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  1. 1. Dr Nisha Singh Guide:Dr S .S RautHLA TYPING AND ITS IMPLICATIONS Dr. Nisha Singh Guide: Dr S S Raut
  2. 2.  History  MHC  Immunity and MHC  MHC & HLA  Structure & Functions  Nomenclature  Typing  Implications History MHC Immunity and MHC MHC & HLA Structure & Functions Nomenclature Typing Implications
  3. 3. History Early work of Gorer (1930) on antigens responsible for allograft rejection in mice led to the discovery of the ‘MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)’. Development of congenic & recombinant strains of mice by Snell enabled the detailed analysis of various loci of this complex.
  4. 4. Dausset pioneered studies on HUMAN LEUKOCYTE ANTIGENS MAJOR HISTOCOMPATIBILITY ANTIGENS in human being. Benacerraf & colleagues established genetic basis of immune response. For their work on MHC & genetic control of immune response, Snell, Dausset & Benacerraf were awarded Nobel Prize for medicine in 1980.
  5. 5. •Human leukocyte antigen (HLA) complex is the locus of genes that encode for proteins on the surface of cells responsible for regulation of the immune system in humans. •Located on short arm of chromosome 6 •Inherited as haplotypes (i.e. set of alleles) one from each parent •They are co-dominantly expressed
  6. 6. HLA proteins found in the membranes (outer coating) of nearly every cell in the body (all cells that have a nucleus). These antigens are in especially high concentration on the surface of white blood cells(leukocytes). The HLA loci are part of the genetic region known as the major histocompatibility complex
  7. 7.  The MHC system in humans was subsequently discovered in early 1950s.  The MHC has genes (including the HLA) that form part of the normal function of the immune system.  The MHC is an extreme gene-dense region of the genome, and it can be divided into three sub-regions; the class I, the class and the class III regions. All encoded by a gene complex located on the short arm of 6. The MHC system in humans was subsequently discovered in early 1950s. The MHC has genes (including the HLA) that form part of the normal function of the immune system. The MHC is an extreme gene-dense region of the genome, and it can be divided into three sub-regions; the class I, the class and the class III regions. All encoded by a gene complex located on the short arm of 6.
  8. 8.  The class I region of approximately 2000 kilobases include;the polymorphic HLA-A, B, C loci; non classical class HLAE,F, G,..  The class II region of approximately 1000 kilobases include;HLA-DR,DQ an DP loci, ……….and non classical class HLA class II HLA-DM, DO,.  The class III region of approximately 1000 kilobases encode genes with diverse functions and does not contain any HLA genes. Contain loci responsible for the complement,hormones,…
  9. 9. Class I Encoded by A,B,C etc loci Expressed on all nucleated cells and platlets  Present antigenic peptides from within the cells ( endogenous) to CD8 + T cell  MHC restriction of cell mediated cytotoxicity , the acceptance and rejection of grafts  These antigens also function as complement of harmone.
  10. 10. Class ІІ: - or D region –DR, DQ, & DP loci Protein that regulate the immune response These are found only on the cell of immune system such as macrophage, dendritic cell activated T cell and B cell. These are the heterodimer consists of an alpha and beta chains. Each chain has two domain – a proximal constant region and a distal variable region .The distal region constitute the antigen-binding site for reorganization by CD4 lymphocyte. The immune response genes, which control immunological response to specific antigen, are situated in this class 2 antigen. These are responsible for the graft versus host response and mixed leucocytes reaction (MLR).
  11. 11. Class ІІІ: - or complement region--- This group contains genes for C2 and C4 of classical pathway, properdin factor of alternative, heat shock protein, and tumor necrosis factor These are important component of complement system and are responsible for various cellular activities. HLA loci are multiallelic, that is the genes occupying the locus can be any one of the of several alternative forms (alleles). As each allele determines a distinct product (antigen), the HLA system is very pleomorphic .For examples, at least 24 distinct allele have been identified at HLA locus A and 50 at B.
