppression of IL-2 production by Th cells. Trn-mediated immune suppression does

not require IL-4, IL-10, or TGFβ since Trn cells function in vitro in the presence of

neutralizing antibodies to these cytokines. In addition, Trn cells from IL4-/-, IL-10-/-,

or TGFβ-/- mice function normally. Moreover, supernatants from cultures of

activated Trn cells do not inhibit T cell responses. However, Trn-mediated

suppression does require cell contact, at least in vitro. Nevertheless, it is still possible

that suppressive cytokines play a role in Trn function in vivo. CD4+CD25+ Trn cells

express a number of cell-surface markers, including CTLA-4, 4-1BB, and neuropilin-

1. However, none are reliable markers for Trn cells since almost all are expressed by

activated CD4+CD25- T cells. Importantly, recent studies indicate that forkhead

transcription factor (Foxp3) is a functional marker for Trn cells since it is required for

their generation in the thymus. CTLA-4 has been proposed to play a functional role in

Trn-mediated immune suppression but this is still controversial. In any case, Trn cells

do not compete for or prevent costimulation of Th cells. Expansion and induction of

suppressor function by peripheral CD4+CD25+ Trn cells does not appear to require

costimulation through CD28. However, nonspecific signals through the TLR4

pathway (in response to LPS) have been shown to directly activate Trn cells. Indeed,

CD4+CD25+ T cells express several different TLR so additional TLR signaling

pathways may also activate Trn cells. (Ref.-Shimon sakaguchi et,al 2004)

ARIBAS 13

Chapter 1: Introduction

1.5.4 Mechanism of T-reg cells action:

Both innate and adaptive immune cells are targets of TREG-cell-mediated suppression, and

TREG cells are known to employ a variety of mechanisms to mediate these effects. TREG

cells directly suppress many functions of CD4+ and CD8+ T cells, ranging from their

proliferation to their differentiation into T-helper (TH)-1, TH2, and TH17 subsets. In some

cases, TREG cells induce apoptosis of responding T cells. TREG cells also suppress the

activation of B cells, thereby inhibiting humoral immune responses. Other prominent

targets of TREG cells include dendritic cells, macrophages, natural killer cells, mast cells,

and osteoblasts. TREG cells are also involved in tissue repair and in the resolution of tissue

inflammation, suggesting a potential role for TREG cells in the regulation of non-immune

cells. In fact, TREG cells can inhibit the development of transplant vasculopathy, a

complex process that involves many immune and non-immune cells, supporting a broad

role for TREG cells in tissue remodeling.

The mechanism by which TREG cells regulate such diverse cell types both inside and

outside the immune system is an area of considerable interest. TREG cells probably employ

several different mechanisms to suppress pathogenic T-effector cells. In vitro assays have

demonstrated that activation of TREG cells via TCR stimulation is required to mediate

suppression of T-effector cells and that this suppression requires strict cell–cell contact.

Under some conditions, TREG cells have been shown to deprive T-effector cells of survival

and growth factors or to directly kill activated T cells via granzyme-dependent

mechanisms. Furthermore, TREG cells express CD39 and CD73, the ectoenzymes that

break down the extracellular ATP into adenosine. This process has been shown to turn an

ATP-rich inflammatory milieu to one that is immunosuppressive, as adenosine inhibits

the activation of dendritic cells and macrophages, which in turn prevents T-cell priming.

TREG cells also express CTLA-4 on their surface, which can directly engage the peripheral

membrane protein B7 on antigen-presenting cells (APCs) to inhibit APC activation via

different mechanisms. In addition, TREG cells can produce copious amounts of immune

suppressive cytokines, including TGF-β, IL-10, and IL-35, which are known to inhibit a

ARIBAS 14

Chapter 1: Introduction

wide spectrum of cellular activities. TGF-β exerts broad antiproliferative and anti-

inflammatory effects. IL-10 strongly inhibits activation of macrophages and dendritic

cells, and IL-35 is a key mediator of TREG-cell-induced immunosuppression. Despite

advances in our understanding of TREG-cell function, the processes by which this catalog

of in vitro mechanisms contributes to in vivo immunosuppression by TREG cells is still not

known.

1.5.6 Basic mechanisms of Treg-cell function

Defining the mechanisms of Treg-cell function is clearly of crucial importance. Not only

would this provide insight into the control processes of peripheral tolerance but it would

probably provide a number of potentially important therapeutic targets. Although this

quest has been ongoing since interest in Treg cells was reignited in 199523, there has

been significant progress in the last few years. From a functional perspective, the various

potential suppression mechanisms of Treg cells can be grouped into four basic ‘modes of

action’: suppression by inhibitory cytokines, suppression by cytolysis, suppression by

metabolic disruption, and suppression by modulation of dendritic-cell (DC) maturation or

function.

ARIBAS 15

Chapter 1: Introduction

Figure: 6 Mechanism of T-reg cell in different part of body.

1.5.7 Function:

The immune system must discriminate between self and non-self. When self/non-self

discrimination fails, the immune system destroys cells and tissues of the body and as a

result causes autoimmune diseases. Regulatory T cells actively suppress activation of the

immune system and prevent pathological self-reactivity, i.e. autoimmune disease. The

critical role regulatory T cells play within the immune system is evidenced by the severe

autoimmune syndrome that results from a genetic deficiency in regulatory T cells.

The molecular mechanism by which regulatory T cells exert their suppressor/regulatory

activity has not been definitively characterized and is the subject of intense research. In

vitro experiments have given mixed results regarding the requirement of cell-to-cell

contact with the cell being suppressed. The immunosuppressive cytokines TGF-beta and

Interleukin 10 (IL-10) have also been implicated in regulatory T cell function.

An important question in the field of immunology is how the immunosuppressive activity

of regulatory T cells is modulated during the course of an ongoing immune response.

While the immunosuppressive function of regulatory T cells prevents the development of

autoimmune disease, it is not desirable during immune responses to infectious

microorganisms. Current hypotheses suggest that, upon encounter with infectious

microorganisms, the activity of regulatory T cells may be down regulated, either directly

or indirectly, by other cells to facilitate elimination of the infection. Experimental

evidence from mouse models suggests that some pathogens may have evolved to

manipulate regulatory T cells to immunosuppress the host and so potentiate their own

survival. For example, regulatory T cell activity has been reported to increase in several

infectious contexts, such as retroviral infections (the most well-known of which is HIV),

mycobacterial infections (like tuberculosis), and various parasitic infections including

Leishmania and malaria.

ARIBAS 16

Chapter 1: Introduction

1.5.8 Molecular characterization:

Regulatory T cells develop in the thymus. The latest research suggests that regulatory T

cells are defined by expression of the forkhead family transcription factor FOXP3

(forkhead box p3). Expression of FOXP3 is required for regulatory T cell development

and appears to control a genetic program specifying this cell fate. The large majority of

Foxp3-expressing regulatory T cells are found within the major histocompatibility

complex (MHC) class II restricted CD4-expressing (CD4+) helper T cell population and

express high levels of the interleukin-2 receptor alpha chain (CD25). In addition to the

Foxp3-expressing CD4+CD25+, there also appears to be a minor population of MHC class

I restricted CD8+ Foxp3-expressing regulatory T cells. Unlike conventional T cells,

regulatory T cells do not produce IL-2 and are therefore anergic at baseline.

A number of different methods are employed in research to identify and monitor T reg

cells. Originally, high expression of CD25 and CD4 surface markers was used

(CD4+CD25+ cells). This is problematic as CD25 is also expressed on non-regulatory T

cells in the setting of immune activation such as during an immune response to a

pathogen. As defined by CD4 and CD25 expression, regulatory T cells comprise about 5-

10% of the mature CD4+ helper T cell subpopulation in mice and humans, while about 1-

2% of Treg can be measured in whole blood. The additional measurement of cellular

expression of Foxp3 protein allowed a more specific analysis of Treg cells

(CD4+CD25+Foxp3+ cells). However, Foxp3 is also transiently expressed in activated

human effector T cells, thus complicating a correct Treg analysis using CD4, CD25 and

Foxp3 as markers in humans. Therefore, some research groups use another marker, the

absence or low-level expression of the surface protein CD127 in combination with the

presence of CD4 and CD25. Several additional markers have been described, e.g., high

levels of CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) and GITR

(glucocorticoid-induced TNF receptor) are also expressed on regulatory T cells, however

the functional significance of this expression remains to be defined. There is a great

interest in identifying cell surface markers that are uniquely and specifically expressed on

ARIBAS 17

Chapter 1: Introduction

all Foxp3-expressing regulatory T cells. However, to date no such molecule has been

identified.

In addition to the search for novel protein markers, a different method to analyze and

monitor Treg cells more accurately has been described in the literature. This method is

based on DNA methylation analysis. Only in Treg cells, but not in any other cell type,

including activated effector T cells, a certain region within the foxp3 gene (TSDR, T reg-

specific-demthylated region) is found demethylated, which allows to monitor T reg cells

through a PCR reaction or other DNA-based analysis methods. Recent evidence suggests

that mast cells may be important mediators of Treg-dependent peripheral tolerance.

