Transplantation: Immunobiology for the Nephrologist

Douglas J. Norman, MDProfessor of Medicine

Director, Transplant MedicineDirector, Laboratory of Immunogenetics and

TransplantationOregon Health & Science University

Topics to be Covered in Lecture

•Why transplanted organs are rejected

•The effector mechanisms of the immune response

•The role of tissue typing and crossmatching in preventing rejection

•Detection and consequences of donor specific antibodies

ARS: What are the most important tissue barriers to successful kidney

transplantation?

1. ABO > HLA > minor histocompatibility antigens 2. HLA > ABO > minor histocompatibility antigens 3. Minor histocompatibility antigens > HLA > ABO 4. Minor histocompatibility antigens > ABO > HLA

Why are Transplanted Organs Rejected?

1. ABO blood group antigens

2. Major Histocompatibility Antigens --HLA

3. Minor Histocompatibility Antigens

ABO Blood Group Typing

•ABO blood group must be compatible•A2 into O and

A2 and A2B into Bare compatible combinations

•Some centers transplant across the ABO barrier

Major Histocompatibility Complex

DP DQ DR B C A #6

Class II Class I

DP DQ DR B C A #6

HLA

MHC Gene Products

•MHC class I molecules•HLA A, B, C•found on all nucleated cells

•MHC class II molecules•HLA DP, DQ, DR•Expressed on antigen presenting cells (and inducible)

Structure and Function of HLA Molecules

StructureRegion

Peptide BindingRegion

Immunoglobulin-like Region

TransmembraneRegion

IntracytoplasmicRegion

Function

AntigenPresentation

CD4 / CD8Binding site

MembraneAnchoring

SignalTransduction?

C

C

Class I

NN

s

s

s

s

C C

Class II

NN

s

s

s

s

Direct Allorecognition

Indirect

Indirect Allorecognition

Example of a Minor Histocompatibility Antigen

In inbred strains of mice male and female animals are immunologically identical except

for expression of the H-Y proteins in the males, not found in the females

Match v. Mismatch

A2A2

B7

B8DR3

DR3

Alice

A1A2

B7

B8DR3

DR4 Bob

A1A2

B27

B8DR3

DR3

Ted

ARS: The following is true regarding allograft survival and HLA mismatching

(HLA mm)

1. HLA mm is important for kidney, liver and heart 2. HLA mm is important for kidney, liver, but not heart 3. HLA mm is important for kidney, heart, but not liver 4. HLA mm is important for heart, liver, but not kidney 5. HLA mm has no relation to graft survival in any organ

Importance of HLA Matching

•Kidney transplantation

•Heart transplantation

•Liver transplantation

Kidney Graft Survival RatesAccording to Level of HLA Mismatch

0

4

8

12

16

20

24

28

LD--Id

LD--mm

DD--Id

DD--mm

HLAMatch

Donor Source

31.8 17.3 14.5 10.1

Key Board Review PointsI. Why Transplanted Organs are

Rejected

ABO is the most important tissue barrier to successful transplantation—because of naturally occurring antibodies

HLA molecules are the major histocompatibility antigens because their purpose is to present peptides. After ABO, HLA is the next most important barrier

Minor histocompatibility differences explain why HLA identical sibling recipients require some, although reduced, immunosuppression

Key Board Review PointsI. Why Transplanted Organs are

Rejected

The direct pathway of T cell activation is dominant immediately following kidney transplantation and dictates the need for strong initial immunosuppression

A recipient’s immune response to a kidney transplant is more related to the HLA mismatch than to the match—the fewer the mismatches, the better the graft survival

One year graft survival is more related to HLA class II mismatching than to class I mismatching (hence UNOS points given for class II and not for class I)

What are the Effector Mechanisms of the Immune

Response?

