Scientific Principles of HLA

antibody characterization

Rajalingam Raja, Ph.D., D (ABHI)

Professor of Clinical Surgery &
Director of Immunogenetics and Transplantation Laboratory
University of California at San Francisco (UCSF)

1
 Outline of this Presentation

• Impact of HLA antibodies in transplantation (Tx)

• Principles of antibody assignment – MFI, CREG
• What antibodies triggered by kidney Tx or by
pregnancies?
• What antibodies found in unsensitized males?
• Hierarchy of antibody provoking HLA types in kidney Tx
• Correlation between MFI and MCS
• Conundrum of HLA-DP DSA
• C3d/C1q-bindings De Novo DSA in kidney Tx
• IVIG infusions deplete DSA in Lung Tx Recipients
• Transfusion-induced HLA antibodies are not stable, and
do not rebound following heart transplantation

2
 Means of HLA Antibody-Mediated Rejection
Activation of
complement
cascade

Allograft
Antibody-dependent
cell cytotoxicity

Donor-Specific NK Cell
HLA Antibodies

Opsonization &
increased antigen Activation of
presentation APC Endothelial Cell

3
 1-domain 2-domain

4
 No antibodies to self-HLA are made.
Individuals alloimmunized by a specific HLA type can make
antibodies to many HLA types.

Allograft
A1, A2, B7, B8

A68 A23
Anti-A2 A28
A69 B57
A24 B58
Recipient
A1, A1, B7, B8
Cross-REactive groups (CREG)
5
 Public and Private Epitopes (antigenic determinants)

A2 A68 A69 B57 B58 A1 A36

Public Epitopes Private Epitopes

6
 HLA Antibody Testing Methods
• Complement-dependent Cytotoxicity
(CDC) Method

• Solid Phase Methods

• Mixed Antigen Beads:
– Positive/Negative
• Multi Antigen Beads:
– PRA and Specificities
• Single Antigen Beads:
– CPRA, Specificities, Strength (MFI)

7
 Solid Phase HLA Antibody Testing: Luminex Technology
Single HLA Antigen Beads
A2

A66
B55

Polystyrene
Microspheres

8
 patient’s serum Single Antigen beads

A2
A2
A66
B55 A66
+ B55

Detection &
Interpretation

9
 Antibodies Against A2 CREG

• Specificities: A2, A68, A69, B57, B58

• CPRA: 62%
• One Antibody
CPRA: 62%

10
 Cross-reactive groups (CREG)
CREG HLA Specificities CPRA vallue

A1 A1,A3,A11,A23,A24,A29,A30,A31,A36,A80 78%
A2 A2,A23,A24,A68,A69,B57,B58 75%
A10 A11,A25,A26,A32,A33,A34,A43,A66,A68, A69, A74 40%
Bw4 A23,A24,A25,A32,Bw4 74%
B5 B18,B35,B46,B49,B50,B51,B52,B53,B57, B58, B62,B63,B71,B72,B73,B75,B76,B77,B78 63%
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 ----

*DP-specific antibodies that are shown to occur frequently together in UCSF waitlist population

11
 Bw6/Bw4 Epitope

Bw6:
B7, B8, B18, B2708, B35, B39,
B40, B4005, B41, B42, B45,
B48, B50, B54, B55, B56, B60,
B61, B62, B64, B65, B67, B70,
B71, B72, B75, B76, B78, B81,
73
76 78 79 B82
77 80 82 83
81
Bw4:
Bw4-T80: B13, B27, B37, B44,
B47
Bw4-I80: B38, B49, B51, B52,
B53, B57, B58, B59, B63, B77,
A23, A24, A25, A32

