HLa分型和組織移植英文版課件

HLA

TYPING

&

ORGANTRANSPLANTATIONScott

BainbridgeElena

CrousonIsrafiel

MohammedSarah

Tucker

Organ

Transplantation

What

is

it?Organs

or

tissues

from

one

human

being

(the

donor)

areput

into

another

person's

body

(the

recipient).Factors

Effecting

Transplantation?HLA

AntigensStatistics

on

Organ

TransplantationThere

are

more

than

91,500

people

on

the

organ

transplantation

waiting

list.Each

day

74

people

receive

an

organ

transplantation,

but

18

people

onthe

waiting

list

die

because

a

donor

is

not

available.

There

are

55,000

people

waiting

for

a

Kidney,

17,000

waiting

for

a

liverand

3,000

waiting

for

either

a

heart

or

liver

transplant.First

Organ

TransplantationIn

1959,

Joseph

Murray

and

his

colleagues

in

Bostonsuccessfullytransplanted

a

Kidney

that

were

donated

by

fraternal

twins

and

it

functionedfor

20

years

without

immunosuppression

drugs.First

successful

Liver

transplant-

In

Denver

on

7/23/1967First

successful

Heart

transplant-

In

Cape

Town,

South

Africa

on

1/2/68First

successful

Bone

Marrow

transplant-

Minneapolis,

MN

on8/25/68Why

Is

it

Difficult?Organ

transplantation

is

difficult

because

of

the

HLA

antigens

locatedon

the

cellsurface.Human

Leukocyte

Antigen

(HLA)

also

referred

to

as

MajorHistocompatibility

Complex

(MHC)

plays

a

role

in

intercellularrecognition

and

discrimination

between

self

and

non-self.Location

of

HLA/MHCThe

MHC

complex

is

a

collection

of

genes

arrayed

within

a

longcontinuous

stretch

of

DNA

on

chromosome

6.

Each

HLA

type

ofassociated

with

a

different

class

of

MHC

molecule.Types

of

MHCThere

are

three

classes

of

MHCmolecules.Class

I-

encodes

glycoproteins

expressed

on

the

surface

of

nearlyallnucleated

cell;

the

major

function

of

the

class

I

gene

is

presentation

ofpeptide

antigens

to

cytotoxic

T-cellsClass

II-

encodes

glycoproteins

expressed

primarily

on

antigen-presenting

cells,

examples:

macrophages,

dendritic

cells

and

B-cells,where

they

are

present

processed

antigenic

peptides

to

T

helper

cells.Class

III-

encodes

various

secreted

proteins

that

have

immune

functionincluding

components

of

the

complement

system;

C2,C4,

Factor

B,&TNF,

and

molecules

involved

in

inflammation.Different

HLA

AllelesClassI-HLA

AHLA

BHLA

C451

alleles782

alleles238

allelesClassII-HLA

DRHLA

DQHLA

DPHLA

DMHLA

DO525

alleles105

alleles147

alleles11alleles21allelesRequirements

for

TransplantEach

transplant

center

has

different

requirements

for

allele

matches.For

the

National

Marrow

Donor

Program:In

order

to

find

a

match

doctors

require

that

at

least

a

minimum

of

3allele

matches.HLA-A,HLA-Band

HLA-DRB1.One

set

of

the

three

antigens

are

inherited

from

your

mother

and

theother

set

is

inherited

from

your

father.

This

makes

6

antigens

tomatchTherefore,

it

is

required

that

4

of

the

6

antigens

match

for

cord

blooddonation

and

5

of

the

6

antigens

for

adult

donation.Requirements

for

TransplantNational

Marrow

Donor

Program:Chance

of

a

MatchMother/Father:

25%

chance

of

full

matchOne

Sibling:

25

%

chance

of

full

matchTwo

Siblings:

44

%

chance

of

full

matchThree

siblings:

58%

chance

of

full

matchThe

chance

to

find

donors

may

be

better

for

morehomogenous

racial

groups.HLA

In

TransplantationThere

are

two

characteristics

of

the

HLA

genes

thatmake

them

special

for

organtransplantation:There

high

degree

of

polymorphismThere

strong

immune

reactions

that

their

products

canproduce

in

other

individuals.

