HLA – Basics (Or How to Recognize “Self”)
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HLA – Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some Definitions – MHC and HLA – Class I and Class II – Nomenclature • Class II extended haplotypes • HLA Typing Technology • HLA Immunogenicity • Solid Phase Antibody detection methods • HLA and Disease Associations • HLA and Pharmacogenetics • Review 2 Human Leukocyte Antigens Polymorphism slide-new alleles each year 3 Major Histocompatibility Complex (MHC) • MHC in humans is named “Human Leukocyte Antigens” (HLA) as they were first defined on the surface of peripheral blood leukocytes • HLA Class I in humans is found on virtually all nucleated cells and platelets • HLA Class II (constitutive expression) is restricted to specialized cells of the immune system (macrophages, B cells, etc.) • HLA genes are highly polymorphic 4 HLA Protein Structure CD8 Tcell cytosol • Class I is heterodimeric with a polymorphic alpha chain and a plasma membrane common beta-2 microglobulin – Alpha chain is composed of 3 TCR extra-cellular domains ( α1, α2, and α3) – α1 and α2 form a groove like structure with a floor of β α2 α1 pleated sheets and ridges of α helices CD8 – presents peptides derived from α3 β2 m internal cellular proteins to the T cell receptor of CD8 T cells. plasma membrane – involved in the immune response cytosol against intra-cellular parasites, Antigen Presenting Cell viruses and cancer 5 HLA Class I Molecule Influenza M1 A view down the peptide groove of an HLA-A2 ααα1 domain molecule ααα2 domain βββ2 microglobulin ααα3 domain 6 HLA Protein Structure (con’t) CD4 Tcell • Class II is also heterodimeric with cytosol a polymorphic beta chain and a plasma membrane much less polymorphic alpha chain TCR – both chains are composed of 2 extra-cellular domains ( α1, α2, and β1, β2) β1 α1 – Together the two first domains create a peptide binding groove which presents processed peptides, from extra cellular CD4 β2 α2 proteins, to CD4+ T cells plasma membrane – involved in the immune response cytosol against extra cellular infectious agents and non-self HLA Antigen Presenting Cell molecules 7 HLA Class II Molecule HLA-A2 peptide A view down the ααα chain groove of an HLA- DR1 molecule βββ chain 8 HLA Polymorphism Serologic methods resolve only a small fraction of all known alleles (est. 3%) Molecular techniques have emerged as the method of choice for HLA typing, since strategies have been developed that resolve most/all known alleles and provide a tool for identification of new alleles. CLASS I CLASS II 3,107 A alleles 1,726 DRB1 alleles 3,887 B alleles 95 DRB3,B4,B5 alleles 2,623 C alleles 54 DQA1 alleles 780 DQB1 alleles 39 DPA1 alleles 520 DPB1 alleles (status April 2015) 9 HLA Polymorphism http://hla.alleles.org HLA Polymorphism HLA sequences and a current list of known HLA alleles are found at the IMGT/HLA database: http://www.ebi.ac.uk/ipd/imgt/hla/ The IMGT/HLA Database provides a specialist database for sequences of the human major histocompatibility complex (HLA) and includes the official sequences for the WHO Nomenclature Committee For Factors of the HLA System. The IMGT/HLA Database is part of the international ImMunoGeneTics project ( IMGT ). Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE The IPD and IMGT/HLA database: allele variant databases Nucleic Acids Research (2015) 43 :D423-431 11 HLA Nomenclature • Assignments are made under the auspices of World Health Organization (WHO) Nomenclature committee • Aim is to provide unique identification for: – Loci – Alleles – Haplotypes • Therefore the system must be very conservative but adaptable to change – Must not serve personal or national vanity – Nomenclature should optimize not maximize the amount of information in the code 12 HLA Nomenclature (con’t) • Haplotype – The combination of alleles from two or more loci located on the same chromosome – Does NOT imply any linkage disequilibrium • Genotype – The combination of two haplotypes; one from each parent, inherited by an individual • Linkage Disequilibrium – Presence of two alleles that are inherited together more frequently than would be expected based on the gene frequencies 13 HLA Nomenclature (con’t) • Each allele is initially identified by a letter(s) indicating “locus” A, B, C, DR, DQ, and DP • Then identified by individual specificity e.g. A1, B27, DR8, etc. (numbered in order of discovery) – Specificities were initially defined by using antisera (antibodies) • Historically these were sera obtained from multiparous women • Initially HLA Labs maintained large banks of specific sera – Patient cells were mixed with various sera, incubated and then Complement and a vital dye were added. • If a cell had the corresponding antigen, then the antibody would bind, complement fixed and cell death ensued. • Most sera were “cross reactive” such that a technologist had to interpret results, e.g.,: One well had