Bw-:
B46, B73

12
 Bw6 Antibodies
A B Cw DR DR DQ
2 13(Bw4) 10 15 51 5
33 38(Bw4) 7 16 51 5

CPRA=85%

13
Women alloimmunized by Bw6 motif can make antibodies to 2/3 of HLA-B types

Spouse HLA
A2-B61(Bw6)-DR4
A2-B39(Bw6)-DR4

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

Self HLA
A2-B44(Bw4)-DR4
A2-B52(Bw4)-DR4
CPRA=85%

14
 Weak Bw6 Antibodies – Risk of memory response

15
 Bw4 Antibodies

A B Cw DR DR DQ
2 35(Bw6) 4 4 53 8
31 35(Bw6) 4 11 52 7

CPRA=61%

16
 Bw4-I80 Antibodies
A B Cw DR DR DQ
11 62(Bw6) 15 15 51 5
34 61(Bw6) 7 15 51 5

Antigen Bw4 aa 77-83

B27 Bw4 T80 DLRTLLR
B37 Bw4 T80 DLRTLLR
B47 Bw4 T80 DLRTLLR
B13 Bw4 T80 NLRTALR
B44 Bw4 T80 NLRTALR
B38 Bw4 I80 NLRIALR
B49 Bw4 I80 NLRIALR
B51 Bw4 I80 NLRIALR
B52 Bw4 I80 NLRIALR
B53 Bw4 I80 NLRIALR
B57 Bw4 I80 NLRIALR
B58 Bw4 I80 NLRIALR
B59 Bw4 I80 NLRIALR
B77 Bw4 I80 NLRIALR
B63 Bw4 I80 NLRIALR
A23 Aw4 I80 NLRIALR
A24 Aw4 I80 NLRIALR
A25 Aw4 I80 SLRIALR
CPRA=54% A32 Aw4 I80 SLRIALR

17
 HLA allele pairs that differ only by Bw4 and Bw6 motif

18
 Bw4 & Bw6 Antibodies

A B Cw DR DR DQ
2 46 1 14 52 5
2 46 1 14 52 5

19
 C1 and C2 Epitope

AA Pos. 10 20 30 40 50 60 70 80 90 100
C*01:02 CSHSMKYFFT SVSRPGRGEP RFISVGYVDD TQFVRFDSDA ASPRGEPRAP WVEQEGPEYW DRETQKYKRQ AQTDRVSLRN LRGYYNQSEA GSHTLQWMCG
C*07:02 -----R--D- A--------- ---------- ---------- ---------- ---------- ---------- --A------- ---------D ------R-S-
C*08:01 -----R--Y- A--------- ---A------ ----Q----- ---------- ---------- ---------- ---------- ---------- ------R-Y-
C*03:03 G----R--Y- A--------- H--A------ ---------- ---------- ---------- ---------- ---------- ---------- R--II-R-Y-
C*03:04 G----R--Y- A--------- H--A------ ---------- ---------- ---------- ---------- ---------- ---------- ---II-R-Y-
C1 C*03:02
C*12:03
G----R--Y-
*----R--Y-
A---------
A---------
H--A------
---A------
----------
----------
----------
----------
----------
----------
----------
----------
----------
--A-------
----------
----------
---I--R-Y-
--------Y-
C*14:02 -----R--S- ---------- ---A------ ---------- ---------- ---------- ---------- ---------- ---------- --------F-
C*16:01 -----R--Y- A--------- ---A------ ---------- ---------- ---------- ---------- ---------- ---------- --------Y-
B*46:01 G----R--Y- AM-------- ---A------ ---------- ----MA---- -I-------- ---------- ---D------ ---------- ------R-Y-
B*73:01 G----R--H- SV-------- ---T------ ---------- ----EE---- ---------- --N--IC-AK A--D--G--- ---------D ----W-T-Y-

C*02:02 -----R--Y- A----S---- H--A------ ---------- ---------- ---------- ---------- ------N--K ---------- ------R-Y-
C*04:01 *----R--S- ---W------ ---A------ ---------- --------E- ---------- ---------- --A---N--K ---------D ------R-F-
C*05:01 *----R--Y- A--------- ---A------ ----Q----- ---------- ---------- ---------- ------N--K ---------- ------R-Y-
C2 C*06:02
C*15:02
-----R--D-
-----R--Y-
A---------
A---------
----------
H--A------
----------
----------
----------
----------
----------
----------
----------
-----N----
--A---N--K
------N--K
---------D
----------
--------Y-
---II-R-Y-
C*17:01 *----R--Y- A--------- ---A------ ---------- ---------- ---------- ---------- --A---N--K ---------- ----I-R-Y-
C*18:01 *----R--D- A--------- ---S------ ---------- ---------- ---------- ---------- --A---N--K ---------D ------R-F-

20
 C1 Antibodies
A B Cw DR DR DQ
1 35(Bw6) 4 10 5
24 37(Bw4) 6 12 52 7

CPRA=77%

21
 C2 Antibodies

A B Cw DR DR DQ
24 7(Bw6) 7 (C1) 15 51 6
34 60(Bw6) 10 (C1) 7 53 2

CPRA=66%
22
 Bw6 and C1

23
 Antibodies to all HLA except to self-HLA

24
 Allele-specific Antibodies

25
 LABXpress™ Pipettor

26
Antibody Binding Sites on HLA (epitopes)

Conformational
epitope

Peptide
epitope

Peptide+HLA
epitope
Linear epitope
 Problems with Solid Phase Assay
(False Positive/Negative Reactions)

