The ABCs of HLA: TCR Cell Beginners to Advanced CD4 IL2, IFN- - - PowerPoint PPT Presentation

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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 Cytotoxic T 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 Depletion

Plasmaphoresis

Antibody Blocking

IVIG Anti-C5a

Unacceptable Antigens

HLA antibodies

Plasma cell

Rejection Lymphocytes

T T T T T T T T T T T T T T T T T T T T NK NK NK NK B B B B B B B B B B B B NK NK NK NK NK NK NK NK

Maintenance Therapy

Immunosuppression

Cyclosporine MMF Steroids

Induction Therapy

Lymphocytes Depletion

Anti-Thymoglobulin → T & NK cells Anti-CD3 → T cells Anti-CD25 → Activated T cells Anti-CD52 → mature lymphocytes Anti-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

APC APC

• 4. Activation of

Endothelial Cell

Organ Allograft Endothelium

HLA Class I HLA Class II

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Histocompatibility Testing for Solid Organ Transplantation Donor Recipient

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

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Complement Dependent Cytotoxicity (CDC) Crossmatch

Fluorescein Diacetate + Ethidium Bromide

Live cells Dead 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)

8 187

Specificity Problem Sensitivity Problem

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Flow Cytometry Crossmatch

Donor Recipient Lymphocytes Serum

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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 > 50 B 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 > 50 B 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 Virtual Crossmatch Positive

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Crossmatch Methods

Crossmatch method Sensitivity Specificity Cost (US $) Turnaround time CDC Low Low 600 3.5 hours Flow Intermediate Intermediate 600 5 hours Pronase >Intermediate >intermediate 600 6.5 hours Virtual 100% 100% 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 5 2 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 8 1 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% cPRA anti-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 Points Old New 0–19 20–29 30–39 40–49 50–59 60–69 70–74 75–79 80–84 4 85–89 4 90–94 4 95 4 96 4 97 4 98 4 99 4 100 4 Regional Sharing National Sharing

Priority points for CPRA>19%

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

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

CPRA

5% 4% 1.6% 0.8% 15.9% 72.7%

Points

202.1 50.09 24.4 4.05-17.3 0.08-2.46

#Patients

216 85 46 273 867 3974

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Sequence-Specific Oligonucleotide (SSO) Hybridization Method

Polystyrene Microspheres Polystyrene 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 technology

A2 A1 A11 A3 A23

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PCR amplification using florochrome-tagged locus-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: Luminex Technology Polystyrene Microspheres Polystyrene Microspheres

A2 A66 B55

Single HLA Antigen Beads

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Detection & Interpretation

A2 A66 B55

patient’s serum

A2 A66 B55

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Single Antigen beads

Single Antigen Bead-based HLA Antibody Testing: Luminex 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

A36 A1 B58 B57 A69 A68 A2

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

A28 A23 A69 A68 B57 A24 B58

No antibodies to self-HLA are made.

Allograft A1, A2, B7, B8

Individuals alloimmunized by a specific HLA type can 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 DQ 2 13(Bw4) 10 15 51 5 33 38(Bw4) 7 16 51 5

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Spouse HLA A2-B61(Bw6)-DR4 A2-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)-DR4 A2-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 DQ 2 35(Bw6) 4 4 53 8 31 35(Bw6) 4 11 52 7

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A B Cw DR DR DQ 2 46 1 9 53 9 2 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 or PXM

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Peptide+HLA epitope 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 – 1st 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 Candidates (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 – 1st Tx (n=364) Female – Re-Tx (n=26) Male – 1st Tx (n=751) Male – Re-Tx (n=41)

48 Re-Tx Female N= 197 Re-Tx Male N= 237 1st Tx Female N=1241 1st Tx Male N=1537

A DP DQ DR C B

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

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Single Class II DSA MFI vs. B cell Crossmatch MCS

2000 120

50 100 150 200 250 300 350 400 450 500 5000 10000 15000 20000 25000 30000

MCS MFI

DPB1 DRB1 DQB1 DQA1 DRB345

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

• Typically >1000 MFI
• CREG with any MFI

VXM- (DSA-)

HLA lab Protocol for Deceased Donor Kidney Transplantation HLA lab Protocol for Deceased Donor Kidney Transplantation

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 XM candidate

Update Unacceptable Antigens in UNet Transplant

Antibody by Single HLA Beads Physical XM candidate

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 candidates

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

32.6% (n=560) Male Female Re-tx 1st-tx

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20 40 60 80 100

DR B DP DQ C A DR53 DR51 DR52 DR B DP DQ C A DR53 DR51 DR52 DR B DP DQ C A DR53 DR51 DR52 DR B DP DQ C A DR53 DR51 DR52 DR B DP DQ C A DR53 DR51 DR52

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

% of candidates

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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10 20 30 40 50 60 70 80 90

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

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

% of Transplants

Pre- 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 11 0% 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 11 0% 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 11

CPRA % Frequency of Candidates Frequency of CREG Antibodies in Kidney waitlist candidates 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 74 B :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 82 Cw :1 2 5 7 8 9 10 12 14 15 16 18 DR :1 4 7 8 9 10 11 12 13 15 16 103 14:02 DR :51 53 DQ :4 6 7 8 9 DQA:02 03 DP :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 18 103 DRw:51 52 DQ :6 7 8 9

100% CPRA 100% CPRA 97% CPRA

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100 79.5 51.5 32.5 27.5 8.5 19.5 15.5 16.5 3.5 6 10.5 5.5 8.5 23 41.5 50.5 10 20 30 40 50 60 70 80 90 100

current >2000 >5000 >8500 >10000

CPRA

MFI Cutoff % Waitlist Candidates (n=200)

<97% 98% 99% 100%

MFI Cutoff and CPRA

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Amount of Antibodies - MFI

De Nova DSA

CDC xM Flow xM DSA

+ + + – + + – – + – – –

Memory Response

HLA Antibodies and Risks of Antibody-Mediated Rejection

Accelerated Rejection Hyperacute Rejection Chronic Rejection

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45 179 21 16 367 136

Living Donor 147 Deceased Donor 220 (Wait list=5198)

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Adult=69 Pediatric=67

1 5 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

• ther

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 Laboratory

Department of Surgery, University of California, San Francisco

Thank You