1. HLA-TYPING Dr Opoola A.O. Registrar,LUTH

2. Outline  Definition of terms  Classification of HLA types  functions  Characteristics  Clinical significance  References

3. Definition  The HLA system is a group of tissue antigens termed Human Leukocyte Antigens governed by a chromosomal region bearing a number of genetic Loci, each with multiple alleles that have relevance to transplantation rejection reactions and that mark the prevalence of several diseases.  This group of genes resides on the short arm of Chromosome 6 and encodes Cell- Surface antigen-presenting proteins and many other genes.

5. BINDING OF ANTIGEN BY MHC AND T CELL RECEPTOR

6. Definition of terms  CHROMOSOME : One of the threadlike structures in a cell nucleus that carry the genetic information in the form of gene.  ALLELES: Variants of a single genetic locus.  MAJOR HISTOCOMPATIBILITY COMPLEX : A cluster of genes located in close proximity e.g. on human chromosome 6, that encode the histocompatibility antigens (MHC Molecules ).

7. Definition of terms  HISTOCOMPATIBLE : Sharing similar antigens i.e in transplantation  ANTIBODY : A protein produced as a result of interaction with an antigen. The protein has the ability to combine with the antigen that stimulated its production.  ANTIGEN : A substance that can react with antibody. Not all antigens can induce antibody production; those that can are also called immunogens.

8. Definition of terms  B LYMPHOCYTES : Strictly, a bursa-derived cell in avian species and by analogy, a cell derived from the equivalent of the bursa in non-avian species. B Cells are the precursors of plasma cells that produce antibody.  T LYMPHOCYTE : A thymus – derived cell that participates in a variety of cell- mediated immune reactions.  ANTIGEN PRESENTING CELLS : Chiefly Macrophages or B Cells that process and present antigens to T Cells.

9. Definition of terms  MACROPHAGES: A phagocytic mononuclear cells derived from bone monocytes and found in tissues and at the site of inflammation.  Macrophages serve accessory roles in immunity, particularly as APCs  COMPLEMENT : A set of plasma proteins that is the primary mediator of antigen – antibody reactions.

10. Functions  The HLA System (MHC MOLECULES) bind peptide antigens and present them to T-Cells.  Thus these transplantation antigens are responsible for antigen recognition by the T- Cell receptor.  While antibody molecules interact with antigens directly, the T-Cell receptor only recognizes antigen presented by MHC molecules on an antigen presenting cells.

11. Function  The T Cell receptor is also specific for the MHC molecule.  If the antigen is presented by another allelic form of the MHC molecule there is no recognition by the T Cell receptor.  This phenomenon is known as MHC RESTRICTION

12. Classification The HLA system is classified into 3 based on their location on the chromosome and cell distribution. These are:- 1. Class 1: 3 major and 3minor 2. Class 2: 3major and 2 minor 3. Class 3: Complement proteins and several cytokines

14. Class 1MHC Proteins  These major class I genes are encoded by the HLA-A, - B, - C genes.  These proteins are made up of two chains:  (i) A transmembrane glycoprotein of MW 45,000, non-covalently associated with a non- MHC encoded polypeptide of MW 12,000 that is known as B2 – macroglobulin.  Class 1 molecules are to be found on virtually all nucleated cells in the body.  Minor genes are HLA – E, HLA – F and HLA-G.

15. HLA Class 1 molecule

16. Class 2 MHC proteins  Major are HLA-DP,DQ and DR and minor are HLA-DM and DO.  The genes of the Class II combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface of antigen presenting cells.