- Protein Miss fold

- Denatured Antigens
- Cryptic Epitope
- Epitope Loss
- High Sensitivity
- Variable Densities
- Allele-Specific Antibodies

28
 HLA Antibody Profile in Kidney Waiting List Candidates
(n=4204)

Kidney waitlist candidates re-tx females – 203

n=4207 re-tx males – 253
1st-tx females – 1477
1st-t males – 2274
Serum: DTT-treated

HLA class I & II Antibody testing by

One lambda Single Antigen Bead Assay

Data : 4207 x 121 antibodies = 509,047 antibodies with MFI

Frequency analysis MFI analysis via Tukey’s test Over reactive beads analysis

29
 HLA antibody constellation varies by the mode of prior sensitization to HLA
A B C DR DQ DP
A1C A2C A10C B5C B7C B8C B12C C1 C2 DR1C DR51C DR52C DR53C DQ1C DQ3C DP1C DP2C

Re-tx
(n=203)
Re-tx
(n=253)
Kidney waitlist candidates (n=4207)

1st-tx
(n=1477)

1st-tx
(n=2274)

30
 What HLA-mismatch is bad for kidney transplantation?

Allograft failure &

Transplant Relist or 2nd Tx

Recipient 4-13 years

(No HLA antibodies)
HLA antibody test

31
 HLA antibodies in patients with kidney graft failure (n=308)

Pre-Tx HLA antibodies

HLA antibodies at the
time of relisting

32
 HLA antibodies Produced following kidney transplantation (n=308)

R2=0.60
Frequency

Strength (mean of composite MFI)

33
 HLA antibodies Produced by pregnancies (n=1477)

Frequency

Strength (mean of composite MFI)

34
 HLA antibodies detected in males waiting for a kidney
transplantation (n=2274)

Strength (mean of composite MFI)

35
 Conclusions
• DQ is the most frequently and strongly encountered Abs and thus
DQs are the most aggressive antibody inducer following KTx.
DR53 group of antigens (except DR4) and A2 CREG are the next
most aggressive Ab inducers.

• All HLA-Cw antigens and a subset of DPB antigens (DP15, DP12,

DP5, DP1, DP23, DP4) induce Ab production at low levels and at
low frequency. Within Cw types, the most poorly immunogenic
seems to be those carrying C1 epitope that binds to KIR2DL2/L3.

• The moderate inducer includes most HLA-B, HLA-A and DR

antigens, and the hierarchy from the strongest inducer within this
group is B7>A10>B5>DR52>DR51 CREG. The B5 CREG was
diverse and observed to have four outliers from the linear fit.

• The strongly immunogenic HLA types should be considered for

matching in Ktx, while the poorly immunogenic HLA antigens are
tolerable.
36
 Single Class I DSA MFI vs. T cell Crossmatch MCS

Cut-off for
Positive
T-FXM = 50
MCS

2000
2000 MFI

37
 Single Class II DSA MFI vs. B cell Crossmatch MCS

Cut-off for
Positive
B-FXM =120
MCS

2000 MFI

38
 Clinical Significance of HLA Antibodies

• Conundrum of HLA-DP DSA

• C3d/C1q-bindings De Novo DSA in
kidney Tx
• IVIG infusions deplete DSA in Lung
Tx Recipients
• Transfusion-induced HLA antibodies
are not stable, and do not rebound
following heart transplantation

39
 • Conundrum of HLA-DP DSA

40
 A Case Study

41
 UNOS donor is mismatched for only DPB1*01:01 and DPA1*02:01

01 02:01 01:03
01 04:01 01:03

42
43
 IVIG suppress HLA-DP antibodies more efficiently than the other HLA antibodies
MFI

