Major H istocompability’ Complex

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transplantation Eric Spierings Laboratory for Translational Immunology Section HLA and Tissue Typing UMC Utrecht

content

•  organ transplantation

•  hematopoietic stem-cell transplantation

•  take home message

transplantation and immunological outcome

patient donor

solid organ graft

Graft Rejection

immunological outcome

the major antigens in transplant rejection

M H C

Major

Histocompa5bility

Complex

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M H C

Major

Histocompa5bility

Complex


H L A

Human

Leukocyte An5gen

function of HLA

•  HLA supports the immune system (T cells) in eliminating pathogens

•  to this end, HLA needs to be able to present a broad repertoire of antigens

•  a large diversity of different HLA molecules within a single individual is essential

HLA is polygenic

•  HLA class I: –  A –  B –  C

•  HLA class II: –  DR (DRB1, DRB3, DRB4, DRB5) –  DQ –  DP

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HLA loci are highly polymorphic HLA loci are highly polymorphic IMGT database sept 2009

Locus alleles (n) proteins (n)

HLA-A 853 652

HLA-B 1249 1046

HLA-C 463 361

HLA-DRB1 659 553

HLA-DQA1 34 25

HLA-DQB1 99 72

HLA-DPA1 27 16

HLA-DPB1 135 118

so theoretically: 652*1046*361*553*25*72*16*118 =

462 685 733 408 102 400 (463 * 1015)

options per haplotype

genetic diversity is a good thing

•  an optimal variation within the HLA-system is a positive situation

•  it has a positive effect on progression of certain infectious diseases

•  a large diversity within the HLA system has a high potential to generate a broad immune response

good for the species, but bad for transplantation! •  the high level of polymorphism in combination with the

multiple loci, leads to difficulties in finding a fully HLA-matched organ donor

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organ transplantation – family donor

patient

kidney graft survival by number of HLA mismatch

types of humoral rejection

•  hyperacute rejection –  occurs when the recipient has previously been sensitized

to the donor tissue –  caused by complement fixation of pre-existing antibodies

against the –  transplanted tissue, causing damage and extensive

necrosis –  cejection starts within minutes of reperfusion –  celatively rare now with routine crossmatching of donor/

recipient serum •  acute humoral rejection •  chronic humoral rejection

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an in vitro test for hyperacute rejection an in vitro test for hyperacute rejection

an in vitro test for hyperacute rejection types of humoral rejection

•  hyperacute rejection •  acute humoral rejection

–  characterized by the production of anti-class-I and –II antibodies against the donor tissue (C4d on PTC)

–  rapid loss of graft function (days), any time after transplantation

–  treatment involves IVIG, plasmapheresis and/or rituximab (=anti-CD20)

•  chronic humoral rejection

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types of humoral rejection

•  hyperacute rejection •  acute humoral rejection •  chronic humoral rejection

–  transplant glomerulopathy is strongly associated with circulating antibodies to donor HLA class-II antigens and a poor prognosis

–  alloantibody, basement membrane multilamination, C4d –  outcome and optimal therapy not yet defined

HLA antibodies

•  pregnancy - antigens from the father •  transfusion - leukocytes •  transplantation - HLA mismatches donor

•  contra-indication for transplantation –  decreased chance finding suitable donor –  some patients have antibodies against >85% of potential

donors –  current antibody status versus historic

histocompatibility testing in organ transplantation

•  purpose - prevention of rejection •  matching - HLA class-I: A and B (and C) antigens

- HLA class-II: DR (and DQ) antigens - ABO

•  HLA-antibodies - sensitization - allo- vs autoantibodies - screening - cross-match - focus on IgG1

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procedure for kidney transplantation

•  patient is typed for HLA •  HLA antibodies in the patient are analyzed (screening) •  positive antigens are marked as unacceptable •  scores are assigned based upon antibodies, expected

waiting time, actual waiting time •  the patient waits for a suitable donor

procedure for kidney transplantation

•  donors are typed for HLA •  Eurotransplant matches the donor with the waiting list •  the patient with the highest score gets the kidney •  the transplant center receives the kidney and performs

tests

innovations in organ transplantation solid phase screening

drawback of CDC screening

•  cells have more than 1 type of HLA-antigen on their surface

•  literature: positive class-I cross matches (T-cells) are OK •  50-75% positive class-II cross matches (B cells) are

incorrect! •  interpretation heavily depends on quality of the cells •  A1, A2, B7, B8 •  patient sera usually contain more than 1 HLA-specificity

posi+ve A23, A24, B13, B15

pega+ve A1, A24, B13, B15 A1, A2, B13, B15 A1, A2, B7, B15

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100 different dye ratios Optics in Luminex