1.5.9 Genetic deficiency:

Genetic mutations in the gene encoding Foxp3 have been identified in both humans and

mice based on the heritable disease caused by these mutations. This disease provides the

most striking evidence that regulatory T cells play a critical role in maintaining normal

immune system function. Humans with mutations in Foxp3 suffer from a severe and

rapidly fatal autoimmune disorder known as Immune dysregulation Polyendocrinopathy

Enteropathy X-linked (IPEX) syndrome.

The IPEX syndrome is characterized by the development of overwhelming systemic

autoimmunity in the first year of life, resulting in the commonly observed triad of watery

diarrhea, eczematous dermatitis, and endocrinopathy seen most commonly as insulin-

dependent diabetes mellitus. Most individuals have other autoimmune phenomena

including Coombs-positive hemolytic anemia, autoimmune thrombocytopenia,

autoimmune neutropenia, and tubular nephropathy. The majority of affected males die

within the first year of life of either metabolic derangements or sepsis. An analogous

disease is also observed in a spontaneous Foxp3-mutant mouse known as “scurfy”.

ARIBAS 18

Chapter 1: Introduction

1.6 TRANSPLANTATION:

Transplantation, as term is used in IMMUNOLOGY, refers to the act of transferring cell,

tissues, or organs from one site to another. Many diseases can cured by transplantation of

a healthy organ, tissues, or cells, or a graft from the donor to recipient (host).

DEFINATION:

Organ transplantation is the moving of an organ from one body to another or from a

donor site on the patient's own body, for the purpose of replacing the recipient's damaged

or absent organ. The emerging field of Regenerative medicine is allowing scientists and

engineers to create organs to be re-grown from the patient's own cells.

(Stem cells, or cells extracted from the failing organs).

1.6 Types of transplant:

1.6.1 Auto graft:Transplant of tissue to the same person. Sometimes this is done with surplus tissue, or

tissue that can regenerate, or tissues more desperately needed elsewhere (examples

include skin grafts, vein extraction for CABG, etc.) Sometimes an autograft is done to

remove the tissue and then treat it or the person, before returning it (examples include

stem cell autograft and storing blood in advance of surgery). In a rotationplasty a

distal joint is use to replace a more proximal one, typically a foot and ankle joint is used

to replace a knee joint. The patient's foot is severed and reversed, the knee removed, and

the tibia joined with the femur.

ARIBAS 19

Chapter 1: Introduction

1.6.2 Allograft and allotransplantation:

Allotransplantation (allo- from the Greek meaning "other") is the transplantation of

cells, tissues, or organs, sourced from a genetically non-identical member of the same

species as the recipient. The transplant is called an allograft or allogeneic transplant or

homograft. Most human tissue and organ transplants are allograft.

An allograft is a transplant of an organ or tissue between two genetically non-identical

members of the same species. Most human tissue and organ transplants are allograft. Due

to the genetic difference between the organ and the recipient, the recipient's immune

system will identify the organ as foreign and attempt to destroy it, causing transplant

rejection.

1.6.3 Isograft:

An Isograft is a graft of tissue between two individuals who are genetically identical (i.e.

monozygotic twins). Transplant rejection between two such individuals virtually never

occurs.

As monozygotic twins have the same major histocompatibility complex, there is very

rarely any rejection of transplanted tissue by the adaptive immune system.

Isografts are differentiated from other types of transplants because while they are

anatomically identical to allograft, they do not trigger an immune response.

1.6.4 Xenograft and xenotransplantation:

A transplant of organs or tissue from one species to another. Examples are porcine heart

valve transplants, which are quite common and successful. Another example is attempted

piscine-primate (fish to non-human primate) transplant of islet (i.e. pancreatic or insular

tissue) tissue. The latter research study was intended to pave the way for potential human

use, if successful. However, xenotransplantion is often an extremely dangerous type of

transplant because of the increased risk of non-compatibility, rejection, and disease

carried in the tissue.

ARIBAS 20

Chapter 1: Introduction

FIGURE 7: Schematic diagram of the process of graft acceptance and rejection:

(a) Acceptance of an auto graft is completed within 12–14 days. (b) First-set rejection of

an allograft begins 7–10 days after grafting, with full rejection occurring by 10–14 days.

(c) Second-set rejection of an allograft begins within 3–4 days, with full rejection by 5–6

days. The cellular infiltrate that

Invades an allograft (b, c) contains lymphocytes, phagocytes, and

Other inflammatory cells.

T-regs are emerging as important cells associated with transplantation tolerance which

means survival of a transplanted organ in the recipients’ body without the recipient’s

immune system rejecting it.

ARIBAS 21

Chapter 1: Introduction

1.7 Major organs and tissues transplanted:

Thoracic organs

Heart (Deceased-donor only),

Lung (Deceased-donor and living-related lung transplantation),

Heart/Lung (Deceased-donor and Domino transplant).

Abdominal organs

Kidney (Deceased-donor and Living-Donor)

Liver (Deceased-donor and Living-Donor)

Pancreas (Deceased-donor only)

Intestine (Deceased-donor and Living-Donor)

Stomach (Deceased-donor only)

Testis

Abdominal organ- Kidney transplantation:

Kidney transplantation or renal transplantation is the organ transplant of a kidney into a

patient with end-stage renal disease. Kidney transplantation is typically classified as

deceased-donor (formerly known as cadaveric) or living-donor transplantation depending

on the source of the donor organ. Living-donor renal transplants are further characterized

as genetically related (living-related) or non-related (living-unrelated) transplants,

depending on whether a biological relationship exists between the donor and recipient.

FIGURE:8 DIAGRAM OF TRANSPLATED KIDNEY IN HUMAN BODY

ARIBAS 22

Chapter 1: Introduction

1.8. FLOW CYTOMETRY:

1.8.1 INTRODUCTION:

Flow-Cytometry refers to the measurement of physical and chemical characteristics of

cells or any other biological particles. Modern flow cytometers are able to analyze several

thousand particles every second, in "real time," and can actively separate and isolate

particles having specified properties. A flow cytometer is similar to a microscope, except

that, instead of producing an image of the cell, flow cytometry offers "high-throughput"

(for a large number of cells) automated quantification of set parameters. To analyze solid

tissues, a single-cell suspension must first be prepared.

A flow cytometry is a method for quantitating components or structural features of cells

primarily by optical means.

FIGURE: 9 Flowcytometer.

ARIBAS 23

Chapter 1: Introduction

1.8.2 A flow cytometer has five main components:

1. a flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that

they pass single file through the light beam for sensing

2. a measuring system - commonly used are measurement of impedance (or

conductivity) and optical systems - lamps (mercury, xenon); high-power water-

cooled lasers (argon, krypton, dye laser); low-power air-cooled lasers (argon

(488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue,

green, red, violet) resulting in light signals

3. a detector and Analogue-to-Digital Conversion (ADC) system - which generates

FSC and SSC as well as fluorescence signals from light into electrical signals that

can be processed by a computer

4. an amplification system - linear or logarithmic

5. A computer for analysis of the signals.

1.8.3 Fluorescence-activated cell sorting

Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It

provides a method for sorting a heterogeneous mixture of biological cells into two or

more containers, one cell at a time, based upon the specific light scattering and

fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides

fast, objective and quantitative recording of fluorescent signals from individual cells as

well as physical separation of cells of particular interest. The acronym FACS is

trademarked and owned by Company. While many immunologists use this term

frequently for all types of sorting and non-sorting applications, it is not a generic term for

flow cytometry. The first cell sorter was invented by Mack Fulwyler in 1965, using the

Coulter principle, a relatively difficult technique and one no longer used in modern

instruments. The technique was expanded by Len Herzenberg, who was responsible for

coining the term FACS. Herzenberg won the Kyoto Prize in 2006 for his work in flow

cytometry.

ARIBAS 24

Chapter 1: Introduction

The cell suspension is entrained in the center of a narrow, rapidly flowing stream of

liquid. The flow is arranged so that there is a large separation between cells relative to

their diameter. A vibrating mechanism causes the stream of cells to break into individual

droplets. The system is adjusted so that there is a low probability of more than one cell

per droplet. Just before the stream breaks into droplets, the flow passes through a

fluorescence measuring station where the fluorescent character of interest of each cell is

measured. An electrical charging ring is placed just at the point where the stream breaks

into droplets. A charge is placed on the ring based on the immediately prior fluorescence

intensity measurement, and the opposite charge is trapped on the droplet as it breaks from

the stream. The charged droplets then fall through an electrostatic deflection system that

diverts droplets into containers based upon their charge. In some systems, the charge is

applied directly to the stream, and the droplet breaking off retains charge of the same sign

as the stream. The stream is then returned to neutral after the droplet breaks off.