  12. 12. Glycoproteins, heterodimers (two chains) Structure of HLA molecules of both classes enables antigen binding and contact with T cell receptors. Extracellulary located peptide binding cleft polymorphic (predominantly in the cleft). Nonpolymorphic part of the molecule contains binding sites for the T cell molecules CD4 and CD8
  13. 13. HLA class 1 and class 2 molecules
  14. 14. HLA is extremely polymorphic. It is one of the most polymorphic genetic system in man. Associated with each of the loci is a set of alleles, each of which give rise to the production of a unique antigenic specificity that is expressed on cell surface and that can be detected by specific antibodies or immunologically activated T cells.
  15. 15. Limited diversity in HLA gene polymorphism due to breeding bottle-neck in recent past leaves cheetahs exceptionally susceptible to viral infections. (6th Ed. P. 206)
  16. 16. Most individuals inherit a set of non recombined HLA alleles from each parent. These genes are codominantly expressed. Thus if the HLA types of family member are determined, segregation of HLA types within the family can be used to construct the HLA types from each chromosome The set of HLA alleles found on one chromosome is called a haplotype. Determination of haplotype is important for identification of HLA identical siblings because sharing of antigen from different haplotypes is common.
  17. 17.  The primary target of immune responses to allogenic transplants.  Critical for response to antigenic stimuli.  Implicated in genetic susceptibility to autoimmune disease.
  18. 18. A. Serological ( microcytotoxicity test) B. Mixed lymphocyte reaction C. Molecular methods
  19. 19. Potential donor’s and recepient’s WBC’s added to the different wells of microtitre plates Antibodies specific for HLA class I and II added After incubation complement is added Cytotoxicity is assessed by uptake or exclusion of dye.
  20. 20. HLA Typing is done serologically by MICROCYTOTOXICITY( microlymphocytotoxicity) which tests for complement mediated lysis of peripheral blood lymphocytes with a standard set of typing sera. Viable peripheral blood lymphocytes are obtained by discontinuous density gradient centrifugation using Ficoll / Tryosil or Ficoll / Sodium Metrizoate at a density of 1.077 at 19º - 22ºC.
  21. 21. Microlymphocytotoxic test: 3 stages 1.Viable lymphocytes are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound. 2.Rabbit serum as a source of complement is added, incubate. If antibody is bound to the HLA antigen on the cell surface it activates the complement which damages the cell membrane making it permeable to vital stains. 3.Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Eosin Y have been used in the past
  22. 22. If the reaction has taken place the EB enters the cell and binds to the DNA. For ease double staining is normally used. A cocktail of Ethidium Bromide and Acridine Orange, quenched using Bovine Haemoglobin to allow simultaneous visualisation of both living and dead cells.
  23. 23.  White blood cells from potential donors and the recipient are added to separate wells of a microtiter plate. The example depicts the reaction of donor and recipient cells with a single antibody directed against an HLA-A antigen. The reaction sequence shows that if the antigen is present on the lymphocytes, addition of complement will cause them to become porous and unable to exclude the added dye.
  24. 24. Presence or absense of various MHC allels determined by antibody mediated cytotoxicity
  25. 25. Because cells express numerous HLA antigens, they are tested separately with a battery of antibodies specific for various HLA-A antigens. Here, donor 1 shares HLA-A antigens recognized by antisera in wells 1 and 7 with the recipient, whereas donor 2 has none of HLA-A antigens in common with the recipient.
  26. 26. Test is left for 10 minutes and then read using an inverted fluorescent microscope. A mixture of T and B lymphocytes can be used for HLA Class I typing. B lymphocytes are required for HLA Class II typing by serology. (Normal population 85-90% T and 10-15% B cells) This can be achieved using a number of methods.