100 Region Map bead identification

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A F H E B G C D

Reporter Signal 100 analyses in one tube

innovations in organ transplantation better matching algorithms

The HLAMatchmaker principle

•  HLAMatchmaker is a structure-based matching program

–  each HLA antigen represents a distinct string of

structurally defined epitopes as potential immunogens that can induce specific antibodies

–  patients cannot make antibodies against epitopes that are expressed by their own HLA molecules

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conventional matching

•  Donor B51 (B*51:01) •  Recipient B35, 41 (B*35:01, *41:01)

10 20 30 40 50 B*51:01 GSHSMRYFYT AMSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASPRTEPRAP B*35:01 ---------- ---------- ---------- ---------- ---------- 60 70 80 90 100 B*51:01 WIEQEGPEYW DRNTQIFKTN TQTYRENLRI ALRYYNQSEA GSHTWQTMYG B*35:01 ---------- ---------- ------S--N LRG------- ---II-R--- 110 120 130 140 150 B*51:01 CDVGPDGRLL RGHNQYAYDG KDYIALNEDL SSWTAADTAA QITQRKWEAA B*35:01 --L------- ---D-S---- ---------- ---------- ---------- 160 170 180 190 200 B*51:01 REAEQLRAYL EGLCVEWLRR HLENGKETLQ RADPPKTHVT HHPVSDHEAT B*35:01 -V-------- ---------- Y--------- ---------- ----------

conventional matching

•  Donor B51 (B*51:01) •  Recipient B35, 41 (B*35:01, *41:01)

10 20 30 40 50 B*51:01 GSHSMRYFYT AMSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASPRTEPRAP B*41:01 --------H- ---------- ---T------ -L-------- T---K----- 60 70 80 90 100 B*51:01 WIEQEGPEYW DRNTQIFKTN TQTYRENLRI ALRYYNQSEA GSHTWQTMYG B*41:01 ---------- --E---S--- ------S--N LRG------- ------R--- 110 120 130 140 150 B*51:01 CDVGPDGRLL RGHNQYAYDG KDYIALNEDL SSWTAADTAA QITQRKWEAA B*41:01 ---------- ---------- ---------- R--------- ---------- 160 170 180 190 200 B*51:01 REAEQLRAYL EGLCVEWLRR HLENGKETLQ RADPPKTHVT HHPVSDHEAT B*41:01 -V---D---- --T------- Y-----D--E ---------- ---I------

including all HLA loci

10 20 30 40 50 B*51:01 GSHSMRYFYT AMSRPGRGEP RFIAVGYVDD TQFVRFDSDA ASPRTEPRAP B*35:01 ---------- ---------- ---------- ---------- ---------- B*41:01 --------H- ---------- ---T------ -L-------- T---K----- 60 70 80 90 100 B*51:01 WIEQEGPEYW DRNTQIFKTN TQTYRENLRI ALRYYNQSEA GSHTWQTMYG B*35:01 ---------- ---------- ------S--N LRG------- ---II-R--- B*41:01 ---------- --E---S--- ------S--N LRG------- ------R--- C*04:01 -V-------- --E---Y-RQ A-A-R----K LRG------D ---T-QR--G A*25:01 ---------- -QE-QIV-TN T--D--S--- ---------A ----I-R--- 110 120 130 140 150 B*51:01 CDVGPDGRLL RGHNQYAYDG KDYIALNEDL SSWTAADTAA QITQRKWEAA B*35:01 --L------- ---D-S---- ---------- ---------- ---------- B*41:01 ---------- ---------- ---------- R--------- ---------- Cw04:01 —L-------- --Y------- ---------- R--------- ----------

topography of the mismatches

97,77,80,81,82,83

171 152

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topography of the mismatches

97

77,80,81,82,83

the HLAMatchmaker principle

extend the acceptable mismatches using the HLA typing and the Luminex negatives in HLAMatchmaker

innovations in organ transplantation predicting T-helper cell responses

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Anti-HLA IgG antibodies in a patient (A1, B8), 2 weeks after nefrectomy of first kidney (A3, B18)

MM A3

MM B18

Self HLA A1, B8

The absence of HLA-B18 antibodies cannot be explained by HLAMatchmaker

IgG production requires isotype switching

isotype switching requires T-cell help direct recognition of mismatched HLA

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indirect recognition of mismatched HLA indirect recognition and t-cell help to b cells

historical indications for a role of indirect recognition of HLA

“We conclude that there are at least two human Ir genes, HLA-DRB1*01 and HLA-DRB1*03, that confer a high risk for both humoral allosensitization and renal allograft failure in situations of HLA-Bw4 incompatibility.” Fuller & Fuller Transplantation 1999

HLA-DRB1*01

HLA-DRB1*03

HLA-DRB1*07

hypothesis: •  indirect antigen presentation of donor HLA fragments by the HLA class II molecules of the recipient is essential for the IgMàIgG isotype switching

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question: •  can we explain the unexplained absence of HLA antibodies by the incapability of the involved HLA allele to bind to HLA class II molecules

do anti-HLA IgG antibodies depend on the presence of T-helper epitopes? •  897 kidney transplants in the UMC Utrecht database

•  not immunized pretransplant –  first transplant –  no blood transfusions –  no pretransplant anti-HLA antibodies –  no antibody-mediated rejection –  nefrectomy analyzed by pathologist

•  post-transplant peak serum available

do anti-HLA IgG antibodies depend on the presence of T-helper epitopes?