1.8.4 Applications of FLOW CYTOMETRY:

The technology has applications in a number of fields, including molecular biology,

pathology, immunology, plant biology and marine biology. It has broad application in

medicine especially in transplantation, hematology, tumor immunology and

chemotherapy, genetics and sperm sorting for sex pre-selection. In marine biology, the

auto-fluorescent properties of photosynthetic plankton can be exploited by flow

cytometry in order to characterize abundance and community structure. In protein

engineering, flow cytometry is used in conjunction with yeast display and bacterial

display to identify cell surface-displayed protein variants with desired properties. It is

also used to determine ploidy of grass carp fry.

Now days it used in the field of immunology and pathology for determination of patients

immune cells examination.

FLOW CYTOMETRY has been the method of choice for monitoring CD4 lymphocytes

levels in the blood of AIDS patients.

ARIBAS 25

Chapter 1: Introduction

Lymphomas and leukemia’s are intensively studied for surface markers of diagnostic and

prognostic values. In the diagnosis of leukemia, flow cytometry may be used for the

immunophenotypic analysis of abnormal cells by focusing on cell lineage.

In renal, cardiac and bone marrow transplants, flow cytometry is used in discriminating

between graft rejection and viral infections in post-operative patients.

ARIBAS 26

CHAPTER-2 Review of literature

Chapter 2: Review of Literature

2.1 History of immunology:

The subject of immunology belongs to the biological and medical sciences. The term

immunochemistry was coined by the Swedish chemist ARRHENIUS who used it for the

first in this “chemical reactions of substances that occur in the blood of animals after

injection of foreign substances. i.e., after immunization.

2.2 History of kidney transplantation:

The first cadaveric kidney transplantation in the United States was performed June 17,

1950, on Ruth Tucker, a 44-year-old woman with polycystic kidney disease, at Little

Company of Mary Hospital in Evergreen Park, Illinois. Although the donated kidney was

rejected ten months later because no immunosuppressive therapy was available at the

time—the development of effective anti-rejection drugs was years away—the intervening

time gave Tucker's remaining kidney time to recover and she lived another five years.

Dr. John P. Merrill (left) explains the workings of a

then-new machine called an artificial kidney to

Richard Herrick (middle) and his brother Ronald

(right). The Herrick twin brothers were the subjects

of the world's first successful kidney transplant,

Ronald being the donor.1954: First successful kidney transplant by Joseph Murray

(Boston, U.S.A.)

The first kidney transplants between living patients were undertaken in 1954 in Boston

and Paris. The Boston transplantation, performed on December 23, 1954, at Brigham

Hospital was performed by Joseph Murray, J. Hartwell Harrison, John P. Merrill and

others. The procedure was done between identical twins to eliminate any problems of an

immune reaction. For this and later work, Dr. Murray received the Nobel Prize for

Medicine in 1990. The recipient died eight years after the transplantation.

ARIBAS 30

Chapter 2: Review of Literature

The first kidney transplantation in the United Kingdom did not occur until 1960, when

Michael Woodruff performed one between identical twins in Edinburgh. Until the routine

use of medications to prevent and treat acute rejection, introduced in 1964, deceased

donor transplantation was not performed. The kidney was the easiest organ to transplant:

tissue typing was simple, the organ was relatively easy to remove and implant, live

donors could be used without difficulty, and in the event of failure, kidney dialysis was

available from the 1940s. Tissue typing was essential to the success: early attempts in the

1950s on sufferers from Bright's disease had been very unsuccessful.

The major barrier to organ transplantation between genetically non-identical patients lay

in the recipient's immune system, which would treat a transplanted kidney as a "non-self"

and immediately or chronically, reject it. Thus, having medications to suppress the

immune system was essential. However, suppressing an individual's immune system

places that individual at greater risk of infection and cancer (particularly skin cancer and

lymphoma), in addition to the side effects of the medications.

The basis for most immunosuppressive regimens is prednisolone, a corticosteroid.

Prednisolone suppresses the immune system, but its long-term use at high doses causes a

multitude of side effects, including glucose intolerance and diabetes, weight gain,

osteoporosis, muscle weakness, hypercholesterolemia, and cataract formation.

Prednisolone alone is usually inadequate to prevent rejection of a transplanted kidney.

Thus other, non-steroid immunosuppressive agents are needed, which also allow lower

doses of prednisolone.

Indications of disease in kidney:

The indication for kidney transplantation is end-stage renal disease (ESRD), regardless of

the primary cause. This is defined as a glomerular filtration rate <15ml/min/1.73 sq.m.

Common diseases leading to ESRD include malignant hypertension, infections, diabetes

mellitus, and focal segmental glomerulosclerosis; genetic causes include polycystic

kidney disease, a number of inborn errors of metabolism, and autoimmune conditions

such as lupus and Good pasture’s syndrome. Diabetes is the most common cause of

ARIBAS 31

Chapter 2: Review of Literature

kidney transplantation, accounting for approximately 25% of those in the US. The

majority of renal transplant recipients are on some form of peritoneal dialysis, or the

similar process of hemofiltration—at the time of transplantation. However, individuals

with chronic renal failure who have a living donor available may undergo pre-emptive

transplantation before dialysis is needed.

In 2004 the FDA approved the Cedars-Sinai High Dose IVIG therapy which reduces the

need for the living donor to be the same blood type (ABO compatible) or even a tissue

match. The therapy reduced the incidence of the recipient's immune system rejecting the

donated kidney in highly sensitized patients.

In 2009 at the Johns Hopkins Medical Center, a healthy kidney was removed through the

donor's vagina. Vaginal donations promise to speed recovery and reduce scarring. The

first donor was chosen as she had previously had a hysterectomy. The extraction was

performed using natural orifice transluminal endoscopic surgery, where an endoscope is

inserted through an orifice, then through an internal incision, so that there is no external

scar. The recent advance of single port laparoscopy requiring only one entry point at the

navel is another advance with potential for more frequent use.

Donors can be divided in two groups:

Brain-dead (BD) donors

Donation after Cardiac Death (DCD) donors

Although brain-dead (or "beating heart") donors are considered dead, the donor's heart

continues to pump and maintain the circulation. This makes it possible for surgeons to

start operating while the organs are still being perfused. During the operation, the aorta

will be cannulated, after which the donor's blood will be replaced by an ice-cold storage

solution, such as UW (Viaspan), HTK, or Perfadex. Depending on which organs are

transplanted, more than one solution may be used simultaneously. Due to the temperature

of the solution, and since large amounts of cold NaCl-solution are poured over the organs

for a rapid cooling, the heart will stop pumping.

ARIBAS 32

Chapter 2: Review of Literature

"Donation after Cardiac Death" donors are patients who do not meet the brain-dead

criteria but, due to the small chance of recovery, have elected via a living will or through

family to withdraw support. In this procedure, treatment is discontinued (mechanical

ventilation is shut off). After a time of death has been pronounced, the patient is rushed to

the operating room where the organs are recovered. Storage solution is flushed through

the organs. Since the blood is no longer being circulated, coagulation must be prevented

with large amounts of anti-coagulation agents such as heparin. Several ethical and

procedural guidelines must be followed; most importantly, the organ recovery team

should not participate in the patient's care in any manner until after death has been

declared.

Compatibility

If plasmapheresis or IVIG is not performed, the donor and recipient have to be ABO

blood group compatible. Also, they should ideally share as many HLA and "minor

antigens" as possible. This decreases the risk of transplant rejection and the need for

another transplant. The risk of rejection may be further reduced if the recipient is not

already sensitized to potential donor HLA antigens, and if immunosuppressant levels are

kept in an appropriate range. The level of sensitization to donor HLA antigens is

determined by performing a panel reactive antibody test on the potential recipient. In the

United States, up to 17% of all deceased donor kidney transplants have no HLA

mismatch. However, HLA matching is a relatively minor predictor of transplant

outcomes. In fact, living non-related donors are now almost as common as living

(genetically)-related donors.

In the 1980s, experimental protocols were developed for ABO-incompatible transplants

using increased immunosuppressant and plasmapheresis. Through the 1990s these

techniques were improved and an important study of long-term outcomes in Japan was

published. Now, a number of programs around the world are routinely performing ABO-

incompatible transplants.

ARIBAS 33

Chapter 2: Review of Literature

Transplantation medicine is one of the most challenging and complex areas of modern

medicine. Some of the key areas for medical management are the problems of transplant

rejection, during which the body has an immune response to the transplanted organ,

possibly leading to transplant failure and the need to immediately remove the organ from

the recipient. When possible, transplant rejection can be reduced through serotyping to

determine the most appropriate donor-recipient match and through the use of

immunosuppressant drugs. (Ref.3)

2.3 History of Flow cytometry

The first impedance-based flow cytometry device, using the Coulter principle, was

disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. The first

fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang

Göhde from the University of Münster and first commercialized in 1968/69 by German

developer and manufacturer Partec through Phywe AG in Göttingen. At that time,

absorption methods were still widely favored by other scientists over fluorescence

methods. Soon after, flow cytometry instruments were developed, including the

Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the

PAS 8000 (1973) from Partec, the first FACS instrument from Becton Dickinson (1974),

the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78).