HLA

Pathways

There

are

two

pathways

that

can

occur

that

causeproblems

in

organ

transplantation

as

a result

of

HLAantigens.Direct

pathway-

the

alloreactive

responses

of

recipient

T-cells

todonor

APC

expressing

incompatible

antigens.Indirect

Pathway-

allogeneic

HLA

antigens

are

taken

up

andprocessed

by

recipient

APC

and

presented

in

context

withautologous

HLA

molecules

to

recipient

T-cells.

Problems

from

HLA

antigensEngraftment-

immunological

rejection

of

donorhematopoietic

cells

by

recipient

T

cells

that

recognizeincompatible

HLA

determinants.Factors:pregnancy

or

transfusionHLA

mismatching

of

the

donorUsing

of

less

intense

preparative

regimen

before

transplantSuboptimal

post-transplant

immunosuppressive

therapyDepletion

of

T

lymphocytes

from

marrowgrafts.Class

I

determinants

govern

graft

acceptanceClass

II

determinants

play

a

role

inGVHD.Problems

from

HLA

antigensAcute

Graft

versus

Host

Disease-

immune

reaction

of

mature

donor

Tlymphocytes

against

HLA

determinants

of

the

recipient.The

reaction

is

directed

toward

normal

tissue

including

skin

andgastrointestinal

mucosa.HLA

matched

unrelated

Bone

Marrow

transplant

Acute

GVHD

is

79%

vs.35%

HLA

matched

sibling

BMT.Matching

donor/recipient

pairs

for

molecular

typing

has

been

showed

toreduce

the

risk

of

acute

GVHD,

48%

vs.

70%IfHLA-DRB1

and

HLA-DQB1

are

matched

with

recipient

than

itreducesthe

risk

of

acuteGVHD.Problems

from

HLA

antigensChronic

GVHD-

is

the

principle

cause

of

morbidity

and

no

relapsemortality

for

patients

reaching

day

100

after

allogeneic

transplant.It

may

involve

skin,

oral

mucosa,

eyes,

liver,

gastrointestinal

tract

andlungs.It

occurs

in

35%

to

70%

of

patients

after

unrelated

donor

BMT.Mortality

rates

range

from

25%

to

70%

,

depending

on

associated

riskfactors.OverviewMethods:

Histocompatibility

test,

consisting

of

three

tests:HLAantigen

typing,

screening

of

the

recipient

for

theanti-HLAantibodies

and

the

lymphocyte

crossmatch

or

compatability

test.Results:

More

allele

mismatch

more

complicationsFurther

Studies/Discussion:

Outcomes

of

unrelateddonor/recipient

transplant.Histocompatibility

testing

consists

of

three

testsHLA

antigen

typing

(also

called

tissue

typing)Screening

of

the

recipient

foranti-HLA

antibodies(alsocalled

antibody

screening)Lymphocyte

cross

matching

(also

called

compatibilitytesting)HLA

antigen

typingTwo

different

methods,

serological

and

DNA

sequencingSerological

methodLymphocytes

are

harvested

from

the

blood

by

densitygradient

centrifugationA

solution

of

Ficoll-Hypaque

is

layer

underneath

the

wholeblood,

and

the

tube

iscentrifugedRed

blood

cells

are

denser

and

go

to

thebottommononuclear

cells

are

less

dense,

and

are

found

in

themiddle,

just

underneath

the

plateletsThe

mononuclear

layer

is

removed,

and

washed.

T-cells

areremoved

usually

by

binding

to

magnetic

beads

coated

withT-cell

antibodies,

and

are

washed

away,

leaving

only

the

B-cells.Serological

methodThis

B

cell

enriched

media

is

added

to

a

microtiter

plate

witheach

well

containing

a

different

antibody

to

a

certain

HLAantigen.

If

a

certain

MHC

cell

is

present,

the

antibodies

willbind,

forming

an

antigen-antibody

complex.After

incubation,

rabbit

complement

is

added

to

each

well.

Ifanantigen-antibody

complex

is

present,

complement

will

beactivated,

and

will

destroy

the

cells

with

an

antigen-antibodycomplex.After

incubation

formalin

is

added

to

fix

the

cells

and

stop

thecomplement

reaction.

Eosin

Y

is

added

to

stain

any

deadcells.Serological

methodCells

are

examined

under

a

phase

contrast

microscope,

andcells

that

are

pink

arepositive.If

60%

or

more

of

the

cells

are

stained

they

areconsideredpositive

for

the

HLAantigen.DNA

typingmethodsGranulocytes

and

lymphocytes

are

separated

from

blood

bylysis

of

the

red

blood

cells

using

ammonium

chloride

andcentrifugation.DNA

is

extracted

from

the

white

cells

by

chloroform

andethanol

and

added

to

the

wells

of

a

microtitertray.Each

well

contains

oligonucleotide

primers

complementaryto

a

small

segment

of

only

one

HLA

allele.