PIRCHE [ˈpercəә] noun, acronym Predicted Indirectly Recognizable HLA Epitope

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how to calculate PIRCHE-II? immunogenic HLA antigens contain more PIRCHE-II than non-immunogenic antigens

… and this difference depends on the HLA-DRB-1 background of the recipient

PIRCHE-II overlaps partly with eplets

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the overlap between PIRCHE-II and eplets is only 38%

conclusions

•  detection of HLA-specific IgG antibodies correlates with a higher number of PIRCHE-II

•  the HLA-DRB1 type of the recipient plays a crucial role

•  PIRCHE-II overlap only partly with eplets (38%)

•  PIRCHE-II are acting independently from eplets in the formation of HLA-specific IgG antibodies

hematopoietic stem-cell transplantation

hematopoietic stem cell transplantation patient donor

hematopoietic stem cells hematological malignancy,

aplastic anemia, SCID

Graft versus Host Disease

Graft Rejection


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cumulative incidence of acute GVHD II-IV after stem-cell transplantation

days after SCT

50

0

10

20

30

40

0 20 40 60 80 100

2 HLA mismatches -‐ related

HLA iden:cal -‐ related

2 HLA mismatches -‐ unrelated

graft-versus-host disease

•  Graft-versus-host disease (GVHD) is a general complication after allogeneic tissue transplantatie.

•  it often occurs after hematopoietc stem-cell transplantation, but also in other cases where immune cells (leucocytes) in the graft recognize the “foreign” host

•  these transplanted immune cells attack the cells of the host

Billingham Criteria for GVHD

there are 3 criteria that should be met :

1.  the transplant needs to be immunocompetent, containing viable and functional immune cells

2.  donor and recipient need to be histo-incompatible.

3.  the recipient needs to be immuno-compromised; the recipients cannot destroy or inactivate the transplant

there are two forms of graft-versus-host disease •  Acute GVHD

–  occurs generally within 100 days after transplantation –  involved tissues: skin, gut, liver

•  Chronic GVHD –  occurs generally at day 100 after transplantation, or later –  involved tissues: skin, gut, liver, and additionally

connective tissue and glands

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a clinical picture of acute GVHD

H&

E

CD

8

a histological picture of acute GVHD

% of d

eaths

100

0

20

40

60

80

causes of death after autotransplants

Primary Disease

GVHD IPn Infec:on Organ Failure

Other

correlation between GvHD and GvL effect after HLA identical SCT

0 10 20 30

Horowitz et al. 1990 Blood

no GvHD

acute GvHD

chronic GvHD

acute and chronic GvHD

relapse percentage

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hematopoietic stem cell transplantation

GraI versus Tumor

pa5ent donor

hematopoie5c stem cells hematological malignancy, aplas5c anemia, SCID

GraI versus Host Disease

GraI Rejec5on


can we predict GVHD?

•  not every HLA-mismatched transplant leads to GVHD •  can we predict GVHD based upon the HLA?

•  can we dissect GVHD from the GVL effect?

HLA mismatches in stem-cell transplantation •  mismatched transplantations are a risk factor for

GVHD •  for 25-40% no HLA-matched donor available

•  which mismatch is preferred?

direct recognition of mismatched HLA

•  T cells are primary reactive against self HLA with a viral peptide

•  the secondary response is directed towards non-self HLA presenting a non-polymorphic peptide

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HLA mismatches in stem-cell transplantation

Kawase et al. Blood 2007

direct recognition of mismatched HLA

•  recognition of self-HLA with a polymorphic peptide (e.g. derived from non-self HLA)

•  case reports in organ transplantation

•  predictable !!!