antibody reactive with A23, A24 (Pos); but another well with anti-A2, A23 might be negative; thus suggesting a patient typing of A24 14 HLA Nomenclature (con’t) • HLA specificities can also be determined by genetic analysis by identifying the presence/absence of the gene encoding the HLA protein. • Techniques include direct DNA sequencing, PCR using Sequence Specific Primers (SSP) and PCR using sequence specific oligonucleotide probes (SSO) – Technique specifics will be discussed later • Again the initial identification is the locus letter(s): A, B, C, etc. – To differentiate from serological identification we use an asterisk (*) and a place marker, such as: A*01, B*27, etc. 15 HLA Nomenclature (con’t) • Class II molecular specificities are identified at the level of the gene encoding a particular chain, that is alpha or beta. But remember most of the polymorphism for Class II is in the beta chain thus common identification reflects that: DRB1*01, DQB1*02, etc. • The molecular two digit specificity is referred to as “Low Resolution” typing e.g., A*01, B*27, etc. • “High Resolution” typing are sub-specificities of the main group e.g., A*01:01, A*01:02, B*27:01, B*27:100 – Each representing a change in at least one AA 16 HLA Nomenclature (con’t) Expanded Allele Nomenclature: Molecular Colon Typing Delimiters LOCUS Allele Level Intron “Subtype” Substitutions Suffix to denote HLA-DRB4 *01 :03 :01 :02N changes in Expression eg N=Null Synonymous “Non- “MHC” Coding” Substitutions Antigen Level or Allele Family 17 http://hla.alleles.org/nomenclature/naming.html HLA Nomenclature (con’t) • Quiz Question - What does the “--” (Blank) in the following typing indicate? A*01, --; B*08, --; DRB1*03, DRB1*15 • Most molecular and certainly all serological typing can NOT distinguish between a single versus a double presence of any particular allele • Given that both A*01 and B*08 are common in the N. Amer. population it is highly likely that the “real” typing is as follows: A*01, A*01; B*08, B*08; DRB1*03, DRB1*15 • Another but much, much less likely possibility is that the individual is carrying an allele that can not be detected by the specific molecular technique employed 18 HLA Nomenclature (con’t) PROBLEM: • Many registry donors have been tested by serological methods – The vast majority of these do not have good documentation of which antigens were tested for and which were not • The majority of HPC transplant candidates have been tested by molecular (DNA-based) methodologies • The nomenclature of antigens (serology) and alleles (DNA) is in some cases NOT concordant 19 HLA Nomenclature (con’t) • Quiz Question – Is this Donor/Recipient pair a good match from an HLA perspective? Donor: A19, A10; B12, B62; DR5, DR2 Recipient: A*29, A*26; B*44, B*15; DRB1*11, DRB1*15 • Answer: Maybe but possibly yes – A19 (serological) was split: A*29, *30, *31, *32, *33, *74 – A10 (serological) was split: A*25, *26, *34, *66 – B12 (serological) was split: B*44, *45 – All the others are also splits although the molecular B*15 includes the very different serological specificities B62, 63, 70, 71, 72, 75, 76 and 77 20 DRB Haplotype Variations Expressed DPA1 DPB1 DQA1 DQB1 Genes DRA Pseudo- genes DR1, DR10 DRB1 DRB6 DRB9 DR15,16, 51 DRB1 DRB6 DRB5 DRB9 (51 ) DR11, 12, 13, 14, 3 (17,18), 52 DRB1 DRB6 DRB2 DRB3 DRB9 (52 ) DR4, 7*, 9, 53 *about 1/3 of DR7 DRB1 DRB6 DRB7 DRB8 DRB4 DRB9 (53 ) haplotypes lack an expressed DR8 DRB4 (DR53) gene DRB1 DRB9 Almost always inherited as a predictable haplotype 21 How do we do HLA typing? Historically, HLA typing has been done at the protein level, microlymphocytoxicity test being the standard method. HLA typing at the DNA level has become the method of choice for clinical laboratories. DNA methods are more robust and reproducible and provide more information. 22 Genomic DNA: HLA-A, B or C locus 5’UTR E1 E2 E3 E4 E5 E6 E7 E8 3’UTR PCR SSP SSO SBT Sequence Hybridization with set of Sequencing Specific oligonucleotide probes PCR amplification corresponding to the polymorphic sites 23 PCR Amplification The majority of molecular assays for HLA typing require the amplification of the target sequence of DNA via Polymerase Chain Reaction, or PCR. A sample of the patient’s DNA, called the template, is allowed to react with specific primers. The primers bind to the template DNA just upstream of the sequence of DNA that is intended for amplification. PCR Amplification Let’s say the DNA sequence below is located on the Major Histocompatibility Complex on the short arm of chromosome 6, at exon 2 of the –DRB1 gene. The region we want to amplify is the sequence ‘GTTTAACGGCAT’. The primer would bind immediately upstream of the target sequence. 3’ 5’ C A T G G C G T T T A A C G G C A T A C A G G G A C Target sequence Template DNA PCR Amplification A DNA polymerase enzyme (most frequently Taq), recognizes the primer bound to the template, and begins to add nucleotides to the 3’ end.