Flow cytometry developed from microscopy. Thus Leeuwenhoek is often cited in any

discussion regarding its history.

F.T.Gucker (1947) builds the first apparatus for detecting bacteria in a LAMINAR

SHEATH stream of air.

L.Kamentsky (IBM Labs) and M.Fulwyler (Los Alamos Nat. Lab.) experimented with

fluidic switching and electrostatic cell sorters respectively. Both described cell sorters in

1965.

ARIBAS 34

Chapter 2: Review of Literature

M.Fulwyler utilized pulse height analyzers to accumulate distributions from a coulter

counter. This feature allowed him to apply statistical analysis to samples analyzed by

flow.

In 1972 L.Herzenberg (Stanford university), developed a cell sorter that separated cells

stained with fluorescent antibodies. The .Herzenberg group coined the term

Fluorescence-activated cell sorting (FACS).

Name of the technology

The original name of the flow cytometry technology was "pulse cytophotometry"

(German: Impulszytophotometrie). Only 20 years later in 1988, at the Conference of the

American Engineering Foundation in Pensacola, Florida, the name was changed to "flow

cytometry", a term that quickly became popular. (Ref.-Wikipedia for history)

ARIBAS 35

CHAPTER-3Objectives

ARIBAS

Chapter 3: Objectives

3.1 AIM OF THE STUDY:

1. To study the role of T-Regulatory cells (T-regs) in kidney transplantation.

2. To evaluate T-regs in Tolerance Induction protocol (TIP) using stem cells (CDB).

3. To evaluate T-regs in TIP without stem cell (MCDB).

4. To evaluate (T-regs) in kidney transplant patients who have not undergone TIP

(control patients).

5. To Compare T-regs in CDB, MCDB and control patients.

6. To evaluate the role of T-reg in kidney transplantation tolerance and autoimmune

diseases.

ARIBAS 36

CHAPTER-4MATERIALS AND METHODS

Chapter 4: Materials and Methods

This was the prospective study carried out for a period of 4 months between 28th April to

24th Aug.2011 at Smt. G.R.Doshi and Smt. K.M.Mehta Institute of kidney diseases and

Research center (IKDRC) - Dr.H.L.Trivedi Institute of Transplantation sciences (ITS),

Ahmedabad.

SELECTION OF PATIENTS:- There were three sets of patients:

Group A: It comprised of the kidney transplant patients who underwent tolerance

induction protocol using stem cells (TIP)

Group B: It comprised of the kidney transplant patients who underwent tolerance

induction protocol without using stem cells

Group C- Group A: It comprised of the kidney transplant patients who did not opt for

any kind of tolerance induction protocol and were transplanted under the standard

tripe drug immune suppression.

Graft function in above groups of patients in terms of serum creatinine and incidence

of rejection was studied.

Serum creatinine levels were evaluated by using Jaffe’s method,

Rejection data was taken from the patient charts

T-regulatory cells (T-regs) in all the three groups were evaluated at different time

intervals after transplantation, in groups A and B they were studied at 1, 3, 6, and 9

months post-transplantation and in group C they were studied as and when patients

allowed to draw blood samples. These were evaluated by using flowcytometer

(FACScan, BD, USA). And basically we analysis the data table of only 9 month.

ARIBAS 37

Chapter 4: Materials and Methods

BLOOD COLLECTION:

Blood was drawn from venipuncture from any arm from cubital fossa and 2 ml

was drawn by technician in to syringe under proper aseptic and antiseptic

precautions.

The test tubes were centrifuged at 2000 rpm for 5 minutes in swinging bucket

centrifuge.

Serum was separated and collected into plastic cups of the auto-analyzer.

EQUIPMENTS:

Flow-cytometer, spectrophotometer, table top centrifuge machine,

Vortex, sterile BD Falcon Centrifuge Tubes and BD Falcon Test Tubes,

Syringe, Niddles.

4.1 CREATININE TEST:

Creatinine is a chemical waste molecule that is generated from muscle metabolism.

Creatinine is produced from creatine, a molecule of major importance for energy

production in muscles. Approximately 2% of the body's creatine is converted to

creatinine every day. Creatinine is transported through the bloodstream to the kidneys.

The kidneys filter out most of the creatinine and dispose of it in the urine.

Because the muscle mass in the body is relatively constant from day to day, the creatinine

level in the blood normally remains essentially unchanged on a daily basis.

ARIBAS 38

Chapter 4: Materials and Methods

The kidneys maintain the blood creatinine in a normal range. Creatinine has been found

to be a fairly reliable indicator of kidney function.

As the kidneys become impaired for any reason, the creatinine level in the blood will rise

due to poor clearance by the kidneys. Abnormally high levels of creatinine thus warn of

possible malfunction or failure of the kidneys. It is for this reason that standard blood

tests routinely check the amount of creatinine in the blood. A more precise measure of the

kidney function can be estimated by calculating how much creatinine is cleared from the

body by the kidneys and it is referred to creatinine clearance.

Significance of creatinine

Creatinine is a natural by-product of muscles doing work in your body. It starts out as

creatine phosphate, and it ends up as a waste product in your blood which is then

eliminated in urine. This waste product can be easily measured in both blood and urine,

and, because it is released at a steady rate by your skeletal muscles, it is an excellent

indicator of kidney function. Unlike urea, which also measures kidney function to some

extent, creatinine is only slightly affected by the meat proteins you eat. As a result, it is a

more precise, more specific measure of your kidney function than urea is.

FIGURE: 10 CREATININE SYNTHESES

ARIBAS 39

Chapter 4: Materials and Methods

Exterminations of creatinine:

Creatinine is estimated by the modified jaffe’s method.

PRINCIPLE

Creatinine react with alkaline picrate solution, forms an orange yellow color complex.

Specificity of the assay has been improved by the introduction of an initial rate method.

However, cephalosporin antibiotics are still a major interferon’s. The absorbance of the

orange yellow color formed is directly proportional to creatinine concentration and is

measured photomerically at 500-520 nm.

FIGURE: 11 CYCLE OF CREATININE

ARIBAS 40

Chapter 4: Materials and Methods

REAGENTS

     Reagent A

Picric acid reagent (25.8 mmol/l)

 Reagent B

Sodium hydroxide (95 mmol/l)

     Reagent C

Creatinine 2 mg/dl (0.166 mmol/l

Reagent preparation

Mix equal volume of reagent 1 and 2 wait for 15 minutes before use.

This reagent is used as working reagent.

Pipette Standard Test

Working reagent 1000 μl 1000 μl

Standard 100μl -

Test - 100μl

Hemolysed sample should be discarded as hemolysis increase non-creatinine

chromogens.

Specimens are stable for 12 hours at room temperature (at 250 C), for 1 week

when refrigerated at 2-60 C and longer if stored frozen (at < -200 C).

ARIBAS 41

Chapter 4: Materials and Methods

Calculations

(A2-A1) of sample

Creatinine=   -------------------------- x Concentration of STD (2.0 mg/dl)

                         (A2-A1) of Standard      

Normal Values of Creatinine:

Serum creatinine: Men: 0.7–1.4 milligrams per deciliter (mg/dL)

Women: 0.6–1.2 mg/dL

Teen: 0.5–1.0 mg/dL

Child: 0.3–0.7 mg/dL

Newborn: 0.3–1.2 mg/dL

ARIBAS 42

Chapter 4: Materials and Methods

4.2 Flow Cytometry:

Flow cytometry uses the principles of light scattering, light excitation, and emission of

Fluor chrome molecules to generate specific multi-parameter data from particles and cells

in the size range of 0.5um to 40um diameter.

Cells are hydro-dynamically focused in a sheath of PBS before intercepting an optimally

focused light source (See Figure). Lasers are most often used as a light source in flow

cytometry. Flow cytometer has ability to perform multi-parameter analyses on a single

cell. The many measurable properties are size, volume, viscosity, the content of DNA,

RNA and enzymes, surface antigens and immunological cells.

Flow cytometers use the principle of hydrodynamic focusing for presenting cells to a

laser (or any other light excitation source). The sample is injected into the center of a

sheath flow. The combined flow is reduced in diameter, forcing the cell into the center of

the stream. This the laser one cell at a time.

This schematic of the flow chamber in relation to the laser beam in the sensing area.

FIGURE: 12 PASSING OF CELL SUSPENSION THROUGH THE NOZZLE.

ARIBAS 43

Chapter 4: Materials and Methods

Principle:

A beam of light (usually laser light) of a single wavelength is directed onto a hydro

dynamically-focused stream of liquid. A number of detectors are aimed at the point

where the stream passes through the light beam: one in line with the light beam (Forward

Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more

fluorescent detectors.

Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters

the ray, and fluorescent chemicals found in the particle or attached to the particle may be

excited into emitting light at a longer wavelength than the light source. This combination

of scattered and fluorescent light is picked up by the detectors, and, by analyzing

fluctuations in brightness at each detector (one for each fluorescent emission peak), it is

then possible to derive various types of information about the physical and chemical

structure of each individual particle.

FSC correlates with the cell volume and SSC depends on the inner complexity of the

particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the

membrane roughness). This is because the light is scattered off of the internal

components of the cell. Some flow cytometers on the market have eliminated the need for

fluorescence and use only light scatter for measurement.

ARIBAS 44

Chapter 4: Materials and Methods

FIGURE: 13 Mechanism of flow-cytometry florescent markers

ARIBAS 45

Chapter 4: Materials and Methods

Working Methodology:The following are the steps involved in FACS:

1. Before any procedure is performed the fluorescence activated cell sorter must be

aligned and calibrated in order to obtain accurate and precise data.

2. The desire single cell suspension is isolated from the blood or tissue and labeled

with a fluorescent monoclonal antibody.

3. For each individual cellular property being analysed, a different “colour”

flurochrome or monoclonal antibody must be used so that the computer can

electronically distinguish the properties of the cell surfaces.

4. These fluorescent markers allow the laser to recognize the cell and record data of

that particular cell.

5. By the application of air pressure, cells are forced through a nozzle, they are met

with a liquid jet of saline or sheath fluid that protect the cells.

6. Vibrations at the tip of the nozzle interrupt the stream in order to break it up into a

series of droplets, and each contain a single cell.

7. in droplet flurocent label cells are negatively charged where as non flurocent label

are positively charged.

8. Flow-cytometric data is primarily displayed as a histogram or plot. The X-axis of

the histogram displays the fluorescence intensity, which is usually measured on a

log scale.

9. The Y- axis displays the number of the cells found within each parameter. When

measuring three or more parameters, the histogram is usually displayed in a three-

dimensional, colour co-ordinated display.(Ref.:6 Wikipedia of flow cytometry)

ARIBAS 46

Chapter 4: Materials and Methods

FIGURE: 14 Flow cytometer - the Becton-Dickinson FACSCalibur

FIGURE: 15 Set-up of the flow-cytometer attach with computer:

ARIBAS 47

Chapter 4: Materials and Methods

Reagent:

1. Florescent dye conjugated monoclonal antibodies are:

CD127 mAb PerCP, CD4mAb PE, CD25 mAb FITC,

CD 8 mAb PE, CD 3 mAb PerCP, CD 4 mAb FITC,

CD45 mAb FITC, CD 33 mAb PerCP, CD 34 mAb PE.

2. FACS LYSING SOLUTION,

3. FACS SHEETH FLUIID.

4.3 BASIC INFORMATION OF MARKERS:

1. PerCP- Pridinin chlorophyll protein complex Peridinin Chlorophyll Protein Complex (PerCP)-conjugated antibodies are convenient

tools for use in flow cytometry experiments. PerCP is a water soluble carotenoid pigment

found in photosynthetic dinoflagellates. It is excited by a 488 nm argon laser, and with a

relatively large Stokes shift, emits at a maximum wavelength of 675 nm. Because of

these spectral characteristics, there is minimal overlap with other commonly used

fluorochromes such as phycoerythrin (PE) or fluorescein. This makes PerCP-labeled

antibodies especially useful for multi-color analysis with PE and fluorescein-conjugated

antibodies. In addition, low cross talk between channels reduces the time spent setting

fluorescent compensation.

2. FITC- Fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging

applications including flow cytometry. FITC is the original fluorescein molecule

functionalized with an isothiocyanate reactive group (-N=C=S), replacing a hydrogen

atom on the bottom ring of the structure. This derivative is reactive towards nucleophiles

including amine and sulfhydryl groups on proteins.

ARIBAS 48

Chapter 4: Materials and Methods

A succinimidyl-ester functional group attached to the fluorescein core, creating NHS-

fluorescein, forms another common amine reactive derivative that has much greater

specificity toward primary amines in the presence of other nucleophiles. FITC has

excitation and emission spectrum peak wavelengths of approximately 495 nm/521 nm.

3. PE-Phycoerythrin

Phycoerythrin is a red protein from the light-harvesting phycobiliprotein family, present

in cyanobacteria, red algae and cryptomonads. Phycoerythrin is composed of a protein

part, organized in a hexameric structure of alpha and beta chains, covalently binding

chromophores called phycobilins. In the phycoerythrin family, the phycobilins are:

phycoerythrobilin, the typical phycoerythrin acceptor chromophore, and sometimes

phycourobilin (marine organisms). Phycoerythrins are the phycobiliproteins that bind

the highest number of phycobilins (up to six per alpha-beta subunit dimer).

Absorption peaks in the visible light spectrum are at 495 and 545/566 nm, depending on

the chromophores bound and the considered organism. A strong emission peak exists at

575 ± 10 nm. (i.e., phycoerythrin absorbs slightly blue-green/yellowish light and emits

slightly orange-yellow light.). R-Phycoerythrin, or PE, is useful in the laboratory as a

fluorescence-based indicator for the presence of cyanobacteria and for labeling antibodies

in a technique called immunofluorescence, among other applications. There are also other

types of phycoerythrins, such as B-Phycoerythrin, which has slightly different spectral

properties. B-Phycoerythrin absorbs strongly at about 545 nm (slightly yellowish green)

and emits strongly at 572 nm (yellow) instead and could be better suited for some

instruments. B-Phycoerythrin may also be less "sticky" than R-Phycoerythrin and

contributes less to background signal due to non-specific binding in certain applications.

R-Phycoerythrin and B-Phycoerythrin are among the brightest fluorescent dyes ever

identified.

The immune system is the body’s natural defence in combating organisms. Immunology

has developed rapidly over the past decade owing to the refinements made in the

molecular tests employed in this area of research.

ARIBAS 49

Chapter 4: Materials and Methods

FIGURE: 16 Photographs of Marker used in Flow-cytometry

ARIBAS 50

Chapter 4: Materials and Methods

Reagents:

1. Florescent dye conjugated monoclonal antibodies are:

CD127 mAb PerCP, CD4mAb PE, CD25 mAb FITC,

CD 8 mAb PE, CD 3 mAb PerCP, CD 4 mAb FITC,

CD45 mAb FITC, CD 33 mAb PerCP, CD 34 mAb PE.

2. FACS LYSING SOLUTION,

3. FACS SHEETH FLUIID.

Sample: Peripheral blood,

Blood is drawn from vein puncture into 2 ml syringe with EDTA anticoagulant.

Method of Flow Cytometry:Using FACScan (Becton-Dickinson, CA, U.S.A), in flow-cytomatry we analyzed by

using this markers CD4+, CD25+, CD4+, CD3+, CD8+,CD33+,CD34+,CD45+ cell lines

every 3 months post-transplantation in peripheral blood.

We used CD3 mAb (PerCP conjugated), CD4 mAb (fluorescein isothiocynate

(FITC conjugated) and CD8 mAb (phycoerythrin (PE) conjugated),

CD127 mAb (PerCP conjugated), CD4 mAb (PE conjugated), CD25 mAb (FITC

conjugated),

CD33 mAb (PerCP conjugated), CD34 mAb (PE conjugated), CD 45 m Ab (FITC

conjugated). These three PE, PerCP, and FITC give the three different colors for

specific cells identification.

The monoclonal antibodies were purchased from B.D.Biosciences, CA, U.S.A.

ARIBAS 51

Chapter 4: Materials and Methods

The method was as under:

1. Take 3 FACS tube so make 3 set ,

2. In first tube Add 20 μl of monoclonal antibody marker CD127,CD4, CD25,

3. In second tube Add 20 μl of monoclonal antibody marker CD8,CD3,CD4,

4. In third tube Add 20 μl of monoclonal antibody marker CD45, CD33, CD34.

5. Add 100 μl of blood in all marker tubes,

6. Incubate in dark for 30 minutes,

7. Add 2ml of 1x lysing solution,

8. Vortex for 5 seconds,

9. Incubate for 10minutes in dark place,

10. Centrifuge tube at 5000rpm for 5 minutes,

11. Discard the supernatant and use the pellet,

12. Add 1ml of sheath fluid,

13. Vortex 5 seconds,

14. Centrifuge tube at 1000rpm for 5 minutes,

15. Discard the supernatant and used the pellet,

16. Add 500ml of sheath fluid and examine the sample in flowcytometer.

ARIBAS 52

Chapter 4: Materials and Methods

The Flow cytometer is connected with the computer system and data entered in the

specific folders for the specific markers.

ARIBAS 53

CHAPTER-5RESULTS

Chapter 5: Results

HOW THE TESTS WERE CARRIED OUT??? The relation between T-reg. cells and serum creatinine (SCR) level in the post-

kidney transplantation patients were studied. Three groups of post-kidney transplantation

patients were identified to observe the rejection and autoimmune diseases. In the study

group A and B were applied tolerance induction protocol and group C was of control

patients and were not applied tolerance induction protocol. In all these groups, each

patient’s blood group and Human Lymphocytes Antigen typing (HLA) were examined

before the transplantation.

