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The ABC’s of HLA:Beginners to Advanced

�Rajalingam Raja, Ph.D, D(ABHI)�Professor of Clinical Surgery�Director of Immunogenetics and Transplantation Laboratory�University of California, San Francisco

� Phone: 415-476-0647� Email: Rajalingam.Raja@ucsf.edu

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HLA class I

Cell

HLA Antibody

23%

In peripheral blood

CytotoxicT cell

TCR

CD8

Every cell expresses a HLA to present antigens to T lymphocytes

Infection

Perforine, Granzyme, Granulysin

HLA class II

APC

Helper T cell

TCR

CD4

48%

B cell

BCR

11%

IL2, IFN-γ

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HLA is the Challenging Barrier to Transplantation

HLA mismatched Allograft

Recipient

• Transplantation• Pregnancy• Transfusion

Antibody DepletionPlasmaphoresis

Antibody BlockingIVIGAnti-C5a

Unacceptable Antigens HLA antibodies

Plasma cell

Rejection Lymphocytes

TTTT

TTTT

TTTT

TTTT

TTTT

NKNKNKNK

BBBB

BBBB

BBBB

NKNKNKNK

NKNKNKNK

MaintenanceTherapy

ImmunosuppressionCyclosporine MMFSteroids

InductionTherapy

Lymphocytes DepletionAnti-Thymoglobulin → T & NK cellsAnti-CD3 → T cellsAnti-CD25 → Activated T cellsAnti-CD52 → mature lymphocytesAnti-CD20 → B cells

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Consequences of Pre-formed Donor-Specific HLA Antibodies

• Hyperacute rejection• Delayed graft function• Accelerated acute rejection• Chronic rejection• Prolonged waiting times• No transplantation

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Means of HLA Antibody-Mediated Rejection

Donor-Specific HLA Antibodies

1. Activation of complement cascade

DSA

C1q

2. Antibody-dependent Cell-mediated cytotoxicity (ADCC)

FcR

NK Cell

3. Opsonization & increased antigen presentation

FcR

APCAPC4. Activation of

Endothelial Cell

OrganAllograftEndothelium

HLA Class I

HLA Class II

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Histocompatibility Testing for Solid Organ Transpla ntation

DonorRecipient

Pre-Transplant

Post-Transplant

HLA Typing HLA Typing HLA Mismatch

HLA Antibodies Preformed-DSA

Crossmatch Compatibility

Serum Cells

HLA Antibodies Donor-specific Antibodies (DSA)

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Complement

Donor Recipient

Lymphocytes Serum+

Complement Dependent Cytotoxicity (CDC) Crossmatch

Fluorescein Diacetate + Ethidium Bromide

Live cellsDead cells

Positive Negative

Membrane Attack Complex (MAC)

Paul Terasaki

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CDC xM(n=225)

Hyperacute or Accelerated

Rejection

Functional Graft

Positive(n=30)

24 6

Negative(n=195)

8 187Specificity Problem

Sensitivity Problem

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Flow Cytometry CrossmatchDonor Recipient

Lymphocytes Serum+

Anti-human IgG F(ab’)2 FITC

• Median Chanel Shift (MCS) –a quantitative readout (Ag+Ab)

• Detects only IgG antibodies• Non-specific reactivity can be

reduced by Pronase digestion

Measure FITC intensity by flow cytometry

T cell MCS > 50B cell MCS > 120

Negative Control

Patient Serum

T cell B cell

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Flow Crossmatch - problems

• ~8% of flow crossmatches are false positive – unneccessary exclusion

• ~7% of flow crossmatches are false negative – risk to patient

Flow Cytometry Crossmatch

Donor Recipient

Lymphocytes Serum

Anti-human IgG F(ab’)2 FITC

+

• Median Chanel Shift (MCS) –a quantitative readout (Ag+Ab)