If

the

primer

canattach,

the

HLA

antigen

is

present

on

the

cells.DNA

typingmethods

DNA

polymerase

andoligonucleotidetriphosphates

are

added

to

each

well

andthe

plate

is

incubated

in

a

thermal

cycler,which

multiplies

the

sequence

between

theprimers

(same

as

PCR)The

DNA

is

removed

and

run

on

agarosegel

byelectrophoresis.Since

the

DNA

was

amplified,

if

there

isany

DNA

detected,

HLA

is

present.

If

noDNA

is

seen,

HLA

is

notpresent.Antibody

screening

for

anti-HLAantibodiesPurpose:

to

detect

antibodies

in

the

recipient’s

serum

that

reactwith

HLA

antigens.

We

know

what

HLA

type

thepersonis,

but

we

don’t

know

what

antibodies

they

have

to

otherHLA

typesAntibody

screening

for

anti-HLA

antibodiesLeukocytes

(neutrophils,monocytes,

basophils,lymphocytes)

are

harvested

fromthe

blood

of

donors

with

a

knownHLA

type

and

are

added

to

amicrotiter

plate.Serum

from

the

recipient

isaddedto

each

well.After

incubation,

cells

are

washedto

remove

any

unbound

proteinsAnti-human

Ab

is

added,incubated,

and

then

rabbitcomplement

is

added.Antibody

screening

for

anti-HLA

antibodiesIf

an

antibody

against

HLA

is

present,

it

will

bind

tothe

cells,

andantigen-antibody

complexes

will

bind

tothe

anti-human

Ab,

which

will

then

activatecomplement.Eosin

Y

is

added,

cells

are

examined

under

amicroscope.Pink

stained

cells

indicates

the

presence

of

anti-HLAantibodies.The

higher

the

number

of

different

HLA

antibodiesthelower

the

probability

of

finding

a

match.Crossmatch

testPurpose:

to

detect

presence

of

preformed

antibodiesin

recipient

that

are

reactive

against

donor

tissues.Crossmatch

testPeripheral

blood

lymphocytes

from

the

donor

are

separated

into

Band

T

lymphocyte

populationsT-cells

are

purified

by

magnetic

beads

coated

with

monoclonalantibodies

for

B-cells.

The

B-cells

bind

and

are

removed

bymagnetic

force.B-cells

are

purified

in

the

same

manner,

but

the

magnetic

beads

arecoated

with

monoclonal

antibodies

for

T-cells.Crossmatch

testB-cell

crossmatch

is

performed

using

the

same

method

as

HLAtypingT-cell

crossmatch

is

performed

using

the

same

method

asscreening

testWhy

do

a

crossmatch

when

screening

seems

sufficient?Antibodies

against

low-incidence

antigens

are

likely

to

bemissed.Acts

as

a

mock

transplantResultsPaper’s

Results

Focus

on

Four

Aspects

of

ExperimentHLA

Typing

serological

methods

for

HLA

Class

I

(A,

B,

C)

alleles

andusing

DNA

methods

forClass

II

alleles

(DRB-1,DQB-1).Graft

FailureUsed

statistical

methods

to

determine

percentage

of

graftfailures

based

on

types

of

mismatches

presentAcute

Graft

vs.

Host

Disease

(GVHD)Also

used

statistical

methods

to

study

this.SurvivalOver

the

course

of

8

years

studied

survival

rates

inpatientsHLA

MatchingHLA

MatchingTested

300

pairs

in

study§

142

matched

for

the

HLA

Alleles

(A,

B,

C,

DRB1,

DBQ1)§

158

mismatched

pairs83

mismatched

at

a

single

locus75

mismatched

at

two

or

more

lociOf

the

transplants,

83%

were

done

using

a

Caucasian

donor

toCaucasian

patient.85%

of

transplants

had

donor

and

recipient

of

samerace.Graft

FailureSource:

Blood,

Vol.

92,

Issue

10,

3515-3520,

November

15,1998§

Graft

failure

was

high

when

one

or

more

HLA

class

I

allele

mismatches

werepresent.§

Class

II

allele

mismatches

did

not

lead

to

graft

failure.§

However

when

both

class

I

and

class

II

mismatches

present,

graft

failure

is

high

Acute

GVHD

Fig

1.