Lymph Node

Naïve T cell Activated T cell

Transplanted organ

Donor and recipient APCs migrate From the graft to LN

Effector T cellsand alloantibodies

track to anddestroy organ

T and B cell activation

Naïve B cell Activated antibody-producing B cell

Y

Y

YY

OVERVIEW Cytotoxic Lymphocytes (CTLs) kill target cells

FasL Fas

Perforin

nucleus

Apoptosis

CTL

Allogeneic Target Cell

Granzyme BRenal Tubule Cell—primary target

Endothelial Cell—also a target

Alloantibodies Damage Endothelium

( C4d staining in Antibody Mediated Rejection: Collins et al, JASN (1999) 10:2208 )

C’ Activation

MAC

CDC

C4d

C4a +C4b

C4C1anti-HLA Ab

Donor HLA

Endothelium

Platelet and fibrinmicrothrombi

Fc ReceptorMediated Binding ADCC

Slide supplied by Peter Nickerson, Manitoba

PMN

PMN PMN

PMN

ARS: Antibody mediated rejection is characterized by the following:

1. Peritubular capillaritis, tubulitis and interstitial edema 2. Peritubular capillaritis, C4d deposits in the peritubular

capillaries and the presence of donor specific antibodies in the patient’s serum

3. Tubulitis, C4d deposits in the peritubular and glomerular capillaries

4. Medullary plasma cell infiltration, glomerular capillary C4d deposits and presence of anti HLA antibodies in the patient’s serum

What are the Pathological Manifestations of Rejection

• Hyperacute rejection

• Acute rejection• Cell mediated• Antibody mediated

• Chronic Rejection

Key Board Review PointsII. Effector Mechanisms of

Rejections

Hyperacute rejection is caused by preformed anti HLA antibodies

Antibody mediated rejection is suggested by C4d deposits in the peritubular capillaries, peritubular capillaritis and finding donor specific antibodies in the patient’s serum

Cell mediated rejection is suggested by an interstitial infiltrate and tubulitis

Key Board Review PointsII. Effector Mechanisms of

Rejections

Peritubular capillary C4d deposits are not always present in antibody mediated rejection

Endothelial damage from sources other than antibodies can mimic antibody mediated rejection (excepting that C4d deposits and donor specific antibodies are absent)

Rarely, antibodies other than anti HLA might be implicated in antibody mediated rejection (anti endothelial antibodies, anti angiotensin receptor antibodies)

How do we Prevent Hyperacute and Early

antibody-mediated Rejection?

ARS: The following is true regarding the pre transplant crossmatch for kidney

transplantation

1. A crossmatch is unnecessary if the HLA typing of both the donor and recipient are known

2. A crossmatch needs to detect only anti HLA class I antibodies

3. A pre transplant Flow crossmatch is sufficient to prevent hyperacute rejection

4. A B cell crossmatch detects HLA class II but not class I antibodies

Pre transplant Crossmatchesused for Kidney Transplantation

•The Standard NIH Crossmatch

•The Antiglobulin enhanced Crossmatch

•The B Cell Crossmatch

•The Flow Crossmatch

•The Virtual Crossmatch

NIH Standard Technique

•The classic assay

•The least sensitive of the tests

•The most specific of the tests

Donor

HanksMononuclear cell layer

Ficoll

ACD tube

Recipient

Clot tube

Serum

23oC

30

Eosin/ formalin 23

oC

60

Standard NIH Crossmatch

Rabbit complement

C

Standard NIH Crossmatch

C

C

C

C

C

C

C

Role of the NIH Standard Crossmatch in Kidney Transplant Outcomes

Graft SurvivalRecipients with Anti-HLA Antibodies Recipients

without Anti-HLA

AntibodiesPositive

CrossmatchNo

CrossmatchNegative

Crossmatch

ImmediateFailure 24 (80%) 6 (26%) 4 (15%) 4 (2.4%)

Failure < 3months

0 6 4 32

Failure > 3months

1 3 7 22

Survival < 3months

2 2 1 6

Survival > 3months 3 (10%) 6 (26%) 11 (41%) 104 (62%)

Total Patients 30 23 27 168

80Patel and Terasaki, NEJM 280:735, 1969

Antiglobulin-Enhanced Technique

•More sensitive

•Can detect non-complement binding antibodies

•Can detect antibodies presentin small amounts

Donor

HanksMononuclear cell layer

Ficoll

ACD tube

Recipient

Clot tube

Serum

Anti-human globulin antibodies

23oC30

Eosin/formalin 23

oC

60

Anti-human Globulin Enhanced Crossmatch

Wash x 3Rabbit complementC

Anti-human globulin (AHG) enhancedCrossmatch

C

C

C

C

C

C

C

Human Globulin Enhanced Crossmatch Role of Anti-Human Globulin Enhanced Crossmatch on Two Year Kidney Transplant Outcomes

All patients had negative NIH standard crossmatches.