• Detects only IgG antibodies• Non-specific reactivity can be

reduced by Pronase digestion

Measure FITC intensity by flow cytometry

T cell MCS > 50B cell MCS > 120

Negative Control

Patient Serum

T cell B cell

Virtual

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Donor Recipient

Lymphocytes Serum

Virtual Crossmatch - Essentials

HLA Typing

HLA Antibody Testing

A2, A24, B7, B18, DR1, DR4

Anti HLA-A2 antibodies

VirtualCrossmatchPositive

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Crossmatch Methods

Crossmatch method

Sensitivity Specificity Cost(US $)

Turnaroundtime

CDC Low Low 600 3.5 hours

Flow Intermediate Intermediate 600 5 hours

Pronase >Intermediate >intermediate 600 6.5 hours

Virtual 100% 100% 0 10 min

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Virtual Crossmatch - Advantages

• Eliminates the physical crossmatch- Saves 4-6 hours – cut downs cold ischemic time - No samples required - Reduces laboratory & OPO workload - Reduces laboratory, OPO, and Tx program cost

• Adds precision to actual crossmatch- CDC/flow XM prediction- DSA identification

• Improves allocation efficiency• Increased rate of transplantation for sensitized

patients• Risk of memory response can be accounted:

Previous transplants & pregnancies

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Virtual crossmatch by listing Unacceptable Antigens in UNet

X

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1 36 7 44 9 17 4 9

2 24 7 18 1 10 5 52 29 13 51 8 14 4 8

2 68 39 71 15 16 5 6

2 34 57 61 11 14 2 4

2 25 39 65 9 17 4 9 2 23 44 45 13 18 7 81 2 8 62 4 17 4 7

Candidate:

Potential Donors, >12,000

A B DR DQ

+ anti-DR4 61% cPRA

+ anti-DQ5 76% cPRA

Unacceptable HLA Antigens & Virtual Crossmatch

1 68 8 13 4 15 2 5

69 74 55 60 4 7 7 8 3 24 18 39 1 4 4 4

24 43 27 45 4 8 4 8

66 68 27 39 4 15 8 5

23 26 49 62 1 17 2 5

11 33 51 64 15 18 5 7

3 29 35 44 1 11 7 6

48% cPRAanti-A2

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• Increase priority for sensitized candidates/CPRA sliding scale

• Replace SCD/ECD with KDPI• Add longevity matching• Include pre ‐registration dialysis time• Incorporate A2/A2B to B• Base pediatric priority on KDPI• Remove payback system• Remove variances

KAS: Major Allocation Components

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CPRA (%) KAS Priority PointsOld New

0–19 020–29 030–39 040–49 050–59 060–69 070–74 075–79 080–84 485–89 490–94 4

95 496 497 498 499 4100 4

Regional SharingNational Sharing

Priority points for CPRA>19%

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Distribution of CPRA scores in UCSF Kidney Transpla nt Waitlist (n=5461)

100%99%98%85-97%20-84%0-19%

CPRA5%4%

1.6%0.8%

15.9%

72.7%

Points

202.150.0924.44.05-17.30.08-2.460

#Patients

21685462738673974

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Sequence-Specific Oligonucleotide ( SSO) Hybridization Method

Polystyrene MicrospheresPolystyrene Microspheres

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• 100 Color-coded Polystyrene beads using a blend of different fluorescent intensities of two dyes• Each bead is conjugated with oligonucleotide probe specific for a HLA allele (s)

Luminex technologyA2

A1

A11

A3

A23

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PCR amplificationusing florochrome-taggedlocus-specific primers

Cell

DNA

*

Amplified DNA

Denatured PCR

products

Detection & Interpretation

Luminex: rSSO Method

A2 A1

A11

A3

A23

A2 A1

A11

A3

A23

Hybridization

+

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Single Antigen Bead-based HLA Antibody Testing: Lum inex Technology

