IMMUNONGENETICS IN TRANSFUSION MEDICINE

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IMMUNONGENETICS IN TRANSFUSION MEDICINE

• Wolfgang R. Mayr
• Division of Blood Group Serology
• Medical University of Vienna
• wolfgang.mayr_at_meduniwien.ac.at

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IMMUNOGENETICS

• Immunogenetics can be defined as studies of
  polymorphic genes and molecules involved in the
  immune response, including regulation and
  expression
• E. Thorsby, President of the EFI (European
  Foundation for Immunogenetics)

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HLA GENE COMPLEX

• gt 400 loci
• gt 300 expressed loci
• 30 HLA class I
• 27 HLA class II
• 43 HLA class III

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HLA-A, B, C, DR and DQ 2.796 alleles,
2.270 proteins
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TYPING AT THE NUCLEOTIDE LEVEL
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HLA Typing - Resolution

• Low resolution - 2 digits phenotyping,
  corresponds to serologically defined factors
• High resolution - 4 digits genotyping

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PEPTIDES BOUND TO MHC

• Class I nonamers, 1 or 2 anchor positions
• Class II longer, promiscuous

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PEPTIDE BINDING MOTIFS

• Amino-acid position 1 2 3 4 5
  6 7 8 9
• A0201 7 2 7 6 8 4 3
  3 2
• B2705 6 1 8 11 8 7 7
  11 6
• A0201 gt 330 000 peptides
• B2705 gt 13 600 000 peptides

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CELLULAR THERAPY

• Cellular therapies depend on the preparation and
  handling of cells in a GMP environment with an
  extensive QM-system.
• Such conditions are found in blood
  establishments.
• Further regulations concerning biovigilance,
  labelling, etc... also can be easily met by blood
  establishments.

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Cellular Therapy Main Directions

• Stem Cell Transplantation
• Dendritic Cells in vitro sensitization by tumour
  antigens and reinjection in order to stimulate T
  cell response
• NK Cells killing of tumour cells that do not
  express the own HLA class I gene products
• Cytotoxic T Lymphocytes stimulation by tumour
  antigens and reinjection into patient where they
  attack tumour cells
• Regenerative Medicine e.g. in vitro preparation
  of autologous skin grafts, infusion of stem cells
  in hearts damaged by infarctions, ...
• Gene Therapy introduction of gene in cells and
  reinfusion

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Cabrera T et al, Cancer Immunol Immunother 2007
56 709 - 171
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HLA Typing Demands for Peptide-Based Anti-Cancer
VaccineNagorsen D Thiel ECancer Immunol
Immunother, 2008, in press

• Taken together, proper tissue typing for cancer
  vaccine therapies represents the main pillar for
  successful immunotherapy. Clinical
  immunotherapists should be more aware of the
  importance of correct HLA determination to avoid
  vaccination of unsuitable patients.
• Correct HLA determination high resolution
  genotyping (at the 4-digit level), in order to
  determine the binding of tumour-derived peptides

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Flomenberg N et al, Blood 2004 104 1923-1930
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Flomenberg N et al, Blood 2004 104 1923-1930
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Effect of Single Mismatches on Mortatility(15th
IHIWS)

• DQB1 1.00
• DRB1 1.08
• C 1.18
• A 1.20
• B 1.29

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Shaw BE et al, Blood 2007 110 4560 - 4566
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Summary of HLA matching in allogeneic stem cell
transplantation

• High resolution HLA typing is standard
• HLA allele mismatching leads to graft rejection,
  GvHD and mortality
• Total number of mismatches is important risk
  factor for complications
• Mismatches for specificities (low resolution)
  confer different risks than mismatches within one
  single allele (high resolution)
• Non-genetic factors (nature and stage of disease,
  timing of transplantation, ) influence the
  tolerability of HLA mismatching

Petersdorf EW, Current Opinions Hemat 11,
386-391, 2004
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• Methods
• Outcomes of 503 children(lt16 years) with acute
  leukemia and transplanted with umbilical cord
  blood were compared with outcomes of 282
  bone-marrow recipients. All transplantation took
  place in the USA.
• Recipients of umbilical cord blood were
  transplanted with grafts that were HLA-matched
  (n35) or HLA-mismatched for one(n201) or two
  antigens (n267) (typing at antigen level for
  HLA-A and HLA-B, and allele level for HLA-DRB1).
  Bone-marrow recipients were transplanted with
  grafts that were matched at the allele level for
  HLA-A, HLA-B,HLA-C, and HLA-DRB1 (n116), or
  mismatched (n166).
• The primary endpoint was 5-year leukemia-free
  survival.

