major h istocompability’ complex
TRANSCRIPT
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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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the major antigens in transplant rejection
M H C
Major
Histocompa5bility
Complex
the major antigens in transplant rejection
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
immunological outcome
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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
immunological outcome
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