Cumulative

incidence

estimates

of

grades

III-IV

acute

GVHD

accordingto

recipient

disparity.Source:

Blood,

Vol.

92,

Issue

10,

3515-3520,

November

15,1998Acute

GVHDWhat

does

the

previous

slide

show?

Risk

of

having

grades

III-IV

GVHD

dependmainly

on

the

class

of

mismatched

allele

andthe

number

of

mismatches.Class

II

allele

mismatches

and

Class

I/IImismatches

combined

have

thehighestprobability

for

GVHD.Also

more

than

one

class

I

mismatchleadsto

an

increased

risk

for

GVHD.SurvivalMultiple

Class

I

MismatchesLower

chance

of

survival

amongst

patients

that

have

morethan

one

class

I

mismatch

or

have

both

class

I

and

class

IImismatches.Multiple

Class

II

MismatchesOf

seven

patients

with

multiple

mismatches,

threediedwithin

100

days-

Other

Four

lived

3-8

years.Overall

MessageHaving

a

single

mismatch

in

either

Class

I

or

Class

II

cansurvive,

but

when

there

is

more

than

one

mismatch

it

couldlead

tofatalities.Survival

(Cont.)What

Do

the

Results

Mean?Results

ExplainedMismatches

in

certain

classes

of

alleles

resulted

in

either

graft

failure

(Class

Ialleles)

or

acute

GVHD

(Class

IIalleles)The

two

classes

of

alleles

are

distinguished

on

a

molecular

level.In

addition,

HLA

class

I

molecules

will

interact

with

natural

killer(NK)cells.This

contributed

to

death

in

individuals

within

an

8

yearspan.To

understand

the

differences

between

the

loci

in

class

I

alleles

will

have

to

bedone

in

future

results.This

is

because

donors

with

a

single

HLA-A,

B,

or

C

mismatch

was

toosmall

to

do

comparisons.Having

one

mismatch

affected

survival

slightlyBeneficial

to

certain

ethnic

groups

which

finding

alleles

that

matchcompletely

might

be

difficult.Why

the

majority

for

this

experiment

were

CaucasianThings

to

considerFor

this

experiment,

the

researched

focused

onindividualswith

chronic

myeloid

leukemia.Ideal

transplantation

would

be

one

where

time

betweendiagnosis

and

transplant

is

minimum.The

results

are

not

representative

of

what

it

couldhappento

patients

with

other

types

ofleukemia.OutcomesAfterUnrelated-DonorTransplantUnrelated

marrow

transplantTransplantation

survival

is

increased

by

matching

Class

I

andClass

IIallelesMultiple

Class

I

disparities

in

the

donor

increase

the

risk

ofgraftfailureMultiple

Class

II

disparities

in

the

recipient

increase

the

risk

ofGVHDSingle

disparities

did

not

appear

to

compromise

survivalBiological

functions

of

Class

I

and

ClassII

molecules

Class

IPresent

peptides

derived

fromendogenously

synthesizedproteinsResponding

T

cells

expressCD8+Complex

interactions

with

NK

cells/jkimball.ma.ultranet/BiologyPages/H/HLA.html#cd8Biological

functions

of

Class

I

and

ClassII

molecules

ClassIIPresent

peptides

derived

fromexogenously

synthesized

proteinsResponding

T

cells

expressCD4+/jkimball.ma.ultranet/BiologyPages/H/HLA.html#class_IIGraft-versus-Host

Disease

Complication

of

bone

marrowtransplants

in

which

T

cells

fromdonor

attack

the

host’s

tissues.Acute

GVHD

and

chronic

GVHDGraft

FailureAntigen-presenting

cell

triggerCD4

and

CD8

cellsLocal

and

systemic

immuneresponse

developCytokine

recruitmentandactivation

of

specific

T

cells,

NKcells

macrophage-mediatedcytotoxicityAllograft

destructionhttp://cnserver0.nkf.med.ualberta.ca/cn/Schrier/Volume5/ch9/ADK5-09_1-3.pdfChimerismImplanted

organ

allografts

become

mixtures

of

donor

andrecipient

cells.1968,

karyotyping

of

livers

transplanted

to

females

from

malecadaversMost

livers

remain

maleKupffer

cells

were

replaced

with

recipient

female

cellsLimitations

on