DSA
C4d Negative
44
 Kidney transplants with HLA-DP DSAs
Sex # Time
Post-Tx Post-Tx
Prev CPRA HLA Antibodies T XM B XM Pre-tx DSA MFI post-Tx Biopsy date C4d
DSA MFI DSA Date
TX (Mos)
F 2 100% A:2 3 11 23 24 25 26 29 30 31 32 33 34 36 43 66 68 69 74 80 NEG POS DP19=23073 20462 3 days 5/25/2015 5/28/2015 Neg
B:13 27 35 38 39 41 42 45 46 47 48 49 50 51 52 53 54 55 56 (MCS=200)
57 58 59 60 61 62 63 64 67 7 71 72 73 75 76 77 78 81 Cw:1
2 4 5 6 9 10 12 15 17 18 DQ:7 8 9 DQA:03:03 DP:1 3 5 6 9 10
11 13 14 17 19 20
F 0 100% A:1 2 3 11 25 26 30 31 33 34 36 43 66 68 69 74 80 B:13 27 NEG POS DP402=15568 NEG 6 7/17/2015 7/17/2015 Neg
2708 46 47 48 57 60 61 62 63 7 73 75 76 77 81 Cw:2 4 5 6 7 (MCS=143)
15 17 18 DR:7 8 9 11 12 13 16 103 DQ:4 5 6 DQA:02 DP:1 2
3 5 6 9 10 11 13 14 15 17 18 19 20 28 402
M 1 99% A:2 11 23 25 26 29 32 34 43 66 69 B:13 27 38 44 49 51 52 NEG POS DP1=10930; 3540; 6 12/17/2015 12/17/2015 Neg
53 57 58 59 63 Bw:4 Cw:1 DR:9 10 DRw:53 DQ:2 4 7 8 9 (MCS=204) DPA1*02=1457 5268
F 1 99% A:1 11 23 24 25 26 29 32 33 34 36 43 66 68 69 80 B:13 18 NEG NEG DP4=6956 NEG 6 7/2/2015 7/2/2015 Neg
35 37 38 39 42 44 45 46 51 54 55 57 59 62 63 64 65 67 75
76 77 78 8 82 Cw:1 10 DR:4 8 11 12 13 14 17 18 DRw:52
DQ:4 6 7 8 9 DQA:03 04 05 DP:2 18 23 28 4
F 2 100% A:1 2 23 24 25 32 68 69 B:13 27 37 44 45 47 48 60 61 7 76 NEG NEG DP2=5853 NEG 7 7/30/2015 7/30/2015 Neg
81 DR:8 11 12 13 14 15 16 17 18 DRw:51 DQ:4 5 6 DP:2 3 6
9 10 14 18 20 28 402
F 0 A98% A:23 24 25 32 B:13 27 37 38 41 44 45 47 49 50 51 52 53 57 NEG NEG DP3=5547 NEG 2 10/8/2015 NA NA
58 59 60 61 63 7 76 77 81 82 Bw:4 Cw:2 5 9 10 15 DR:4 11
DQ:2 DP:2 3 6 9 10 14 17 18 20 28 04:02
F 0 99% A:1 2 23 24 25 29 32 43 68 69 B:13 27 37 38 44 45 47 49 50 NEG NEG DP3=4667 1182 6 10/8/2015 10/8/2015 Neg
51 52 53 57 58 59 62 63 72 76 77 82 Bw:4 DR:7 15 16 DQ:2
DP:2 3 6 9 14 17 18 20 28 4
M 1 A70% B:2708 42 54 55 56 67 7 81 Cw:2 5 6 15 17 18 DR:7 DP:5 9 NEG NEG DP5=4144 NEG 6 10/21/2015 10/21/2015 Neg
10 14 17 DPA:02
M 0 0% DP:1 3 5 6 9 11 13 14 19 20 NEG NEG DP1=3186 1378 6 7/1/2015 6/19/2015 Neg
M 1 A100% A:11 B:49 52 56 62 71 72 Cw:7 17 DR:4 7 8 9 11 12 13 14 17 NEG NEG DP3=2823 NEG 8 10/6/2015 10/6/2015 Neg
18 DRw:52 53 DQ:2 4 6 7 8 9 DP:2 3 6 9 10 14 17 18 20 28
0402
F 0 98% A:2 25 26 29 31 32 33 34 43 66 68 69 74 B:7 8 42 54 55 57 NEG NEG DP2=1916; NA NA NA NA NA
58 81 82 Cw:17 DR:7 8 9 11 12 13 14 17 18 DRw:52 DP6=2766
DQA:0501 DP:3 6 9 20
F 0 A99% Cw:2 4 5 6 15 17 18 DR:4 8 10 11 12 13 14 15 16 17 18 NEG NEG DP2=2387 NA NA NA NA NA
DRw:52 DQ:7 8 9 DQA:05 DP:2 3 6 9 10 14 17 18 20 28
04:02
F 1 100% DR:4 7 9 10 DRw:53 DQ:2 5 6 7 8 9 DQA:03 DP:1 3 5 6 9 10 NA NA DP3=2003 NEG 6 9/10/2015 9/10/2015 Neg
11 13 14 15 17 18 19 20
F 2 A100% A:2 3 23 24 29 66 68 69 B:13 27 37 41 44 45 47 48 49 50 56 NEG NEG DPA1*02=1000 1610 3 9/19/2015 9/21/2015 Neg
57 58 60 61 65 67 73 8 82 Cw:5 DR:1 9 10 15 16 103
DRw:51 DQ:4 5 6 8 9 DQA:02 03 04 06 DP:5