Cadaveric Kidney Donor

Recipients

Negative NIH Standard Crossmatch

P-ValueAll

PatientsAnti-Human Globulin Crossmatch

Negative Positive

Two Year Graft Survival

1st transplants

81%(N = 166)

82%(N = 151)

67%(N = 15)

< 0.01

Re-Transplants

64%(N = 70)

77%(N = 48)

36%(N = 22)

< 0.01

Kerman et.al. Transplantation. 51:316, 1991

B cell Crossmatch

•Can detect anti-class I or anti-class II antibodies

•Can detect anti-class I antibodies even when the standard crossmatch is negative

Flow Crossmatch

•Most sensitive of all the crossmatches

•Can be useful if the donor lymphocytes are dead

Laser

Cells

Flow chamber

Laser activated fluorochromes

emit light in red or green spectrum

Flow Crossmatch

Donor cells are incubated with

recipient serum and then fluorochrome-coated antihuman

antibodies

FLOW Crossmatch

Patient SerumNegative Control

Channels Channel Shift

≥ 30 = positive

Crossmatches Used for Kidney Transplantation—Different Scenarios

Cytotoxic Flow

Standard AG B Cell T Cell* B Cell**

+ + + + +

- + - + +

- + - + -

- - + - +

- - + + +

- - - + +

- - - + -

- - - - +

- - - - -

* Class I **Class I and II

Is the Virtual Crossmatch The Future?

Is the Virtual Crossmatch the Future?Different Scenarios

HLA Recipient Antibodies HLA Donor Antigens

A A 2, 26

B B 38, 52

C C 1, 3

DP DP 2, 8

DQ DQ 1, 5

DR DR 13, 15

OK to transplant without a physical XM? Yes

1. All Donor Antigens are KnownNo anti HLA antibodies

Is the Virtual Crossmatch the Future?Different Scenarios

HLA Recipient Antibodies HLA Donor Antigens

A 1, 23, 32 A 2, 26

B 7, 8 B 38, 52

C 4, 5 C 1, 3

DP 3, 7 DP 2, 8

DQ 2, 4 DQ 1, 5

DR 3,4 DR 13, 15

OK to transplant without a physical XM? Probably but worrisome

2. All Donor Antigens are KnownNo donor specific antibodies

Is the Virtual Crossmatch the Future?Different Scenarios

HLA Recipient Antibodies HLA Donor Antigens

A A 3, 26

B B 38, 52

C C 1, 3

DP 3, 7 DP

DQ DQ 1, 5

DR DR 13, 15

OK to transplant without a physical XM? No

3. Some Donor Antigens are KnownAnti HLA antibodies are present

Is the Virtual Crossmatch the Future?Different Scenarios

HLA Recipient Antibodies HLA Donor Antigens

A 2 A 2, 26

B B 38, 52

C C 1, 3

DP DP 2, 8

DQ DQ 1, 5

DR DR 13, 15

OK to rule out transplant? Maybe but worrisome

4. All Donor Antigens are KnownDonor specific antibodies present

Is the Virtual Crossmatch the Future?Different Scenarios

HLA Recipient Antibodies HLA Donor Antigens

A 02:01 A 2, 26

B B 38, 52

C C 1, 3

DP DP 2, 8

DQ DQ 1, 5

DR DR 13, 15

OK to rule out transplant? Probably but worrisome

5. Allele Level Donor Antigens are Unknown

Is the Virtual Crossmatch the Future?Different Scenarios

HLA Recipient Antibodies HLA Donor Antigens

A 02:01 A 02:08, 26

B B 38, 52

C C 1, 3

DP DP 2, 8

DQ DQ 1, 5

DR DR 13, 15

OK to transplant? Probably—need more data

6. Allele Level Donor Antigens are KnownNo allele specific antibodies present

Key Board Review PointsIII. The Pre-transplant Crossmatch

The standard NIH cytotoxic crossmatch is the most specific but the least sensitive of the crossmatches

The B cell crossmatch detects both anti HLA class I and anti class II antibodies

The T cell crossmatch detects only anti class I antibodies The Flow crossmatch is the most sensitive crossmatch and

can detect antibodies that will shorten graft survival but not cause hyperacute rejection (if the standard NIH is negative)

Key Board Review PointsIII. The Pre-transplant Crossmatch

A virtual crossmatch is possible if all antibodies are identified and donor HLA antigens are known (no need for a physical crossmatch)

What is the Role ofDetecting the Presence of

Anti-HLA Antibodiesin Preventing Rejection?