Polystyrene MicrospheresPolystyrene Microspheres

A2

A66B55

Single HLA Antigen Beads

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Detection & Interpretation

A2

A66B55

patient’s serum

A2A66

B55+

Single Antigen beads

Single Antigen Bead-based HLA Antibody Testing: Lum inex Technology

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HLA class I antibody test results: Antibodies to A2 CREG

• Specificities: A2, A68, A69, B57, B58• CPRA: 62%• One Antibody

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α1-domain α2-domain

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Public and Private Epitopes (antigenic determinants)

Public Epitopes

A36A1B58B57A69A68A2

Private Epitopes

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• Specificities: A2, A68, A69, B57, B58• CPRA: 62%• One Antibody

CPRA: 62%

HLA class I antibody test results: Antibodies to A2 CREG

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Recipient

A1, A1, B7, B8

A28A23

A69

A68

B57

A24 B58

No antibodies to self-HLA are made.

Allograft

A1, A2, B7, B8

Individuals alloimmunized by a specific HLA type ca n make antibodies to many HLA types.

Anti-A2

Cross-REactive groups (CREG)

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Cross-REactive Groups (CREG)CREG HLA Specificities CPRA value

A1 A1,A3,A11,A29,A30,A31,A36,A80 65%

A2 A2,A23,A24,A68,A69,B57,B58 75%

A10 A25,A26,A32,A33,A34,A43,A66,A74 22%

Bw4 A23,A24,A25,A32,Bw4 74%

B5 B18,B35,B46,B49,B50,B51,B52,B53,B62,B63,B71,B72,B73,B75,B76,B77,B78 56%

Bw6 Bw6 85%

B7 B7,B8,B13,B27,B41,B42,B47,B48,B54,B55,B56,B59,B60,B61,B67,B81,B82 59%

B8 B8,B18,B38,B39,B59,B64,B65,B67 36%

B12 B13,B37,B41,B44,B45,B47,B49,B50,B60,B61 48%

C1 Cw1,Cw7,Cw8,Cw9,Cw10,Cw12,Cw14,Cw16,B46,B73 77%

C2 Cw2,Cw4,Cw5,Cw6,Cw15,Cw17,Cw18 66%

DR1 DR1,DR10,DR103 21%

DR51 DR51,DR15,DR16 29%

DR52 DR52,DR11,DR12,DR13,DR14,DR17,DR18 62%

DR53 DR53,DR4,DR7,DR9 50%

DQ1 DQ5,DQ6 64%

DQ2 DQ2 37%

DQ3 DQ7,DQ8,DQ9 56%

DQ4 DQ4 10%

DP1c DP2,DP3,DP4,DP6,DP9,DP10,DP11,DP14,DP17,DP18.DP20,DP28 ----

DP2c DP1,DP5,DP13,DP15,DP19,DP23 ----

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Bw6 Antibodies

CPRA=85%

A B Cw DR DR DQ2 13(Bw4) 10 15 51 533 38(Bw4) 7 16 51 5

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Spouse HLAA2-B61(Bw6)-DR4A2-B39(Bw6)-DR4

Women alloimmunized by Bw6 motif can make antibodies to 2/3 of HLA-B types

B7, B8, B14, B18, B22, B35, B39, B40, B4005, B41, B42, B45, B46, B48, B50, B54, B55, B56, B60, B61, B62, B64, B65, B67, B70, B71, B72, B73, B75, B76, B78, B81, B82

A2-B44(Bw4)-DR4A2-B52(Bw4)-DR4

Self HLA

CPRA=85%

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Weak Bw6 Antibodies – Risk of memory response

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Bw4 Antibodies

CPRA=61%

A B Cw DR DR DQ2 35(Bw6) 4 4 53 831 35(Bw6) 4 11 52 7

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A B Cw DR DR DQ2 46 1 9 53 92 46 1 14 52 5

Bw4 & Bw6 Antibodies

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Antibodies to all HLA except to self-HLA

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Allele-specific Antibodies

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LABXpress™ Pipettor HLA Antibody Report