  27. 27.  .  In the past neuraminidase treated sheep red blood cell rosetting and nylon wool have been used.  Immunomagnetic bead separation is the current method of choice.  It utilises polystyrene microspheres with a magnetisable core coated in monoclonal antibody for a HLA Class II b chain monomorphic epitope. Positive selection
  28. 28. It has been observed that lymphocytes from one donor, when cultured with lymphocytes from an unrelated donor, are stimulated to proliferate. It has been established that this proliferation is primarily due to a disparity in the class II MHC (DR) antigens and T cells of one individual interact with allogeneic class-II MHC antigen bearing cells (B cells, dendritic cells, langerhans cells, etc.). This reactivity was termed mixed leukocyte reaction (MLR) and has been used for studying the degree of histocompatibility
  29. 29. In this test, the test lymphocytes (responder cells)are mixed with irradiated or mitomycin C treated leukocytes from the recipient, containing B-lymphocytes and monocytes (stimulator cells). The cells are cultured for 4 6 days. The responder T cells will recognize the foreign class II antigens found on the donor and undergo transformation (DNA synthesis and enlargement: blastogenesis) and proliferation (mitogenesis). The T cells that respond to foreign class II antigens are typically CD4+ TH-1 type cells. These changes are recorded by the addition of radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA.
  30. 30.  Pros:  Easily performed does not require expensive equipment.  Takes around three hours to perform.  Low level resolution, with good antisera reliable results.  Cons:  Requires large volumes of blood  Requires viable lymphocytes  Difficult to find good antisera for rarer antigens in different populations
  31. 31. Allele A Recipient cell sharing Class II MHC of donors No reaction Irradiation Allele A Donor cells Allele B Recipient cells lacking Class II MHC of donor Activation & proliferation of recipient cells [ 3H] thymidine Incorporation of radioactivity into Cell Nuclear DNA
  32. 32. DNA based procedures has increased the accuracy of HLA typing and lead to the identification of serologically undetected alleles and of many subtypes of serological specificities. DNA based methods to type for HLA alleles have therefore focused in the analysis of nucleotide variation occurring in both exon 2 and 3 of class I genes and exon-2 of class II genes.
  33. 33. Polymorphism is identified directly as part of the PCR process, although there are post amplification steps e.g.(SSP). Product containing internally located polymorphism that can be identified by a second technique e.g. PCR sequence specific oligonucleotide probing (SSOP), PCR-RFLP, PCR followed by sequencing. Conformational analysis
  34. 34. Extraction of genomic DNA Amplification of the genes of interest and  Detection of sequence polymorphism that define the alleles.
  35. 35. . Genomic DNA extracted from nucleated cells. The purity of the DNA extracted may be an important factor for successful results
  36. 36.  The specificity of the amplification can be locus specific e.g.(HLA-A, HLA-B, HLA-DRB1), group specific. e.g.(DRB1- 01 , DRB1-02) or allele specific (DRB1-0401 ,DRB1*0402) The primers are selected to amplify a single allele or a group of alleles For PCR, the specificity is determined by the sequence of the primers and amplification condition. Most typing scheme require conditions that avoid the coamplification of pseudogene.
  37. 37.  Amplification of exon 2 (Approximately 270 bp) of HLA class II is sufficient to achieve the highest resolution level. For typing HLA class I, both exon 2 and 3 and the intervening intron (fragment longer than 900 bp) are amplified by single pair of primers.
  38. 38. Sequence specific primers (SSP) (group and alleles specific primers). Hybridization with Sequence specific oligonucleotide probes (SSOP). Sequence based typing (SSP)
  39. 39. SSP is a rapid method of typing that uses sets of primer pairs to amplify specific region of genomic DNA. The efficiency of the amplification reaction is controlled by the primers that amplify conserved sequences of a selected gene.
  40. 40. SSOP: this method involves selective amplification of target followed by hybridization to a panel of oligonucleotide probes. Specificity for a particular HLA locus was achieved by selecting PCR primers specific for a sequence in the conserved region of the second exon.
  41. 41. Sequence based HLA typing involves determining the nucleotide sequence of an amplified segment of an HLA gene. SBT presents the advantages over the other procedure because of the relatively fast (24-48)hrs, high level resolution. More reliable and specific method
  42. 42. Different mutation generates specific conformational changes in PCR products. These are identified by electrophoresis analysis. e.g. heteroduplex analysis, single strand conformation polymorphism (SSCP),denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE).