Why Do Patients Make Anti-HLA Antibodies?

Antibodies occur as a result of exposure to:

--blood products--pregnancies--transplants

Terminology:

“PRA”

(Panel Reactive Antibodies, Expressed as a percent)

Purpose of Determining the PRA

•Triggers different management

•Defines unacceptable donor antigens—virtual crossmatch

•Determines organ allocation

•Predicts when a patient might be transplanted

ARS: The following is true regarding detection of anti HLA antibodies before and

after transplantation

1. Cell based detection methods are superior to bead based methods

2. Highly sensitive techniques for detecting anti HLA antibodies are not yet available

3. Techniques are available to detect antibodies to specific HLA antigens

4. Mean fluorescence intensity (MFI) is not a measure of antibody titer (strength) in the Luminex single antigen bead anti HLA antibody detection assay

Techniques for Detecting Anti HLA Antibodies

•Cytotoxic—the classic

•Solid Phase—the present

Multiple PanelMembers

Mononuclear cell layer

RecombinantHLA

Molecules

Whole Cells

Terasaki TrayMulti Antigen Beads

Single Antigen Beads

Digested CellsSoluble HLA molecules

Cells vs. Beads for PRA and antibody specificity determination

Cells have multiple HLA antigens

Use of donor cells determines if a patient has antibodies to donor antigens (OK for the crossmatch)

But cells do not allow identification of specific antigens to which a patient has antibodies

Beads have only HLA and their use can determine PRA and identify antibodies to specific antigens

Principle of Flow Antibody Assays

Laser

Beads

Flow chamber

Laser activated fluorochromes

emit light

The intensity of light indicates

the PRA

Flow PRA Determination

HLA antigen coated beads are incubated with patient serum

and then with a fluorochrome tagged anti human antibody

FLOW PRA value Percentage of HLA antigen

coated beads in a pool that react with antibodies in a patient’s serum

Negative control

Patient SerumBackground fluorescence

of a negative control

Increased fluorescence

indicatespresence of antibodies

DONOR SPECIFIC ANTIBODIES

How to identify them and the consequences of having them

40

50

60

70

80

90

100

0 1 2 3 4

NDSA (152)

DSA (66)

no antibodies (550)

89%

Years after testing

% G

raft

sur

viva

l

p < 0.0001

p < 0.0001 70%

51%

Lachmann, Terasaki, et al. Clinical Transplants 2006, p. 189

Impact of DSA on Graft Survival

Patients were tested once, post

transplantation in 2002, and followed for 4 years

HLA Antibody Identification

•Single antigen beads are used•Beads have HLA molecules of a single specificity•Can identify unacceptable donor antigens•Can identify acceptable donor antigens

B7

A2

Laser

Beads

Flow chamber

Laser activated fluorochromes emit light and

beads have intrinsic colors

identifying which antigens

are bound

HLA antigen SpecificityDetermination

Single HLA antigen coated beads are

incubated with patient serum and then with a fluorochrome tagged anti human antibody B7

B7

B7

B7

B7 B7

B7B7

B7

B7

A2A2

A2

A2

A2 A2

A2A2

A2

A2

The Calculated PRA (cPRA)

The PRA determines if a patient will receive additional points in the UNOS allocation point schema

UNOS does not allow a transplant center to enter the PRA of a patient onto the national computer

Transplant centers must input into the national computer “unacceptable antigens” for patients with anti HLA antibodies

An unacceptable antigen is defined by the strength of the antibody

The Calculated PRA (cPRA)

The strength of an antibody is related to the mean fluorescence intensity (MFI) observed from antigen specific beads used in the assay to detect specific antibodies

B7B7

B7

B7

B7 B7

B7B7

B7

B7

MFI

1000

6000

4000

3000

2000

Unacceptable Antigen Designation

0

1000

2000

3000

4000

5000

6000

7000

8000

A2 A3 B7 B8 B38 B39

Patient X

Patient Y

Mea

n Fl

uore

scen

ce I

nten

sity

(M

FI)