Page-1 of 2 Page-2 of 2

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HLA Lab

HLA lab updates VXM & PXM qualification weekly

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VXM orPXM

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Antibody Binding Sites on HLA (epitopes)

Peptide+HLAepitope

Conformational epitope

Peptide epitope

Linear epitope

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- Protein Miss fold- Denatured Antigens- Cryptic Epitope- Loss of Epitope- High Sensitivity- Variable Densities- Not all Alleles are Covered

Problems with Single Antigen Assay(False Positive/Negative Reactions)

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Female – 1 st Tx (n=1522)Female – Re-Tx (n=209)Male – 1st Tx (n=2109)Male – Re-Tx (n=259)

HLA Antibody Profile in Kidney Waiting List Candida tes (n=5281)

Negative (n=1182)

HLA Antibody Testing by Single Antigen Beads

Positive (n=4099)

HLA Antibody Screen by Mixed Beads/Phenotype Beads

4099 candidates X 123 antibodies = 504,177 antibodies with MFI

Female – 1 st Tx (n=364)Female – Re-Tx (n=26)Male – 1st Tx (n=751)Male – Re-Tx (n=41)

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Re-Tx FemaleN= 197

Re-Tx MaleN= 237

1st Tx FemaleN=1241

1st Tx MaleN=1537

A DPDQDRCB

Candidate

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� Locus-specific (<Cw)� Allele-specific (<B44)� Tissue-specific (<neuronal tissue)� Cytokine-induced (IFN- γ)� Down regulation by viral infection

and tumor transformation.

HLA Expression Variation

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Single Class I DSA MFI vs. T cell Crossmatch MCS

2000

50

51

Single Class II DSA MFI vs. B cell Crossmatch MCS

2000

120

0

50

100

150

200

250

300

350

400

450

500

0 5000 10000 15000 20000 25000 30000

MCS

MFI

DPB1DRB1DQB1DQA1DRB345

Few / well defined HLA-A,B,C, DR, DQB and/or DQAAntibodies only

- Typically >1000 MFI- CREG with any MFI

VXM- (DSA-)

HLA lab Protocol for Deceased Donor Kidney Transpla ntationHLA lab Protocol for Deceased Donor Kidney Transpla ntation

Single antigen bead HLA antibody identification (At least 2 sera are tested that are drawn within a year)Single antigen bead HLA antibody identification (At least 2 sera are tested that are drawn within a year)

HLA Antibodies Negative

HLA Antibodies Positive

None

VXM- (DSA-)

Unacceptable HLA Antigens

Crossmatch

All VXM are retrospectively confirmed by FXM

PXM - Call Lab/ Director PXM - Call Lab/ Director

Well defined antibodies and/or• DPβ, DPα Ab• Allele-specific Ab• Unstable Ab• Too many weak Ab

>2000 MFI

VXM FXM (pronase)

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Tray List ; quarterly sera

~900 Active Candidates• with total points of >7• all AB-blood group• consented for KDPI

>85%

Virtual XMcandidate Update

Unacceptable Antigens in UNet

Transplant

Antibody by Single HLA Beads

Physical XMcandidate

every 3 months

every 3 months

Waiting List (n=5416)

New Kidney Allocation System

New Candidate

HLA Typing

Antibodies by Single HLA Beads

& List Unacceptable Antigens;Receives points per CPRA

<20% CPRA

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5.5%17.9%

31.7% 28.8%

61.2%37.6%

74.8%60% 62.5%

33%56.9%

7.3% 8.3% 8.7% 5.8%

0%

20%

40%

60%

80%

100%

CPRA 100%(n=209)

99%(n=80)

95-98%(n=120)

80-94%(n=151)

0-79%(n=1159)

% of

cand

idates

Re-Tx candidates with total points >7 (n=1719)

32.6% (n=560)

