Chapter 15

1. Chapter 15 DNA-Based Tissue Typing

2. Objectives • Describe the structure of the MHC locus that encodes the HLA antigens. • Explain the role of these antigens in tissue engraftment and rejection. • Describe the laboratory methods used to identify HLA antigens by serology testing. • Describe the DNA-based testing methods that are used for the identification of HLA antigens.

3. The Major Histocompatability Complex (MHC) • The MHC is located on chromosome 6. • The MHC contains the human leukocyte antigen (HLA) and other genes. 1 Mb 2 Mb 3 Mb 4 Mb TNF b a b a b a b b b b a a b HLA- DP DQ DR B C A Class II Class III Class I

4. Genes of the Major Histocompatibility Locus

5. a b a a 1 1 2 1 HLA-D a b 2 2 a 3 b 2 microglobulin Cell membrane a b a chain chain chain The Human Leukocyte Antigens (HLA) Human leukocyte antigens, the MHC gene products, are membrane proteins that are responsible for rejection of transplanted organs and tissues.

6. The Human Leukocyte Antigens (HLA) • HLA-gene sequences differ from one individual to another. • Also written as: • Each sequence is a different allele.

7. Subregion Gene region HLA-DRB1 a-or b-chain polypeptide Gene locus HLA Allele Nomenclature • A standard nomenclature has been established by the World Health Organization (WHO) Nomenclature Committee. • A small “w” is included in HLA-C, HLAB-4, and HLAB-6 allele nomenclature: HLA-Cw, HLABw-4, HLABw-6.

8. Subregion Allele family 25 Generegion HLA-DRB1*2503 Third allele a-or b-chain polypeptide Gene locus HLA Allele Nomenclature • HLA-typing at the DNA level requires nomenclature for specific DNA sequences. • There are over 900 HLA alleles identified so far in all loci.

9. HLA Alleles are Inherited in Blocks as Haplotypes

10. HLA-Typing • Every person (except identical twins) has different sets of HLA alleles. • Transplanted organs are allografts, in which the donor organ and the recipient are genetically different. • Compatibility (matching) of the HLA of the donor and the recipient increases the chance for a successful engraftment. • Matching is determined by comparing alleles. • Resolution is the level of detail with which an allele is determined.

11. Complement antibody Positive reaction to antibody kills cells. Dead cells pick up dye. Buffy coat from patient Negative reaction to antibody: cells survive and exclude dye. Serological Typing Lymphocytes are HLA-typed by crossmatching to panel reactive antibodies (PRA) using the complement-dependent cytotoxicity (CDC) test.

12. Lymphocytes from organ donor or lymphocytes of known HLA types Positive reaction to antibody kills cells. Dead cells pick up dye. Recipient serum Negative reaction to antibody: cells survive and exclude dye. Serological Typing Recipient antihuman antibodies are assessed by crossmatching to donor lymphocytes.

13. Serological Typing Using Bead Arrays Recipient antihuman antibodies are assessed by crossmatching to known lymphocyte antigens conjugated to microparticles. Results are assessed by flow cytometry. Serum antibodies Beads conjugated to different lymphocyte antigens Positive for antibody (Wash) Fluorescent reporter antibodies Negative for antibody

14. Other Serological Typing Methods • Cytotoxic and noncytotoxic methods with flow cytometry detection. • Enzyme-linked immunosorbent assay (ELISA) with solubilized HLA antigens. • Mixed lymphocyte culture measuring growth of lymphocytes activated by cross-reactivity. • Measure of HLA-protein mobility differences in one-dimensional gel isoelectric focusing or two-dimensional gel electrophoresis.

15. DNA-Based Typing Methods • DNA typing focuses on the most polymorphic loci in the MHC, HLA-B, and HLA-DRB. • Whole-blood patient specimens collected in anticoagulant are used for DNA typing. • Cell lines of known HLA type are used for reference samples.

16. Specimen 1 (Type A*0203) Specimen 2 Type A*0501 TAG C GAT TAG A GAT ATC G CTA ATC T CTA Amplify, denature, and spot onto membranes Specimen 1 Specimen 2 Probe with allele-specific probes ...TAGCGAT..(A*02) ...TAGAGAT…(A*05) Specimen 1 Specimen 2 Specimen 1 Specimen 2 DNA-Based Typing Methods: SSOP Sequence-specific oligonucleotide probe hybridization (SSOP, SSOPH)

17. DNA-Based Typing Methods: SSP-PCR Sequence-specific PCR is performed with allele-specific primers. Amplification controls SSP= Sequence-specific primer Allele-specific product SSP matches allele Amplification SSP No amplification SSP SSP does not match allele

18. DNA-Based Typing Methods: SSP-PCR Primers recognizing different alleles are supplied in a 96-well plate format. Reagent blank Amplification control Allele-specific product Agarose gel

19. Reverse PCR primer Forward PCR primer Sequencing primers HLA-B DNA-Based Typing Methods: Sequence-based Typing • Sequence-based typing (SBT) is high resolution. • Polymorphic regions are amplified by PCR and then sequenced.

20. Sequence-based Typing Sequences are compared to reference sequences for previously assigned alleles.

21. Typing Discrepancies • DNA sequence changes do not always affect epitopes. • Serology does not recognize every allele detectable by DNA. • New antigens recognized by serology may be assigned to a previously identified parent allele by SBT. • Serology antibodies may be cross-reactive for multiple alleles. • Due to new allele discovery, retyping results may differ from typing performed before the new allele was known.

22. Resolution Levels of HLA Typing Methods

23. Combining Typing Results • SSP-PCR followed by PCR RFLP. • SSOP followed by SSP-PCR. • SBT results clarified by serology.

24. Summary • The MHC is a polymorphic locus encoding the HLA genes. • Antigens encoded by the HLA genes are responsible for allograft tissue and organ rejection. Identifying and matching alleles increases the chance of successful organ and tissue transplant. • HLA antigens and their corresponding sequence alleles are determined by serological- and DNA- based methods. • Serology identifies functional antigen recognition, while sequence analysis identifies genetic alleles with high resolution.