Antigen processing and presentation Antigen processing and presentation

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Antigen processing and presentation presentation by HLA class II

NetMHCIIpan

presentation by HLA class I

NetChop

NetMHCPan

PIRCHE [`percə] (peertje) noun, acronym Predicted Indirectly Recognizable HLA Epitope

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sources for hematopoietic stem cells

•  bone marrow •  peripheral blood stem cells •  umbilical cord blood (single or double)

Materials & Methods

•  n = 92 •  source is PBSC •  high-resolution HLA-typing 5 loci (A, B, C, DRB1, DQB1) •  retrospective HLA-DP typing •  8 (9%) HLA-DP matched (excluded)

•  determined HLA-derived peptide presentation via –  class I: NetChop, NetMHCpan –  class II: NetMHC-IIpan

•  extract self from non-self peptides

indirect recognition and the effect on acute GVHD

*

* = p-value < 0,05 for 1-4 and > 4 vs 0 PIRCHE-I, > 5 vs ≤1 PIRCHE-II

Factor OR 95%CI p-‐value

PIRCHE-‐I 1-‐4 vs 0 3,600 1,114-‐11,637 0,032

PIRCHE-‐I > 4 vs 0 4,800 1,305-‐17,658 0,018

PIRCHE-‐II 2-‐5 vs <= 1 2,105 0,657-‐7,302 NS

PIRCHE-‐II > 5 vs <= 1 4,000 1,139-‐13,461 0,025

PIRCHE-I > 4, N = 10/20

PIRCHE-I 1-4, N = 15/35

PIRCHE-I = 0, N = 5/29

PIRCHE-II > 5, N = 15/30

PIRCHE-II 2-5, N = 10/29

PIRCHE-II ≤ 1, N = 5/25

*

indirect recognition and the effect on relapse-related mortality

* = p-value < 0,05 for > 4 vs 1-4 PIRCHE-I p-value = 0.05 for >4 vs 0, p-value < 0,05 > 5 vs lower PIRCHE-II

Odds ratios could not be determined due to zero events in the high PIRCHE groups.

PIRCHE-I > 4, N = 0/20

PIRCHE-I 1-4, N = 7/35

PIRCHE-I = 0, N = 5/29

PIRCHE-II > 5, N = 0/29

PIRCHE-II 2-5, N = 5/26

PIRCHE-II ≤ 1, N = 6/25

* *

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conclusion

•  indirectly recognizable HLA-DP mismatches lead to: –  an increased risk of acute GVHD –  a decreased risk of relapse related mortality –  no effect on TRM (data not shown)

HLA mismatches and umbilical cord blood transplantation

•  HLA mismatches are a risk factor for graft-versus-host disease (GVHD)

•  For up to 40% of patients a completely HLA-matched donor is not available

•  Some mismatches are better permissible than others1 •  Indirect recognition of HLA mismatches predicts2

alloreactivity after single mismatched MUD SCT •  Transplantion with cord blood (CB) leads to a reduced

GVHD risk •  Patients are often transplanted with HLA-mismatched CB

donors •  Can we predict permissible mismatches after CB

transplantation? 1.  Kawase et al. Blood 2007 2.  Thus et al. Submitted to BBMT

HLA mismatches and umbilical cord blood transplantation

•  HLA mismatches are a risk factor for graft-versus-host disease (GVHD)

•  For up to 40% of patients a completely HLA-matched donor is not available

•  Some mismatches are better permissible than others1 •  Indirect recognition of HLA mismatches predicts2

alloreactivity after single mismatched MUD SCT •  Transplantion with cord blood (CB) leads to a reduced

GVHD risk •  Patients are often transplanted with HLA-mismatched CB

donors •  Can we predict permissible mismatches after CB

transplantation? 1.  Kawase et al. Blood 2007 2.  Thus et al. Submitted to BBMT

PIRCHES do not predict acute GVHD

PIRCHE-I PIRCHE-II

PIRCHE-I ≤ 3, N = 6/24

PIRCHE-I > 3, N = 7/31

PIRCHE-II ≤ 11, N = 7/25

PIRCHE-II > 11, N = 6/30

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PIRCHE-II predict chronic GVHD

PIRCHE-I PIRCHE-II

PIRCHE-I ≤ 3, N = 3/20

PIRCHE-I > 3, N = 4/24

PIRCHE-II ≤ 11, N = 1/20

PIRCHE-II > 11, N = 6/24

PIRCHE-I predict relapse-related mortality

PIRCHE-I PIRCHE-II

PIRCHE-I ≤ 3, N = 4/7

PIRCHE-I > 3, N = 2/14

PIRCHE-II ≤ 11, N = 3/12

PIRCHE-II > 11, N = 3/9

conclusions

•  a high number of PIRCHE-II correlates to chronic GVHD in the CB setting

•  a higher number of PIRCHE-I correlates to reduced risk of relapse

•  we are able to select donors that lead to an increased anti-tumor effect without the detrimental increased GVHD risk

•  a feasibility study showed that we could find such a donor for 73%

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synopsis

•  antibodies play a crucial role in organ transplantation •  all patients are therefore continuously screened for HLA

antibodies •  new techniques as Luminex and HLAMatchmaker

facilitate histocompatibility testing

•  T-cell responses play a key role in immune responses after HSCT

•  these responses can be detrimental or beneficial •  both direct and indirect recognition of mismatched HLA

are involved •  in CB, chronic GVHD and the GVL effect can be

dissected based upon the presenting HLA molecule

Major H istocompability’ Complex

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