For the group A, stem cells therapy called clonal deletion bortezomide protocol (CDB)

was used. The group, after transplantation, was administered the immunosuppressive

drugs and stem cells for decreasing the chances of rejection.

While the patients of group B, which were treated without stem cell therapy, called

Modified clonal deletion protocol (MCDB), only immunosuppressive drugs were given

to the patients. The dose of immunosuppressive drug was somewhat higher compared to

the dose of that of control patients. In MCDB, drug used are the modified drug -mofetil

(MMF) or calcineurin inhibitors (CNI).

The last groups C which comprised of control patients, only conventional

immunosuppressive drugs were administered to these patients. They were not applied the

tolerance induction protocol. The drugs used were- mofetil (MMF) or calcineurin

inhibitors (CNI) and prednisone.

ARIBAS 54

Chapter 5: Results

5.1 THE GROUP A- CLONAL DELETION BORTEZOMIDE

(CDB)

The Group A (CDB) comprised of 12 patients with mean age of 32.7± 6.7 (range:

24-45) years, with 3 females and 9 males. The most common original disease to cause

kidney failure was chronic glomerulonephritis (CGN). The mean HLA match was 2.09 ±

1.58. The donors were spouses, parents or siblings with mean age of 43 ± 11.8 (range:

26-60) years. Over a mean follow-up of 0.79 ± 0.36 (range: 0.35-1.15) years the mean

serum creatinine (SCR) was 1.39 ± 0.43 (range: 0.87-2.4) mg/dL.

In total 4 (four) patients were on no conventional immunosuppression and 8 (eight)

patients were rescued with mycophenolate mofetil (MMF) or calcineurin inhibitors

(CNI). There were 3 (three) cases of rejection. Their T-reg mean levels on the 9 th month

was 4.74 ± 2.99 %.

The T-reg. cell values and serum creatinine values were analysed for 9 (nine)

months.

The rejections in the patients are marked by the red colour in the TABLE 1

ARIBAS 55

Chapter 5: Results

TABLE: 1: GROUP: A- “CDB- PATIENTS”CDB'09

TX

DONE PT.NAME AGE GEN DISEASE HLA DONOR AGE

TX

DATE

F.UP

Yrs SCR T-reg

1 RS 36 M CGN 2 WIFE 32 1-May-10 1.12 1.33 2.91

2 RD 31 M CGN 0 WIFE 26 9-Apr-10 1.34 1.37 2.84

3 LP 42 M CGN 1 WIFE 39 2-Jul-10 1.12 1.3 2.74

4 MS 29 M CGN 4 FATHER 51 25-Jan-10 1.13 1.71 7.62

5 GA 29 F CGN 3 FATHER 54 2-Aug-10 1.03 1.11 5.72

6 JK 40 F

SINGLE KIDNEY

CGN 1 HUSBUND 43

30-Nov-

10 0.71 0.87 1.98

7 MA 24 M MPGN 4 MOTHER 58

17-Nov-

10 0.75 1.97 3.48

8 AP 28 M HT 3 MOTHER 50

22-Dec-

10 0.65 1.26 7.16

9 TS 30 M LUPUS 0 MOTHER 60 7-Mar-11 0.44 2.24 5.83

10 GP 45 M CGN 0 WIFE 40 4-Apr-11 0.37 1.24 3.16

11 BJ 33 M CGN 1 WIFE 32

14-Apr-

11 0.34 1.36 3.96

12 SS 25 F CGN 4 BROTHER 28

20-Apr-

11 0.33 0.97 4.66

MEAN 32.7 1.9 43 0.7775 1.394 4.74

ARIBAS 56

Chapter 5: Results

1.33

2.91

1.37

2.84

1.3

2.74

1.71

7.62

1.11

5.72

0.87

1.98 1.97

3.48

1.26

7.16

2.24

5.83

1.24

3.16

1.36

3.96

0.97

4.66

0

1

2

3

4

5

6

7

8

LEVEL

1 2 3 4 5 6 7 8 9 10 11 12

PATIENTS NO.

CDB PATIENT'S PROTOCOL

SCR T-reg

Graph: 1 CDB patients SCR and T-reg values

ARIBAS 57

Chapter 5: Results

5.2 THE GROUP B - MODIFIED- CLONAL DELETION

BORTEZOMIDE (MCDB)

The Group B (MCDB) was comprised of 13 patients with mean age of 34.15 ± 8.51

(range: 19-45) years. All were males. The most common original disease to cause kidney

failure was CGN. The mean HLA match was 2.5 ± 1.33. The donors were spouses,

parents or siblings with mean age of 42.3 ± 12.1 (range: 25-60) years. Over a mean

follow up of 0.81 ± 0.5 (range: 0.2-1.72) years the mean SCR was 1.57 ± 0.36 (range:

1.16-2.23) mg/dL. Their mean T-reg levels at 9 month 5.8 ± 2.3 % respectively.

(Table-2).

From the total 13 patients of the group B, 5(five) patients were on no conventional

immunosuppression and 8 (eight) patients were rescued with mycophenolate mofetil

MMF or calcineurin inhibitors CNI.

There were 2 (two) cases of rejection, marked by the red colour (Table-2).

ARIBAS 58

Chapter 5: Results

TABLE: 2: GROUP: B- “MCDB- PATIENTS”

MCDB'09 TX

DONE PT.NAME AGE GEN DISEASE HLA DONOR AGETX

DATEF.UP

Yrs SCR

T-Reg Cell

1 PG 42 MBENIGN

NEPHROSCLEOSIS 0 WIFE 355-Dec-

09 1.68 1.37 6.92

2 NJ 32 M CGN 2 WIFE 3026-

Nov-09 1.71 1.16 3.55

3 RY 34 M CGN 3 WIFE 2529-Jan-

90 1.12 2.21 5.33

4 GS 29 M HT 3 WIFE 255-Aug-

10 1.02 1.53 6.61

5 GY 44 M CGN 0 WIFE 389-Aug-

10 1.01 1.26 7.31

6 DP 30 M HTN+CGN 4 MOTHER 5028-Oct-

10 0.8 1.91 6.90

7 IR 39 M CGN 3 MOTHER 581-Nov-

10 0.79 2.23 7.08

8 RM 19 M CGN 3 MOTHER 4212-Feb-

11 0.51 1.34 4.09

9 KM 21 M CIN 3 MOTHER 4515-Feb-

11 0.5 1.32 3.58

10 SP 40 M HTN+CGN 3 MOTHER 605-Mar-

11 0.45 1.4 6.72

11 SS 41 M CGN 3 WIFE 4221-Apr-

11 0.32 1.39 4.76

12 HT 45 M CGN 1 WIFE 402-Jun-

11 0.21 1.4 7.21

13 HM 28 M MN 4 FATHER 609-Jun-

11 0.19 1.6 3.53

MEAN 34.2 2.5 42 0.79 1.57 5.8

ARIBAS 59

Chapter 5: Results

1.37

6.92

1.16

3.55

2.21

5.33

1.53

3.23

1.26

7.31

1.91

6.90

2.23

7.08

1.34

4.09

1.32

3.58

1.4

6.72

1.39

4.76

1.4

7.21

1.96

3.53

0

1

2

3

4

5

6

7

8

LEVEL

1 2 3 4 5 6 7 8 9 10 11 12 13

PATIENTS NO.

MCDB PATIETS PROTOCOL

SCR T-reg

Graph:2 MCDB patients SCR and T-reg values

ARIBAS 60

Chapter 5: Results

5.3 THE GROUP C - CONTROL PATIENTS

The Group C comprised of 21 patients with mean age of 32.2 ± 11.9 (range:11-53)

years and there were 5 females and16 males in this group. The most common original

disease to cause kidney failure was CGN. The mean HLA match was 2.4 ± 1.4. The

donors were spouses, parents or siblings with mean age of 47 ± 9.6 (range: 32-68) years.

Over a mean follow up of 0.97 ± 1.04 (range: 0.44-2.27) years the mean SCR was 1.28 ±

0.4 (range: 0.69-2.24 ) mg/dL. The mean T-reg levels of patients with SCR <1.5 mg/dL

was 5.14 ± 1.46 and the mean T-reg levels of patients with SCR >1.5 mg/dL was 3.14 ±

2.02 (Table-3).

All the patients of this group were on the three conventional drugs used for

immunosuppression - CNI, MMF and prednisone. There were 5 rejection marked by red

colour in the table. In this group no tolerance induction protocol is applied so rejection is

more compare to group A and B.

In this group conventional immunosuppressant drug and treatment given to the

patients.