  43. 43. DNA is isolated and amplified in multiple wells, each containing specific primer complementary to particular HLA allele Contents of the well run by electrophoresis through agarose gel If DNA is amplified with the specific primer, appear as band on gel
  44. 44. Pros:  Does not require viable cells.  Samples do not have to arrive in the lab the day they are taken.  PCR SSOP good for batch testing.  Can be semi automated. Cons:  Requires good quality DNA.  Require a degree of redundancy within the primers used.  Sequence of alleles must be known.
  45. 45.  In organ transplantation  In transfusion therapy  Disease association  Disputed paternity  In cancer prevention  Anthropological studies
  46. 46. •HLA typing is performed for kidney, bone marrow, pancreas and heart transplants etc. •HLA typing provides evidence of tissue compatibility. • HLA antigens expressed on the surface of the lymphocytes of the recipient are matched against those from various donors. • HLAA, HLA B and HLA DR: major transplantation antigens.
  47. 47. .
  48. 48.  Transplant success rate increases with the number of identical HLA antigens. Reduces the risk of complications after transplant, especially graft-versus-host disease (GVHD). GVHD is a potentially serious complication. GVHD occurs when the immune cells, which are part of the donated marrow or cord blood, attack your body.
  49. 49. Repeated transfusions may lead to alloimmunization( i.e. development of antibodies after exposure to non self antigens) Alloimmunization may lead to platelet refractoriness
  50. 50. Platelet refractoriness can be avoided by prior: ABO compatibility testing HLA typing( screening for anti HLA antibodies) Transfusion-Related Acute Lung Injury donor HLA antibodies react against recipient antigens Can also be avoided by HLA typing
  51. 51. •An assosciation has been observed between HLA types and certain autoimmune diseases like ankylosing spondilytis. •Evidences show that these autoimmune disease have multiple genetic and environmental factors. •Thus HLA plays an important role not exclusive role in disease causation. •Thus HLA typing supports the diagnosis of a disease.
  52. 52. Disease HLA Relative risk* Ankylosing spondyloarthritis B27 87.4 Uveitis B27 10 Goodpasture syndrome DR2 15.9 Multiple sclerosis DR2 4.8 Graves-Basedow disease DR3 3.7 Systemic lupus erythematodes DR3 5.8 Myasthenia gravis DR3 2.5 Pemphigus DR4 14.4 Rheumatoid arthritis DR4 4.2 Hashimoto thyreoiditis DR5 3.2
  53. 53.  To determine the parentage (paternity testing). The HLA antigens of the mother, child, and alleged father are compared. When an HLA antigen of the child cannot be attributed to the mother or the alleged father, then the latter is excluded as the father of the child.
  54. 54.  HLA-mediated diseases are directly involved in the promotion of cancer. Gluten-sensitive enteropathy is associated with increased prevalence of enteropathy- associated T-cell lymphoma HLA molecules play a protective role, recognizing increases in antigens that are not tolerated because of low levels in the normal state. Abnormal cells might be targeted for apoptosis, which is thought to mediate many cancers before diagnosis.
  55. 55.  Immunology –by – Janis Kuby  Essential immunology ---Ivan M. Roitt  Clinical immunology ---STITES WELLS  Text book of Microbiology – Paniker  Pathology –Robins  Lawlor DA, Zemmour J, Ennis PD, ET AL. Evolution of Class I MHC genes and proteins; from natural selection to thymic selection. Annu Rev Immunol 1990;8:23-63.  So A. Genetic polymorphism and regulation of expression of HLA region genes. In: Lecher A. HLA and Disease. San Diego; Academic Press. Inc., 1994: 1-34.  Daniel P. Stites,Abba T. Terr. Basic Human Immunology: ISBN. 0838505430.  Dyer P, Middleton D Histocompatibility testing .A practical Approach. IRL Press,1993.  Annia Ferrer et al., Genomic structure of the human MHC. Biotechnologia Applicada. 2005,vol.22,No.2