45
IVIG suppress HLA-DP DSAs more efficiently in kidney transplant recipients
25000
3 Patients: AMR

Pat.1 - DP1
MFI of Donor-specific HLA-DP Antibodies

20000 Pat.2 - DP4

Pat.3 - DP1
Pat.4 - DP4
Pat.5 - DP3
15000 Pat.6 - DP4
Pat.7 - DP5
Pat.8 - DP3
Pat.9 - DP19
10000 Pat.10 - DP3
Pat.11 - DP3
Pat.12 - DP4
Pat.13 - DP4
5000 Pat.14A - DP4
Pat.14B - DP14
Pat.15 - DP4
Pat.16 - DP2
0
PRE-TX 1 WK 1 MO 6 MOS
Post Transplant DSA Monitoring

46
 Conclusions

 The DP DSAs <7000 MFI do not cause positive

cross match.

 Kidneys transplanted with positive cross match

with strong DP DSA showing no AMR.

 IVIG infusion is more suppressive on DP

antibodies than other HLA antibodies.

47
 • C1q/C3d-bindings De Novo DSA
in kidney transplantation

48
 Study Subjects
Kidney Transplant Recipients (n=123) who underwent a protocol
or for-cause kidney biopsy, and had de novo DSA (by One Lambda SAB) at the
time of biopsy (median 3.8 yrs post-tx).
Age (Mean + SD) 47 + 18 years
Gender Female; n=53 (43%)
Transplant Primary; n=109 (89%)
Re-graft; n=14 (11%)
Donors
Age (Mean + SD) 41 + 15 years
Type of donor Living; n=46 (37%)
Deceased; n=77 (63%)
Type of transplant Kidney only; n=117 (95%)
Kidney/Pancreas; n=6 (5%)
Number of HLA MM 7.7 + 2.3 (A/B/Cw/DR/DQ)
 HLA Antibody Testing Methods (Luminex®)
Serum sample collected at the time of biopsy
(median 3.8 yrs post-tx) were tested for HLA
antibodies using …

• IgG Antibodies:
– One Lambda Single Antigen Beads.
– LIFECODES Single Antigen Beads.
• C1q-binding Antibodies:
– One Lambda C1q Screen.
• C3d-binding Antibodies:
– LIFECODES C3d Detection assay.
 IgG DSA MFI (OL) vs. C3d and C1q-binding activity
P=0.95
P<0.0001

P<0.0001

51
 • IVIG infusions deplete Donor-
specific HLA Antibodies in Lung
Transplant Recipients

52
 Impact on IVIG treatment on HLA Antibodies

Tx
MFI

IVIG infusions

53
IVIG infusions deplete donor-specific HLA antibodies in
lung transplant recipients
14000
Cw10

12000
A3

10000 Cw7
DQ2
B44
8000
Cw7
MFI

DQ2
6000
DQ4
A2
A1
4000
B8
DQ6
2000 A68

0
pre-IVIG 3 months 6 months 10 months current
Post - IVIG
Post - IVIG

54
 Summary
• Most de novo DSA are specific to HLA
class II, most frequently to HLA-DQ.
• IVIG infusion effectively depletes DSA in
Lung Tx recipients.
• Long term follow up is undergoing to
determine whether IVIG treatment prolongs
lung allograft survival.

55
 • Transfusion-induced HLA
antibodies are not stable, and do
not rebound following heart
transplantation

56
cPRA activity over time in patients with allosensitization associated
with the left ventricular assist device (LVAD).

57 Ko B et al. JHLT 2016, 35:1024–1030

 Transfusion-induced HLA antibodies are not stable, and do not rebound
following heart transplantation
Recipient-1

VAD/Transfusions
Heart Transplantation

Recipient-2

Heart Transplantation

VAD/Transfusions

VAD/Transfusions

58
Immunogenetics and Transplantation Laboratory
Department of Surgery, University of California San Francisco

Thank You