Demo Practicals for hla typing and other immuno

Demo Practicals for hla typing and other immuno

1. Demonstration of HLA typing Aim: To demonstrate HLA typing using T-lymphocytes

2. Major histocompatibility complex Group of genes coding for a set of host surface molecules that bind to a peptide fragments derived from pathogens and foreign antigens, and display them on host cell surface for recognition by the appropriate T cells. Also called human leukocyte antigens (HLA). Serves as a unique identification marker for every individual. Following transplantation of a graft the recipient mount an immune response against the graft’s MHC molecules and vice versa. Also called histocompatibility antigens.

3. HLA complex (MHC genes) and their products In humans, HLA complex coding for MHC proteins are located on short arm of chromosome 6. Around 4000 kbp in length covering >100 genes. Genes are clustered in three regions namely MHC region-I, II and III.

4. MHC region I About 2000 kbp in length. Comprises of three class I genes called HLA-A, HLA-B and HLA-C genes encoding HLA-A, HLA-B and HLA-C proteins respectively. Each protein is capable of forming the α-chain of MHC class I molecules. MHC class I molecules are present on the surface of all nucleated cells (except sperm cells) and platelets. Present the peptide antigen to CD8+ T cells.

5. MHC region II About 1000 kbp length. Comprises of three genes namely DP, DQ and DR genes encoding DP, DQ and DR proteins respectively. Each protein is capable of forming α and β-chain of MHC class II molecules. In addition MHC region also contain non classical genes such as DM, DO, LMP and T AP that help in ant i gen process i ng and presentation. MHC-II proteins are located on the surface of APC. Present the peptide to CD4+ T cells.

6. MHC region III About 1000 kbp in length. Not involved in Ag presentation. Comprises of genes that code for complement factors, heat shock proteins (HSP), tumor necrosis factor (TNF α and β), steroid 21 hydroxylases etc.

7. Difference between MHC I and MHC II molecules MHC class I MHC class II Present on All nucleated cells (except sperm cells) and platelets Ag presenting cells Pept i de Ag is pr e sent e d to CD8+ T cells CD4+ T cells Nature of pept i de Ag Endogenous or intracellular (vir a l / tumor Ag) Exogenous General size of bound antigens 8-10 amino acids 13-18 amino acids Peptide binding site α 1 and α 2 groove α 1 and β 1 groove CD4 or CD8 binding site α 3 binds to CD8 molecules on T c cells β 2 binds to CD4 molecules on T H cells. Ag presentation pathways Cytosolic pathway Endocytic pathway

8. HLA TYPING In this test, donor’s antigens expressed on the surface of leukocytes or their genes are matched with that of the recipient. The closer the HLA antigens on the transplanted organ match the recipient, the more likely that the recipient’s body will not reject the transplant. Value of HLA matching between donor and recipient varies in different solid organ transplantation. In kidney transplants, there is substantial be n ef i t if all the p o ly m or p hic H L A a l l e l e s are m a t ched.

9. Every person inherits each of the following antigens from each parent: HL A -A anti g en HLA-B antigen HLA-C antigen HLA-DR antigen HLA-DQ antigen and HLA-DP antigen Fig: Major histocompatibility complex at chromosome 6

10. When performing an HLA typing test for a kidney transplant, the following HLA antigens are looked at: HLA-A HLA-B HLA-DR Six HLA antigens are looked at for each person.

11. Each person has two of each of the antigens (one inherited from the mother and one inherited from the father).

12. By analyzing which six of these HLA-antigens both the donor and recipient have, scientists are able to determine the closeness of tissue matching. A six-antigen match is the best compatibility between a donor and recipient. This match occurs 25% of the time between siblings who have the same mother and father.

13. MET H ODS O F HLA TYPING Phenotypic method: Serology: Microcytotoxicity Tissue typing: Mixed lymphocyte reaction Genotypic methods: PCR detecting HLA genes PCR-RFLP (restriction fragment length polymorphism) Variable number tandem repeat (VNTR) typing Short tandem repeat (STR) typing DNA sequence based typing Karyosome analysis

14. Serology: Microlymphocytotoxic test: Viable WBC’s of the individual to be typed are incubated with HLA (class I and II) specific antibodies. If the specific Ag is present on the cell, the antibody is bound. Complement is added and incubated. If the antibody is bound, it will activate the complement which damages the cell membrane making it permeable to vital stains.

15. Pros Cons Easily performed, does not require expensive equipments Require large volume of blood 3 hrs Require viable WBC’s With good antisera results are reliable Difficult to find good antisera for rarer antigens.