• Findings
• In comparison with allele-matched bone-marrow
  transplants, 5-year leukaemia-free survival was
  similar to that after transplants of umbilical
  cord blood mismatched for either one or two
  antigens and possibly higher after transplants of
  HLA-matched umbilical cord blood.
• Transplant-related mortality rates were higher
  after transplants of two-antigen HLA-mismatched
  umbilical cord blood (relative risk 231,
  p00003) and possibly after one-antigen
  HLA-mismatched low-cell-dose umbilical-cord-blood
  transplants (188, p00455).
• Relapse rates were lower after two-antigen
  HLA-mismatched umbilical-cord-blood transplants
  (054, p00045).

Eapen et al, Lancet 369, 1947, 2007
Division of Blood Group Serology
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Eapen et al, Lancet 369, 1947, 2007
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Allorecognition by T cells and NK cells
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KIR (Killer-cell Immunoglobulin-like Receptor)

• KIR3DL1-2, KIR3DS1, KIR2DL1-5, KIR2DS1-5
• 2D 2 Ig-like domains
• 3D 3 Ig-like domains
• L Long cytoplasmatic tail inhibitory
• S short cytoplasmatic tail - activating

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HLA-C DIMORPHISM

• C1 - HLA-C Ser77Asn80 Cw1, w3, w7, w8
• C2 - HLA-C Asn77Lys80 Cw2, w4, w5, w6

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KIR ligand incompatibility in GVH
direction(absence in recipients of donor class I
allele groups e.g. for HLA-C)
C1 - HLA-C Ser77Asn80 Cw1, w3, w7, w8 C2 -
HLA-C Asn77Lys80 Cw2, w4, w5, w6
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KIR haplotypes

• Group A absence of the genes KIR2DL5, KIR2DS1,
  KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1
• Group B presence of one or more of the genes
  KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and
  KIR3DS1
• B haplotypes more activating genes

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Ruggieri L et al Role of natural killer cell
alloreactivity in HLA-mismatched hematopoietic
stem cell transplantation. Blood 94, 333-9, 1999

• ... The alloreactive NK clones also killed the
  allogeneic leukemia. Transplants from these KIR
  epitope incompatible donors had higher
  engraftment rates. Therefore, a GVL effector and
  engraftment facilitating mechanism, which is
  independent of T-cell-mediated GVH reactions, may
  be operational in HLA mismatched hematopoetic
  cell transplants.

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Ruggieri L et al, Science 295, 2097-2100, 2002
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KIR - Differences in studies

• Disease type (AML?)
• Conditioning regimen
• Type of hematopoietic stem cell graft
• Study population
• Education of NK-cells
• Genetic differences other than HLA-C possibly
  influencing NK-cell alloreactivity
• Allele polymorphism might influence surface
  expression, ...
• Specificity for ligand

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KIR effects

• The effects of KIR on NK or T cell activity is
  probably a continuum ranging from strong
  inhibition to strong activation.
• This should be considered in studies of KIR
  effects on transplantation / disease outcome.

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HLA Class I vs Diseases
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HLA Class II vs Diseases
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HLA AND DISEASES

• 1. Restriction HLA class I
• HLA class II
• 2. Linkage disequilibrium with mutated genes

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PRESENTATION OF PEPTIDES

• DR3 Autoimmunity
• Birdshot-Retinopathy
• A29 binds peptide of a soluble retinal antigen
• A29-transgene mice similar symptoms
• Rheumatoid Arthritis
• DR4 binds peptides of collagen II
• DR4-transgene mice symptoms like RA

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HLA ASSOCIATED IMMUNE RESPONSE AGAINST PLATELET
SPECIFIC ANTIGENS

• HLA imm. pat. controls
• HPA-1a DR3 71-95 23
• DR52 100 68
• DRB30101 100 55
• HPA-5b DR6 63 29
• DR52 87 60

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SIDE-EFFECTS IN THERAPY