ARIBAS 61

Chapter 5: Results

TABLE: 3: GROUP: C -“CONTROL PATIENT”

Pt. NO, Pt. NAME Rg.NO. TX DATESAMPLE DT. CREATININE mg/dl T-reg

1 AC 174727 6-May-09 18-Feb-11 0.85 2.972 VA 207055 15-Dec-10 18-Feb-11 1.9 1.333 GD 207263 10-Jan-11 18-Feb-11 1.18 2.964 HH 190578 10-Jan-11 18-Feb-11 0.94 3.285 AN 209442 31-Jan-11 28-Feb-11 1.41 1.296 RM 209190 21-Feb-11 11-Mar-11 0.95 6.817 VK 209899 21-Feb-11 11-Mar-11 0.88 6.128 GR 190880 2-Mar-11 18-Mar-11 0.98 5.099 SJ 185314 1-Jun-10 18-Mar-11 1.64 3.37

10 AS 207136 4-Feb-11 18-Mar-11 1.7 4.4411 AK 207389 13-Jan-11 18-Mar-11 1.12 5.0112 HG 155189 27-May-10 18-Mar-11 2.24 0.0913 MP 132971 1-Aug-06 18-Mar-11 1.58 5.3914 VP 161813 6-Nov-09 18-Mar-11 1.31 5.9715 RP 197479 23-Feb-11 21-Mar-11 1.54 4.2316 SD 202879 23-Feb-11 22-Mar-11 1.18 0.4217 SF 207193 1-Dec-10 23-Mar-11 0.69 1.118 AM 195553 12-Mar-11 28-Mar-11 1.17 2.1719 M 201220 25-Feb-11 28-Mar-11 1.08 5.8320 MS 207787 22-Mar-11 4-Apr-11 1 5.7321 JC 210847 14-Mar-11 4-Apr-11 1.56 6.92

MEAN 1.28 3.8

ARIBAS 62

Chapter 5: Results

0.85

2.97

1.91.331.18

2.96

0.94

3.28

1.411.29

0.95

6.81

0.88

6.12

0.98

5.09

1.64

3.37

1.7

4.44

1.12

5.01

2.24

0.09

1.58

5.39

1.31

5.97

1.54

4.23

1.18

0.42 0.69

1.11.17

2.17

1.08

5.83

1

5.73

1.56

6.92

0

1

2

3

4

5

6

7

LEVEL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21PATIENTS NO.

CONTROL PATIENTS

CREATININE mg/dl T-reg

ARIBAS 63

Chapter 5: Results

RESULTS IN BRIEF…….

All the groups were fairly balanced demographically. Group A showed better graft

function in terms of SCR, rejection episodes and immunosuppression requirement. The

T-regs showed sustained rise in group A compared to group B. Both these groups were

functionally better than the group C. The study of T-regs in the group C showed that if

SCR was high T-regs were low and if SCR was low T-regs were high

. Thus the role of T-regs as immunomodulators in achieving and sustaining transplant

tolerance was well established by this study.

ARIBAS 64

Chapter 5: Results

FIGURE: 17 FLOWCYTOMETRY REAULT SHOWN IN THE COMPUTER

The graph at the top is the graph as can be seen in the computer attached with the flow- cytometre .The red coloured part shows the lymphocytes

The graph at the centre shows T-cells. When CD 127 marker is used, the T-cells are seen green colored. The graph at the bottom shows T-reg cells The CD 4+ and CD 25+ are the two markers specifically used to identify the T-reg cells. The markers CD4+ gives blue and CD25+ gives red colour to the T-reg cells. In the upper right part of the graph at the bottom, Treg cells are identified using CD4+ And CD25+ markers

ARIBAS 65

CHAPTER-6CONCLUSION

Chapter 6: Conclusion

As it is well established now that the, T-regulatory cells are the new research area of

the in organ transplantation.

It is widely accepted that Tregs play a pivotal role in tolerance induction. Therefore, an

increase in circulating Tregs may be beneficial to the grafted kidney in terms of immune

tolerance. The results of our study revealed that the frequency of circulating Tregs is

significantly reduced by strong immunosuppressant such as mycophenolate mofetil

(MMF) or calcineurin inhibitors (CNI), and that further reduced the frequency of

circulating Tregs. The decrease in frequency of Tregs seen at one and two weeks after

renal transplant and gradually disappeared by eight weeks after transplant. The findings

suggest that high doses of CNIs at the time of transplantation prevent the development of

Tregs and that tapering of CNI may prevent a further decrease in circulating Tregs. Thus,

treatment with the early post-transplant period should be used cautiously and high doses

of CNIs should be avoided because it may inhibit the development of immune tolerance.

Further evaluation of the long-term effect of CNIs on circulating Tregs and the optimal

frequency of circulating Tregs during the early transplant period in kidney transplantation

is needed. So, due to this reason in the Group A CDB using stem cell, rejection is more

compare to MCDB because of high doses of CNI and MMF.

FOXP3+CD25+ CD4+ Treg. Cells are naturally present in the normal immune system

as a phenotypically and functionally distinct T cell subpopulation. They are therefore a

good target for designing way to teat and prevent the immunological diseases and to

control pathological and physiological immune responses. The molecular basis of their

development and function of T-reg cells, especially the molecular mechanism of T- reg

cells- mediated suspension, is also needed for reliable control of their function in clinical

settings.

In summary, monitoring of circulating Tregs in peripheral blood is helpful for

evaluating the immune status of kidney transplant recipients during the early post-

transplant period.

The experiments were carried out on basis of the trial and error. This is a new research in the field of kidney transplantation.

ARIBAS 67

CHAPTER-7DISCUSSION

Chapter 7: Discussion

THE ROLE OF T-REG CELLS IN TRANSPLANTATION.

In the context of allograft transplantation, the induction of a regulatory T cell phenotype

in otherwise alloresponsive T cells has been proposed as a major contributing factor for

the maintenance of tolerance achieved through selected strategies. Indeed it has been

reported that repetitive stimulation of naive T cells with immature allergenic DCs results

in the development of a suppressive phenotype by responding T cells. The maturation

status and types of stimulating DCs present in the grafted tissue is undoubtedly a critical

factor in determining the outcome of an alloimmune response. Phenotypically, immature

DCs do not stimulate optimal effector T cell responses, due to low expression of T cell co

stimulatory factors and proinflammatory cytokines. In fact, such cells are often able to

induce a Treg phenotype in responding T cells. Beyond their maturational state, however,

it is also important to consider the multiplicity of existing DC subtypes, as a number of

recent reports demonstrate that particular DC subsets can induce a Treg phenotype (e.g.,

Th3 or Tr1 cells) irrespective of their maturation state.

While induced Tregs represent a subset distinct from their naturally occurring

CD4+CD25+ counterparts, there is considerable evidence indicating that CD4+CD25+ T

cells play an important role in the “development” of these cells, promoting otherwise

potentially graft-destructive effector T cells to adopt a Tr1 suppressor phenotype.

The mechanism for this activity is not known and could involve either direct cell-cell

interaction, involvement of a third cell (such as an APC), soluble mediators, or some

combination of the three. As noted above, it has recently been demonstrated that non-

regulatory T cells may also convert to a CD4+CD25+ suppressor phenotype, under the

influence of TGF-β.

Interestingly, TGF-β has been found in tolerated grafts, which suggests that induced

Tregs may develop and exert their influence directly at the site of the graft. Karim et al.

have also shown that CD4+CD25+ Tregs can develop from CD25– precursors in

ARIBAS 68

Chapter 7: Discussion

thymectomized mice and that these Tregs can suppress skin allograft rejection. These

data suggest that inducible Treg subsets can prolong allograft survival without newly

formed innate Tregs entering the periphery. Although appropriate strategies were

employed to deplete innate CD4+CD25+ Tregs, one cannot completely exclude the

possibility that residual non-depleted cells contributed to tolerance. A role for

CD4+CD25+ T cells in the induction of a regulatory phenotype in otherwise non-

suppressive T cells provides an attractive hypothesis bringing together the observations

of numerous groups concerning the respective roles of both innate and induced Treg

subsets in promoting transplantation tolerance. This suggests a model in which the 2

subsets act in a cooperative fashion to suppress potentially inflammatory immune

responses directed toward transplanted tissues. The ability of these cells to induce

regulatory function in other populations would also explain a paradox that has been

raised regarding the potency of CD4+CD25+ Tregs. In vitro, meaningful suppression of

activated T cells by CD4+CD25+ Tregs generally requires at least a 1:3 ratio of Tregs to

effectors; lower ratios yield little suppression. However, the frequency of CD4+CD25+

Tregs in vivo is only approximately 10% that of CD4+ T cells, and approximately 3% of

all T cells. Thus, some combination of selective homing and/or induction of suppressive

function in other cells must be occurring in vivo.

Figure: 18 function of T-reg cell in transplantation tolerance

ARIBAS 69

Chapter 7: Discussion

REJECTION

Mechanisms of rejection

The immune response to a transplanted organ consists of both cellular and humoral

(antibody mediated) mechanisms. Although other cell types are also involved, the T cells

are central in the rejection of grafts. The rejection reaction consists of the sensitization

stage and the effectors stage.