17. Class 2 MHC proteins 1. HLA-DP α- chain encoded by HLA-DPA1 locus β-chain encoded by HLA-DPB1 locus 2. HLA-DQ α-chain encoded by HLA-DQA1 locus β-chain encoded by HLA-DQB1 locus 3. HLA-DR α-chain encoded by HLA-DRA locus 4 β-chains (only 3 possible per person) encoded by HLA-DRB1, DRB3, DRB4, DRB5 loci

18. MHC Class 2 Molecule

19. MHC Class 2 molecule

20. Class 2 MHC proteins  DM and DO are used in the internal processing of antigens, loading the antigenic peptides generated from pathogens onto the HLA molecules of antigen-presenting cell.  The HLA – D Locus encoded proteins are made up of non-covalently associated transmembrane glycoprotein of about MW 33,000 and MW 29,000

21. Class 2 MHC proteins  Unlike Class I proteins, they have a restricted tissue distribution and are chiefly found on macrophages, B-Cells, and other APCs.  Their expression on the other Cells – e.g., endothelial cells can be induced by interferon-gamma.

22. CLASS 1CLASS 11 GENETIC LOCI (PARTIAL LIST) HLA – A, - B, - CHLA-DP, - DQ, - DR POLYPEPTIDE COMPOSITION MW 45,000 + B 2 M (MW 12,000) α CHAIN (MW 33,000) β CHAIN (MW 29,000) CELL DISTRIBUTIONALL NUCLEATED CELLS (SOMATIC) APCs (Macrophages, B- Cells etc) Activated human T-Cells PRESENT ANTIGENS TOCD 8 T CELLSCD 4 T CELLS SIZE OF PEPTIDE BOUND 8 – 11 RESIDUES10 – 30 OR MORE RESIDUES TABLE SHOWING FEATURES OF CLASS I AND CLASS II PROTEINS

23. CHARACTERISTICS OF HLA  THEY ARE POLYGENIC : There are several genes for each class of molecule.  THEY ARE POLYMORPHIC : They have a large number of alleles in the population for each of the genes.  THEY ARE HAPLOTYPIC : Genes tend to be inherited as a block as there are relatively in-frequent cross-over events at the Locus.

24. CHARACTERISTICS OF HLA  LINKAGE DISEQUILLIBRIUM : This is defined as a deviation from Hardy – Weinberg equilibrium for alleles at linked loci. One consequence of this has been the resulting deficiency in assigning HLA- disease associations to a single allele at a single Locus.

25. Characteristics  The proteins encoded by the HLAs are the proteins on the outerpart of body cells that are unique to that person.  The immune system uses the HLAs to differentiate self cells and non-self cells.  Any cell displaying that persons HLA type belongs to that person

26. Clinical Significance  1. In DISEASE DEFENSE  2. AGENTS OF HUMAN DISEASE IN AUTOIMMUNITY: Known to mediate many autoimmune diseases  3. AS ANTIGENS: Responsible for organ transplant rejection  4. IN REPRODUCTION – may be involved in male selection  5. IN CANCER – may be protective or fail to protect.

27. Clinical Significance IN INFECTIOUS DISEASE :  When a foreign pathogen enters the body, specific cells called antigen-presenting cells (APCs) engulf the pathogen through a process called phagocytosis.  Proteins from the pathogen are digested into small peptides and loaded onto HLA antigens (Specifically MHC Class II).  They are then displayed by the APCs for the T Cells to produce a variety of effects to eliminate the pathogen.

28. Clinical Significance  Through a similar process, proteins (both native and foreign, such as the protein of viruses) produced inside most cells are displayed on HLA antigens (specifically MHC Class I) on the cell surface.  Infected cells can be recognised and destroyed by components of the immune system (Specifically CD8+ T Cells).

29. DR PROTEIN WITH BOUND STAPHYLOCOCAL ENTEROTOXIN LIGAND

30. Clinical Significance IN AUTOIMMUNITY  HLA types are inherited, and some of them are connected with autoimmune disorders and other diseases.  People with certain HLA antigens are more likely to develop certain autoimmune diseases such as Type 1 Diabetes, Ankylosing Spondylitis, celiac disease, SLE, myasthenia Gravis, inclusion body myositis and Sjogren’s syndrome.

31. Clinical significance  HLA typing in autoimmunity is being increasingly used as a tool in diagnosis.  In Gluten Sensitive Enteropathy (GSE), it is the only effective means of discriminating between first degree relatives who are at risk from those who are not at risk, prior to the appearance of sometimes irreversible symptoms such as allergies and secondary autoimmune disease.

