Yash Project Work Thesis

Role of T-ReGUlAToRY CellS IN CeRTAIN
AUToIMMUNe DISoRDeRS AND GRAfT ReJeCTIoN IN
CASe of KIDNeY TRANSPlANTATIoN:
A floWCYToMeTRY APPRoACH
A
DISSeRTATIoN RePoRT
SUBMITTeD
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
SUPeRvISoRSUPeRvISoR
DR. DevJANI CHAKRABoRTYDR. DevJANI CHAKRABoRTY
ASST. PRof. IN BIoCHeMISTRYASST. PRof. IN BIoCHeMISTRY
ASHoK AND RITA PATel INSTITUTe of INTeGRATeD STUDY ANDASHoK AND RITA PATel INSTITUTe of INTeGRATeD STUDY AND
ReSeARCH IN BIoTeCHNoloGY & AllIeD SCIeNCeS (ARIBAS),ReSeARCH IN BIoTeCHNoloGY & AllIeD SCIeNCeS (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, 2011
CERTIFICATECERTIFICATE
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oF Any oThER woRk publIshEd oR unpublIshEd.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 dIRECToR
ARIbAs 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 72
AbbREvIATIon
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

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

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

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

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)
B cell types:
ARIBAS 4

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.
1.4.3 T lymphocytes or T-cells:
ARIBAS 5

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
virally infected target cells or tumor cells. Most TC are CD8 +. TC is the major effectors in
allograft organ rejection.
ARIBAS 6

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 Immunity
Mechanism Antibody-mediated Cell-mediated
Cell Type B Lymphocytes T Lymphocytes
Mode of action Antibodies in serum
Direct 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.
There are two major types of MHC molecules:
ARIBAS 7

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
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
ARIBAS 8

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

FIGURE: 5 MECHANISM OF T-LYMPHOCYTE CELLS
ARIBAS 10

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

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
develop T cell-mediated autoimmune disease. Trn cell development in the thymus
ARIBAS 12

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

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
wide spectrum of cellular activities. TGF-β exerts broad antiproliferative and anti-
inflammatory effects. IL-10 strongly inhibits activation of macrophages and dendritic
ARIBAS 14

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.
Figure: 6 Mechanism of T-reg cell in different part of body.
ARIBAS 15

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

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 Treg
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

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, Treg-
specific-demthylated region) is found demethylated, which allows to monitor Treg 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

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

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

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

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.
ARIBAS 22

FIGURE:8 DIAGRAM OF TRANSPLATED KIDNEY IN HUMAN BODY
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

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
ARIBAS 24

coining the term FACS. Herzenberg won the Kyoto Prize in 2006 for his work in flow
cytometry.
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.
ARIBAS 25

FLOW CYTOMETRY has been the method of choice for monitoring CD4 lymphocytes
levels in the blood of AIDS patients.
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

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

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

"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

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

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 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-4
MATERiAls 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

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

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

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

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.
• 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
Pipette Standard Test
Working reagent 1000 μl 1000 μl
Standard 100μl -
Test - 100μl

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
4.2 Flow Cytometry:
ARIBAS 42

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

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

FIGURE: 13 Mechanism of flow-cytometry florescent markers
ARIBAS 45

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

FIGURE: 14 Flow cytometer - the Becton-Dickinson FACSCalibur
FIGURE: 15 Set-up of the flow-cytometer attach with computer:
ARIBAS 47

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

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

FIGURE: 16 Photographs of Marker used in Flow-cytometry
ARIBAS 50

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

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

The Flow cytometer is connected with the computer system and data entered in the
specific folders for the specific markers.
ARIBAS 53

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”
ARIBAS 56
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

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 AGE
TX
DATE
F.UP
Yrs SCR
T-
Reg
Cell
1 PG 42 M
BENIGN
NEPHROSCLEOSIS 0 WIFE 35
5-Dec-
09 1.68 1.37 6.92
2 NJ 32 M CGN 2 WIFE 30
26-
Nov-09 1.71 1.16 3.55
3 RY 34 M CGN 3 WIFE 25
29-Jan-
90 1.12 2.21 5.33
4 GS 29 M HT 3 WIFE 25
5-Aug-
10 1.02 1.53 6.61
5 GY 44 M CGN 0 WIFE 38
9-Aug-
10 1.01 1.26 7.31
6 DP 30 M HTN+CGN 4 MOTHER 50
28-Oct-
10 0.8 1.91 6.90
7 IR 39 M CGN 3 MOTHER 58
1-Nov-
10 0.79 2.23 7.08
8 RM 19 M CGN 3 MOTHER 42
12-Feb-
11 0.51 1.34 4.09
9 KM 21 M CIN 3 MOTHER 45
15-Feb-
11 0.5 1.32 3.58
10 SP 40 M HTN+CGN 3 MOTHER 60
5-Mar-
11 0.45 1.4 6.72
11 SS 41 M CGN 3 WIFE 42
21-Apr-
11 0.32 1.39 4.76
12 HT 45 M CGN 1 WIFE 40
2-Jun-
11 0.21 1.4 7.21
13 HM 28 M MN 4 FATHER 60
9-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 DATE
SAMPLE
DT. CREATININE mg/dl T-reg
1 AC 174727 6-May-09 18-Feb-11 0.85 2.97
2 VA 207055 15-Dec-10 18-Feb-11 1.9 1.33
3 GD 207263 10-Jan-11 18-Feb-11 1.18 2.96
4 HH 190578 10-Jan-11 18-Feb-11 0.94 3.28
5 AN 209442 31-Jan-11 28-Feb-11 1.41 1.29
6 RM 209190 21-Feb-11 11-Mar-11 0.95 6.81
7 VK 209899 21-Feb-11 11-Mar-11 0.88 6.12
8 GR 190880 2-Mar-11 18-Mar-11 0.98 5.09
9 SJ 185314 1-Jun-10 18-Mar-11 1.64 3.37
10 AS 207136 4-Feb-11 18-Mar-11 1.7 4.44
11 AK 207389 13-Jan-11 18-Mar-11 1.12 5.01
12 HG 155189 27-May-10 18-Mar-11 2.24 0.09
13 MP 132971 1-Aug-06 18-Mar-11 1.58 5.39
14 VP 161813 6-Nov-09 18-Mar-11 1.31 5.97
15 RP 197479 23-Feb-11 21-Mar-11 1.54 4.23
16 SD 202879 23-Feb-11 22-Mar-11 1.18 0.42
17 SF 207193 1-Dec-10 23-Mar-11 0.69 1.1
18 AM 195553 12-Mar-11 28-Mar-11 1.17 2.17
19 M 201220 25-Feb-11 28-Mar-11 1.08 5.83
20 MS 207787 22-Mar-11 4-Apr-11 1 5.73
21 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
1.9
1.331.18
2.96
0.94
3.28
1.41
1.29
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
0.420.69
1.1
1.17
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 21
PATIENTSNO.
CONTROLPATIENTS
CREATININEmg/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 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 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

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

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

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

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.
ARIBAS 72

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
ARIBAS 73

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.
ARIBAS 74

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.
ARIBAS 75

Yash Project Work Thesis

Recommended

Recommended

More Related Content

Viewers also liked

Viewers also liked (10)

Similar to Yash Project Work Thesis

Similar to Yash Project Work Thesis (20)

Yash Project Work Thesis