16. Organ and tissue transplantation In organ and tissue transplantation, HLA antigens of the donor identified as invaders by the recipient causing rejection. Careful selection of the matched donor and recipient critically affect the outcome of transplantation. Diagnosing some disease : In autoimmunity: Many HLA combination are potentially indicative of autoimmune disorders, e.g. Application of HLA typi n g

17. HLA al l ele Associated disease H L A B 2 7 Ankylosing spondylitis, Reactive arthritis, Reiter’s syndrome DR2 Multiple sclerosis, Good pasture’s syndrome DR3 Myasthenia gravis, SLE DR3/DR4 Insulin dependent DM DR4 Rheumatoid arthritis A3/B14 Hereditary hemochromatosis

18. Susceptibility to viral infections: There is a link between certain HLA antigens and susceptibility to some viral infections such as AIDS (HIV virus), Hepatitis B (Hep B), Hepatitis C (Hep C), Infectious mononucleosis (EVB), Rubella (Rubella virus) etc. Paternal testing HLA typing can be used alongside other test for paternity testing.

19. Infertility (recurrent pregnancy loss): Infertility due to recurrent pregnancy loss can be attributed to immune factors (40%) one of which is presence of certain common HLA antigens between the parents. Phylogenetic studies: Some HLA haplotypes have distinctive geographical distribution and are found only in some population. These haplotypes can be used to trace human migration.

20. Requirements Donor- T lymphocytes Recipient- serum Complement –rabbit serum Stains – trypan blue and eosin/ethidium bromide Multi-well plate Pippettes Microscope

21. Protocol Lymphocyte from donor are isolated and separated in to T and B cells Serum from recipients is mixed with t-lymphocyte in multi-well plate Complement is added (from rabbit serum) If donor specific antibody is present it will bind to donor cells, then complement cascade will be activated and results in lysis of T-lymphocytes Stain the cells Observe cells under microscope Red color indicates dead cells Greenish yellow indicates live cells Results can be calculated on the basis of score (calculate % of dead cells to live cells) 0-no dead cells 2,4, 6- level of lysis

22. Results HLA typing was studied theoretically

23. Assay for plaque forming cells Aim: to perform plaque assay using B-lymphocytes

24. Principle Antibody secreting B lymphocytes can be enumerated by their ability to bind to antigens present on RBCs. In presence of complement the antibody bound RBCs are lyased The plaques thus formed can be detected using benzidine reagent The peroxidase activity of Hb (released by the lysed cells) decomposes hydrogen peroxidase and the liberated active oxygen oxidizes benzidine to give blue color

25. Requirements B cells from person with Blood gr. O Sterile petri dish Sterile 1% agar RBCs from a person with Blood gr A or B Complement (serum from O blood group person) Benzidine reagent (Benzidine +acetic acid+H2O2)

26. Protocol Separate lymphocytes by Ficoll hypaque using blood group O Separate B-cells by Fenwall wool Remove test tube of agar from 50 0C water and place them in a beaker filled with warm water Add 0.5 ml of the B-cell suspension to the agar and mix the contents Add 0.1 ml of RBCs from A or B blood group person to each tube and mix again Quickly pour the contents into a petri dish that already has a bottom layer of agar. Swirl the petri dishes in a figure eight to obtain an even top layer, allow that agar to harden and then incubate the plates at 37 0C for one hour

27. Add 2 ml of complement to the petri dish, swirl the dish to distribute solution evenly and re-incubate at 37 0C for 30 min Remove plates store at RT for 30-60 min, rinse and store overnight Stain by 8 ml benzidine reagent Pour off stain and rinse and count plaques (light areas on dark blue background) Calculate number of plaques/ml of blood Results: Assay was performed theoretically

28. Raising antibodies in lab animals

29. Rodents and rabbits are often used to produce antibodies for various research activities Certain guidelines have to be followed: The immunizing material must be virtually free of toxic substances (e.g. urea, acetic acid). It should present no risk of pathogenicity or toxicity to the host animals in the colony, or personnel Injections for routine antibody production should be administered subcutaneously in 2-4 sites per animal All guidelines must be given for when and how all test must be done For bleeding, 10 % of blood volume It is possible to produce substantial amounts of polyclonal antibodies Harvesting ascites

30. 7. Dis-infection is necessary with 70% ethanol or betadine 8. Three taps per mouse may be performed 9. Mice that fail to produce ascites within 25 days after hybridoma injection should be euthanized Results: Practical was performed theoretically

31. https://www.indiascience.in/videos/national-centre-for-cell-science-e