32. DiseasesMarkerGene Spondyloarthropathies Ankylosing spondylitis,Reiter’s syndrome, Acute anterior uveitis, Reactive arthritis(Yersinia, Salmonella, Shigella, Chlamydia), Psoriatic Spondylitis B27B*2702, –04, –05 Collagen-Vascular Diseases Juvenile arthritis, pauciarticularDR8,DR5 Rheumatoid arthritisDR4DRB1*0401, –04, –05 Sjögren's syndromeDR3 Systemic lupus erythematosus WhiteDR3 JapaneseDR2

33. DiseaseMarkerGene Autoimmune Gut and Skin Gluten-sensitive enteropathy (celiac disease) DQ2DQA1*0501 DQB1*0201 Chronic active hepatitisDR3 Dermatitis herpetiformisDR3 Psoriasis vulgarisCw6 Pemphigus vulgarisDR4DRB1*0402 DQ1DQB1*0503 Bullous pemphigoid variantDQ7DQB1*0301

34. DiseaseMarkerGene Autoimmune Endocrine Type 1 diabetes mellitusDQ8DQB1*0302 DR4DRB1*0401, –04 DR3 DR2DQB1 *0602 Hyperthyroidism (Graves')B8, DR3 Hyperthyroidism (Japanese)B35 Adrenal insufficiencyDR3 Autoimmune Neurologic Myasthenia gravisB8, DR3 Multiple sclerosisDR2DRB1*1501 DRB5*0101

35. Miscellaneous DiseasesMarkersGene Behçet's disease B51 Congenital adrenal hyperplasiaB4721·OH (Cyp21B) NarcolepsyDR2DQB1*0602 Goodpasture's syndrome (anti- GBM) DR2 Abacavir hypersensitivityB57B*5701

36. HLA and autoimmunity diseases diseases HLA alleleDiseases with increased risk HLA-B27 Ankylosing spondylitis, post gonococcal arthritis, anterior uveitis HLA-B4721-hydroxylase deficiency HLA-DR2Systemic Lupus Erythematosus HLA-DR3 Autoimmune Hepatitis, Primary Sjogren syndrome, Diabetes mellitus type 1, SLE HLA-DR4 Rheumatoid Arthritis, Diabetes mellitus type 1

37. Clinical Significance IN GRAFT REJECTION  Any cell displaying some other HLA type is “non-self” and is an invader resulting in the rejection of the tissue bearing those cells.  Because of the importance of HLA in transplantation, the HLA Loci are among the most frequent typed by serology or PCR relative to any other autosomal alleles.  HLA-A,B &DR-Important in Kidney transplant

38. HLA/TISSUE TYPING  1.HLA- A1*,A2,B8*,B44,DR4*,DR15  2.HLA-A1*,A3,B7,B8*,DR4*,DR12  1A, 1B, 1DR mismatch 1.HLA- A2 - ; B27, B13, DR17, DR4  2.HLA- A2 A3, B8, B14, DR17 -  Once there is double DR mismatch-No transplant*

39. Clinical Significance IN CANCER:  Some HLA mediated diseases are directly involved in the promotion of cancer.  Gluten sensitive enteropathy is associated with increased prevalence of enteritis associated T- Cell Lymphoma and DR3 – DQ2 homozygotes are within the highest risk group with close to 80% of gluten sensitive EATL cases.

40. Clinical Significance  More often; however, HLA molecules play a protective role, recognizing the increase in antigens that were not tolerated.  Abnormal cells may be targeted for apoptosis mediating many cancers before clinical diagnosis.  Prevention of cancer may be a portion of heterozygous selection acting on HLA

41. References  The HLA sysyem and its value in clinical medicine lecture by Dr Mankwe  The HLA system and its products. Harrison Principles of Internal Medicine 18 th edition pages kasper et al pages 2691-2694