Male

Female

Re-

tx1

st-t

x

55

0

20

40

60

80

100

DRB DPDQ

CA

DR53

DR51

DR52 DRB DPDQ

CA

DR53

DR51

DR52 DRB DPDQ

CA

DR53

DR51

DR52 DRB DPDQ

CA

DR53

DR51

DR52 DRB DPDQ

CA

DR53

DR51

DR52

Antibody profile of candidates with total points >7 (n=1719)

% of

cand

idates

CPRA 100%(n=209)

99%(n=80)

95-98%(n=120)

80-94%(n=151)

0-79%(n=1159)

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85.4%(n=108)

14.6%(n=44)

FXM

VXM

Most transplants are preformed using VXM approach in new KAS era

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0

10

20

30

40

50

60

70

80

90

Pre-KAS (1/1/2014 to 12/3/2014) n=235Post-KAS (12/4/2014 to 7/31/2015) n=152

CPRA 100%99%95-98%80-94%0-79%

% of

Tran

splan

tsPre- vs. Post-KAS: Transplant rate

58

59

99%n=60

100%n=202

98%n=49

80-97%n=276

20-79%n=939

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

1 3 5 7 9 110%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

1 3 5 7 9 110%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

1 3 5 7 9 11

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

1 3 5 7 9 110%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

1 3 5 7 9 11

CPRA

% F

requ

ency

of C

andi

date

s

Frequency of CREG Antibodies in Kidney waitlist can didates with different CPRA Groups

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Most 100% CPRA candidates are sensitized to large number of HLA antigens

Candidate#2:A :1 2 11 24 25 26 29 30 31 32 33 34 36 43 66 68 69 74B :13 18 27 37 38 39 41 42 44 45 46 47 49 51 52 53 54 55 56 57

58 59 61 62 63 64 65 67 7 71 72 73 75 76 77 78 8 81 82Cw :1 2 5 7 8 9 10 12 14 15 16 18DR :1 4 7 8 9 10 11 12 13 15 16 103 14:02DR :51 53DQ :4 6 7 8 9DQA:02 03DP :2 3 6 9 10 14 17 18 20 28 04:02

Candidate#1:DR :4 7 8 11 12 13 14 15 16 17 18 103DRw:51 52DQ :6 7 8 9

100%CPRA

100%CPRA

97%CPRA

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100

79.5

51.5

32.527.5

8.5

19.5

15.516.5

3.5

6

10.5

5.5

8.5

23

41.5

50.5

0

10

20

30

40

50

60

70

80

90

100

current >2000 >5000 >8500 >10000

CPRA

MFI Cutoff

% W

aitli

st C

andi

date

s (n

=200

)

<97%

98%

99%

100%

MFI Cutoff and CPRA

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Am

ount

of A

ntib

odie

s -

MF

I

De Nova DSA

CDC xMFlow xMDSA

+++

–++

––+

–––

Memory Response

HLA Antibodies and Risks of Antibody-Mediated Rejec tion

Accelerated Rejection

HyperacuteRejection

Chronic Rejection

63

45

179

21

16

367

136

Living Donor 147Deceased Donor 220

(Wait list=5198)

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Adult=69Pediatric=67

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Number of Transplants Performed in UCSF in 2015 (n= 779)

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Waiting List

University of California San Francisco (UCSF)Kidney Transplant Program (7/1/2014-6/30/2015)

Transplants

95.1% (n=105,743) 98% (n=17,425)

2.0% (n=359)4.9% (n=5,198) UCSF

All other

centers

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CIBMTR monitoring of 1-year Overall Survival for First Allogeneic HCT (performed 2011-2013) suggests that, based on the complexity of the HCTs performed at UCSF:

Our predicted OS rate should be 78.9% (95% CI: 71.5-86.6%)

Our actual OS was 85.4% (N = 103)

UCSF Tops North America in Pediatric Hematopoietic Stem Cell Transplant Outcome

Slide: Christopher C. Dvorak

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Immunogenetics and Transplantation LaboratoryDepartment of Surgery, University of California, San Francisco

Thank You