Sanitization stage

In this stage, the CD4 and CD8 T cells, via their T-cell receptors, recognize the

alloantigen expressed on the cells of the foreign graft.

Two signals are needed for recognition of an antigen;

The first is provided by the interaction of the T cell receptor with the antigen presented

by MHC molecules,

The second by a co stimulatory receptor/legend interaction on the T cell/APC surface. Of

the numerous co stimulatory pathways, the interaction of CD28 on the T cell surface with

its APC surface ligands, B7-1 or B7-2 (commonly known as CD80 or CD86,

respectively), has been studied the most. In addition, cytotoxic T lymphocyte–associated

antigen-4 (CTLA4) also binds to these ligands and provides an inhibitory signal. Other co

stimulatory molecules include the CD40 and its legend CD40L (CD154).

Typically, helices of the MHC molecules form the peptide-binding groove and are

occupied by peptides derived from normal cellular proteins. Thymic or central tolerance

mechanisms (clonal deletion) and peripheral tolerance mechanisms (eg, anergy) ensure

that these self-peptide MHC complexes are not recognized by the T cells, thereby

preventing autoimmune responses.

ARIBAS 70

Chapter 7: Discussion

At least 2 distinct, but not necessarily mutually exclusive, pathways of all recognition

exist, the direct and indirect pathways. Each leads to the generation of different sets of all

specific T cell clones.

Transplant-rejection

Classification & Morphology

On the basis of the morphology and the underlying mechanism, rejection reactions are

classified as:

1. Hyper acute

2. Acute.

3. Chronic

Clinical Stages of Rejection

Hyper acute rejection

In hyper acute rejection, the transplanted tissue is rejected within minutes to hours

because visualization is rapidly destroyed. Hyper acute rejection is humorally mediated

and occurs because the recipient has pre-existing antibodies against the graft, which can

be induced by prior blood transfusions, multiple pregnancies, prior transplantation, or

xenografts against which humans already have antibodies. The antigen-antibody

complexes activate the complement system, causing massive thrombosis in the

capillaries, which prevents the visualization of the graft. The kidney is most susceptible

to hyper acute rejection; the liver is relatively resistant, possibly because of its dual blood

supply, but more likely because of incompletely understood immunologic properties.

ARIBAS 71

Chapter 7: Discussion

Figure: 19 diagram of hyper acute rejection figure

Acute rejection

Acute rejection manifests commonly in the first 6 months after transplantation. Acute

cellular rejection is mediated by lymphocytes that have been activated against donor

antigens, primarily in the lymphoid tissues of the recipient. The donor dendritic cells

(also called passenger leukocytes) enter the circulation and function as antigen-presenting

cells (APCs).

Chronic rejection

Chronic rejection develops months to years after acute rejection episodes have subsided.

Chronic rejections are both antibody- and cell-mediated. The use of immunosuppressive

drugs and tissue-typing methods has increased the survival of allograft in the first year,

but chronic rejection is not prevented in most cases. Chronic rejection appears as fibrosis

and scarring in all transplanted organs, but the specific histopathological picture depends

on the organ transplanted. For example, In heart transplants, chronic rejection manifests

as accelerated coronary artery atherosclerosis.

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Chapter 7: Discussion

In kidney recipients, chronic rejection (called chronic allograft nephropathy) manifests

as fibrosis and glomerulopathy. The following factors increase the risk of chronic

rejection:

Previous episode of acute rejection

Inadequate immunosuppression

Initial delayed graft function

Donor-related factors (eg, old age, hypertension)

Reperfusion injury to organ

Long cold ischemia time

Recipient-related factors (eg, diabetes, hypertension, hyperlipidemia)

Post-transplant infection (eg, cytomegalovirus [CMV])

Its underlying theme is that regulatory T-cells interact with the tissues they serve to

promote the creation of transient but privileged microenvironments. Normally they

prevent autoimmunity, and also immunopathology from exaggerated responses to

microbes. Therapeutically, they may have potential to turn off unwanted responses, were

we able to exploit their capacity to establish protective microenvironments in tissues.

Scope for regulatory cell therapy:Many of the experimental models that have identified a role for Treg, are themselves

disease models. The benefits coming from reconstitution with Treg predict potential in

reversal of inflammatory bowel disease, autoimmunity, prevention of graft versus host

disease, and in organ transplantation. In some of these models therapeutic efficacy likely

derives from the capacity of CD4+CD25+ to homeostatically regulate the full expansion

and differentiation of other T cells. There is a great deal of interest in identifying ways of

expanding T-cell lines from individual patients and stem cell donors, so as to be able to

use these therapeutically. Past experience of cellular therapies suggests that such studies

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Chapter 7: Discussion

may provide academic proof of principle, but rarely do they become adequately refined to

be attractive for commercialization or licensing as procedures by regulatory bodies. In

the end, such therapies become the domain of specialist centres with the capacity to

generate the significant financing required, but tend not to be available to all who need

them. The most realistic appraisal of this know-how is to accept that therapeutic

harnessing of regulatory T cells is most likely to succeed through conventional

pharmacological and or vaccination approaches for which routes to licensing are

Well defined. The pharmacological approach requires that we identify drugs that

empower regulatory T cells in vivo, while immobilising elements of the immune system

that do damage. This may be possible with judicious combinations of drugs already

licensed, or may require the discovery of new drugs designed for the purpose. The

necessary information for such drug design should emerge from science directed at

identifying any unique signalling and growth/survival requirements of Treg. Therapies

designed to reprogram the immune system towards better-self-regulation may also

require aids for diagnostic monitoring. Again, simplicity and clinical utility, dictate that

such diagnostic aids should be aimed at readily available body fluids. The complex

Interactions of regulatory T cells with other hemopoietic cells and tissues may provide a

basis for PCR or serum proteomic-based assays.

Figure: 20 . Treg work in conjunction with tissues to establish a state of privilege in the tissue microenvironment. Regulatory T cells need not be responsible for all protective events within a protected tissue.

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Chapter 7: Discussion

In the shorter term there do seem to be realistic therapeutic opportunities for negative

vaccination to selectively induce/expand regulatory T cells in autoimmune disease,

allergy, transplantation and other forms of immunopathology. Past efforts to desensitise

allergic individuals, may have already made unwitting use of the therapeutic power of

Treg, but have left too few rules established to guide generic protocols. If we can

establish good phenotypic markers for the Treg that matter, and are able to follow the

expansion of antigen-specific regulatory T cells in the blood, then we may be in much

stronger position to design trials in negative vaccination. In autoimmune disease there is

good evidence that autoimmunity may involve many different tissue antigens at the time

of disease presentation. The main attraction of negative vaccination is the possibility that

one need only use one or a limited set of antigens for any disease target, allowing for

linked suppression and infectious tolerance to amplify the therapeutic effect. The

challenge of reprogramming where the immune system is already in heightened attack

mode, May in the future is met by judicious use of appropriate immunomodulatory drugs

given with the vaccine. Where a vaccine can be given in advance of an anticipated

immune challenge, then diagnostic monitoring may allow one to decide when dominant

tolerisation has reached an adequate level. Such may be the case for individuals with a

known high risk for autoimmune disease such as Type I diabetes. This may also be

relevant to enabling successful xenotransplantation, where negative vaccination to a

defined pig tissue protein may be able to harness the power of linked suppression to limit

cellular responses to other antigens in the same tissue.

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Chapter 7: Discussion

LIMITS OF TREG CELL FUNCTION IN TRANSPLANTATION

TOLERANCE:

We now know that CD4+CD25+ TReg cells play a central role in the induction of

peripheral allograft tolerance. In the absence of this regulatory T cell population, a

variety of potent tolerising therapies lose their ability to create tolerance. Indeed, many of

these therapies appear to be acting, at least in part, by directly modulating the function of

TReg cells. However, it is also clear from data generated in several experimental animal

models that tolerogenic therapies can result in CD4+CD25– T cells also acquiring

immunoregulatory properties. In fact, under some circumstances, CD4+CD25+ TReg

cells can even be shown to arise from CD4+CD25– T cell precursors in the periphery.

However, we lack a clear understanding on how these different regulatory populations

interact, and how is the mechanism of infectious tolerance mediated. In fact, despite the

importance of CD4+CD25+ TReg cells during the initial (induction) phase of tolerance

acquisition, it is currently unknown which specific CD4+ regulatory T cell subset is

responsible for the long term maintenance phase of the tolerant state. Unpublished

observations from our laboratory indicate that, once tolerance has been achieved, the

depletion of CD4+CD25+ Treg cells does not result in graft rejection, even after the

injection into tolerant recipients of fresh T cells from naïve mice. These preliminary

results suggest that while Treg cells may be critical for tolerance induction, other

regulatory populations may be ensuring the long-term survival of the tolerised allograft.

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Chapter 7: Discussion

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CHAPTER-8REFERENCES

Chapter 8: References

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Chapter 8: References

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