HLA Antigen

Patient X PRA = 90%Patient Y PRA = 13%

Patient X PRA = 90% Patient Y PRA = 80%

Key Board Review PointsIV. Detecting Anti-HLA Antibodies

Bead-based technology has replaced cell-based technology for detecting anti HLA antibodies

Bead-based technology is more sensitive than cell-based technology

The presence of anti HLA antibodies in recipient’s serum is associated with poorer graft survival than the absence of antibodies

The presence of donor specific antibodies is associated with even poorer graft survival

Key Board Review PointsIV. Detecting Anti-HLA Antibodies

It is possible to confidently know if any anti HLA antibodies are present in a patient’s serum

UNOS uses a calculated PRA (cPRA) that is based on “unacceptable antigens”

Unacceptable HLA antigens are determined by identifying antibodies present in an amount above a threshold that is assigned by the transplant center

It has not yet been established what amount of anti HLA antibody is biologically important

Topics that were Covered in Lecture

•Why transplanted organs are rejected

•The effector mechanisms of the immune response

•The role of tissue typing and crossmatching in preventing rejection

•Detection and consequences of donor specific antibodies

THE END

CASE STUDY OF A HIGHLY SENSITIZED PATIENT WITH SEVERAL DONOR SPECIFIC ANTIBODIES

Challenging Case that demonstrates most of the principles discussed in this talk

OHSU

CASE STUDY PATIENT L.C.

L.C. is a 49 year old woman with ESRD caused by ADPKD. She had a high PRA and required crossmatches with several potential donors before finding a suitable donor.

Specific antibodies had been identified to A 24, 25; B27, 38, 44, 49, 51, 57; DR 7.

Eventually, she underwent a living unrelated donor kidney transplant at OHSU on 5/29/2007.

OHSU

Potential Donors for Patient LC*

OHSU

Donor ABOMolecular Typing Results

A A B B DR DR

#1 B

#2 A 2 27 44 1 4

#3 O 2 25 7 44

#4 O 3 11 7 27 9 15

#5 O 26 31 49 60 1 4

#6 O 3 23 7 44 17 15

#7 A 2 26 27 49 1

#8 O 25 31 7 60 4 8

#9 O 1 25 7 57 15 8

*ABO = A; HLA A 3,11 B 7,65 DR 15,13

Potential Donors for Patient LC*

OHSU

Donor ABOMolecular Typing Results

A A B B DR DR

#10 A 1 31 7 60 7 15

#11 O 26 33 38 55 11 13

#12 A 24 26 51 38 11 13

#13 A 11 30 18 60 17 7

#14 B

#15 O 24 33 39 65

#16 A 1 3 7 60 13 15

#17 A 2 29 51 44

*ABO = A; HLA A 3,11 B 7,65 DR 15,13

Crossmatching of Donors for Patient LC*

DonorCytotoxic Flow

Standard AG B Cell T Cell B Cell

#2 - + - +++ +++

#3 - + - +++ ++

#4 - + - ++ +

#5 - - - + +

#6 - - - +++ +++

#7 - + - ++ +++

#8 - - - + +

#9 - - - ++ ++

#10 - - - - +

#16 - - - - ±

#16 - - - + +

Posttransplant Antibody Formation—LC ABO

Molecular Typing Results

A A B B DR DR DQ

LC A 3 11 7 65 15 13 6

LURDonor A 3 1 7 60 15 13 6

DateCrmg/dl

Flow %Biopsy Treatment

Donor Specific Ab

I II HLA MFI

Transplant5/24/07

5.3 73 12 NONE

6/4/07 0.9 94 NDglomerular capillary fibrin

thrombi; weak C4dPP&IVIG x 3 B60 3,800

6/18/07 0.9 89 24 PP&IVIG x 3A1 3,528

B60 4,877

7/5/07 1.0 82 33CCTT 1 cellular rejection; no fibrin thrombi; C4d neg

Steroid pulseA1 2,318

B60 4,466

8/8/07 1.0 71 23 no rejection; C4d neg NONE

6/16/08 0.9 81 15 no rejection; C4d neg NONE

5/10/10 0.7

THE END