Basel - 6 eptember 2012 J.-M. Tiercy National Reference Laboratory for Histocompatibility (LNRH) University Hospital Geneva
Outline the HLA system is (a) complex anti-hla immunisation and alloreactivity detection of anti-hla antibodies HLA in transfusion HLA in organ and HC transplantation
The major histocompatibility complex 6 MHC (>200 genes) HLA class II HLA class I DP DQ DR B C A B1 A1 B1 A1 B1 B3 B4 B5 A1 2 Jean Dausset 1 (1916-2009) 1 1 2 1 1 3 2m 2 2 3 2m 2 membrane
HLA-A2 Biological role: defense against pathogens, presentation of peptides from foreign organisms to T lymphocytes Björkman et al. Nature 1987; 329:506
C serotypes ( 100) HLA class II HLA class I DP DQ DR B C A B1 A1 B1 A1 B1 B3 B4 B5 6 9 A1 19 46 10 21
C serotypes ( 100) alleles (>7800) HLA class II HLA class I DP DQ DR B C A B1 A1 B1 A1 B1 B3 B4 B5 A1 6 9 19 46 10 21 189 223 1262 2605 1551 2013
serotypes A1 A2 alleles defined by DNA sequencing A*02:01 A*02:02 A*02:03 etc A*01:01 A*01:02 A*01:03 etc
complexity of HLA haplotypes allele 1 allele 2 allele 3 HLA-A HLA-B HLA-DRB1 1 2 3 1 2 3 27 haplotypes
Nomenclature EROLOGY A2 DNA A*02 low resolution organ transplantation platelet transfusion DNA A*02:01 A*02:02 A*02:03 A*02:374 high resolution stem cell transplantation
HLA is (a) complex 7843 alleles 5764 proteins 252 null alleles
1/10 5 naive T-cells specific for 1 Ag Alloreactivity 1-10 CTLs/10 2 infected cell self HLA TCR non-self HLA TCR cytotoxic T-lymphocyte
Humoral response : anti-hla antibodies incompatible donor HLA molecules self HLA dendritic cell anti-hla Ab TCR CD4+ Thlymphocyte B lymphocyte
How to detect anti-hla antibodies? Complement-dependant cytotoxicity (CDC assay) lymphocytes + patient s serum + complement panel of HLA-typed lymphocytes + complement no lysis Ab binding => complementmediated cytoxicity lysis
at the cutting edge of technology Luminex technique: purified HLA molecules on microbeads AB (single antigen beads assay) pos Ag: A31,A33,A66,A74, A26,A43,A30,A33
Luminex technology HLA molecules on microbeads 1 2 HLA-A1 HLA-A2 incubation with patient serum 3 HLA-A3 100 - fixation of HLA-specific Ab on microbeads - detection by R-phycoerythrin-conjugated anti-human IgG
antigens Why do we often observe a broad anti-hla immunisation? - HLA antigens share allodeterminants - 70-90% HLA Ab against public epitopes epitopes
matched platelet support Immune factors for platelet refractoriness HPA (10-20%) HLA class I (80-90%) HPA+HLA (5%) autoimmune (unknown) 3 strategies: CREG matching, but does not prevent immunisation against private epitopes Permitted antigens, requires precise Ab identification (Luminex!) = Ab-specificity prediction HLAMatchmaker
Cross-reacting group (CREG) A2 (A2, A68, A69) NTRN63-66 pecific aa residues: A2: H74 A2+B57: A2+A69: A2+A68+A69: A68+A69: - A68+A69+A26: G62 W107 T142-H145 NTRN63-66 G62 H74 T142-H145 W107
The HLA Matchmaker concept René Duquesnoy (Pittsburgh) Donor HLA mismatches are defined by linear sequences of amino acid residues (triplets) on alloantibody accessible sites of the HLA molecules the HLA type of the recipient will determine what structural components of an immunizing HLA antigen can be seen as non-self
Polymorphic residues on HLA-B51 This polymorphism should be considered in the context of self HLA epitopes of the antibody producer
Polymorphic residues on HLA-B51 seen by A2, A68 B27, B44 seen by A2, A68 B35, B44 seen by A2, A24 B7, B8 www.ncbi.nlm.nih.gov R. Duquesnoy
trong correlation between the number of triplet mismatches and antibody production in 144 patients who rejected their kidney transplant 94% patients produced donor-specific Ab in 11-12 triplet mismatched transplants Dankers et al. Transplantation 2004; 77: 1236
Different matching requirements in organ vs. hematopoietic stem cell transplantation organs platelets rejection HLA-A,B,C/DR,DQ serotypes epitopes recognized by Ab stem cells GVHD HLA-A,B,C,DRB1,DQB1,(DPB1) allele level matching
Histocompatibility testing in organ transplantation 1. Detection of pre-formed anti-hla antibodies in patients on the waiting list (immunological priority) 2. ABO compatibility, HLA-A,B,DR matching 3. Lymphocytotoxic crossmatch: recipient serum tested against donor T/B lymphocytes
The CDC crossmatch C d d C d d donor T/B lymphocytes -lymph nodes -spleen -peripheral blood patient s serum complement C CDC = complement-dependant cytotoxicity
Role of HLA compatibility >15% HLA-A,B,DR
HLA compatibility in stem cell transplantation Compatibility is defined by: - the level of resolution of HLA typing allele mismatches are efficiently recognized by T-cells! - the HLA loci analysed ( C,DQB1,DPB1)
Compatibility criteria related donor DP DQ DR B Cw A HLA genotypically identical sibling unrelated donor DP ( ) DQ DR B C A A/B/C/DR/DQ (>7800 alleles) 10/10
www.bmdw.org 19 700 000 volunteer donors 540 000 cord blood units 66 donor registries (48 countries) 46 cord blood banks (30 countries) HLA typed at different levels of resolution A2 A*02:01 A*02:XX A*02:01/02/04/12/14/ 15/18/19/24/27/28
Impact of HLA incompatibilities 2399 patients, 13th IHW A,B,C,DRB1,DQB1 0 mismatch 1-2 mismatches 3 mismatches Petersdorf et al. (2006)
wiss study 1990-2006 matched (10/10) vs. mismatched unrelated HCT 62 ± 10% vs. 46 ± 14% (p=0.022) 10/10 match 7-9/10 match days Chalandon et al. BMT (2006) Tiercy et al. BMT (2008)
HLA-DPB1 is a clinically relevant alloantigen DP DQ DR B C A 10/10 or 12/12 match? 1.0 0.8 DP compatible 246 patients O 0.6 0.4 DP incompatible 0.2 0.0 P=0.036 0 1 2 Years 12/12 or 1 MM rejection: 71 ± 12% 1-2 MM or 1 MM GvH: 48 ± 10% 3 4 5 Bettens, Passweg, chanz et al. BBMT 2012
Concluding remarks Organ transplantation and platelet transfusion: - sensitive assays for anti-hla Ab (e.g. luminex) - immunization against one Ag is rare (public epitopes) - platelet support: HLAMatchmaker if no Ab defined - threshold for Ab detection? C1q-fixing Ab? Hematopoietic stem cell transplantation: - single HLA mismatches are well recognized by T cells - high diversity of HLA impact on matching rate - 70% patients: 9/10 or 10/10 matched donor (BMDW) - permissive MM?
Thank you Alpine world polymorphism