HLA in Health & Disease

This is an extensive overview of the role of HLA in determining various health and disease predispositions.

1. 1 ROLE OF HLA IN HEALTH & DISEASE Presented by Dr. Anuja Joshi

2. 2 Components of the Immune response Introduction to HLA Historical background of discovery of HLA HLA genome HLA nomenclature HLA in health: MHC proteins & Role HLA in Organ transplant HLA typing Associations with Autoimmune disorders & infections Future: Newer associations and learning PRESENTATION FLOW

3. 3 Physical barriers Innate response Adaptive response COMPONENTS OF THE IMMUNE RESPONSE

4. 4 Human Leucocytic Antigen – HLA complex Highly  polymorphic gene complex located on chromosome 6p in humans encoding the Major Histocompatibility Complex (MHC) proteins in humans Allo -antigens MHC Class 1, 2 & 3 MHC class 1 : HLA- A, B & C ; E,F,G MHC class 2: HLA- DP, DQ & DR ; DM, DO MHC class 3 – Complement system – C2,C4, TNF & HSP INTRODUCTION TO HLA

5. 5 1950s 1960s . 1970s 1980s 1990s Peter Medawar Father of Transplantation Graft rejection Non-self antigens Macfarlane Burnet Clonal Selection theory Tolerance of self Discovery of Thymus, T & B lymphocytes Jacques Miller Jean Dausset Identification of HLA & gene complex MHC Restriction Peter Doherty & Rolf Zinkernagel 1967 – WHO sets up first committee for naming & classifying HLA HISTORY – DISCOVERY OF HLA

6. 6 Letter Significance N Null allele (produces a non-functional protein) L Lower than normal cell surface expression S Soluble protein not found on cell surface Q Questionable (allele may affect normal expression) C Protein that is present in cytoplasm but not cell surface A Aberrant expression (uncertain if protein is expressed) NOMENCLATURE OF HLA

7. 7 HLA GENOME & POLYMORPHISMS

8. 8 HLA IN HEALTH

9. 9 MHC: Major Histocompatibility proteins Major MHC class I There are 3 major and 3 minor MHC class I genes in HLA. HLA-A HLA-B HLA-C Minor genes: HLA-E, HLA-F and HLA-G.  Major MHC class II There are 3 major and 2 minor MHC class II proteins encoded by the HLA. HLA-DP, HLA-DQ, HLA-DR Minor: HLA- DM and HLA- DO Major MHC class III Complement C2, C4, TNF, HSP MHC CLASSES

10. 10 MHC CLASS 1 & 2

11. 11 MHC 1 MHC 2 Structure MHC class I molecules consist of one membrane-spanning α chain (heavy chain) produced by MHC genes, and one β chain (light chain or β2-microglobulin) produced by the β2-microglobulin gene MHC class II molecules consist of two membrane-spanning chains, α and β, of similar size and both produced by MHC genes. Type of APC Nucleated cells, and platelets but not RBCs (* Bg ) Professional APCs: macrophages, dendritic cells Nature of antigen Endogenous peptides & intracellular viruses Extracellular bacteria, parasites Presented to Cytotoxic T cells - CD8+ cells Helper T cells - CD4+ cells Enzymes Responsible for peptide generation  Cytosolic proteasome  Endosomal and lysosomal proteases Site of peptide loading of MHC Endoplasmic reticulum Specialized vesicular compartment End Result Presentation of  foreign-intracellular antigens or altered self-antigens;  targets cell for destruction Presentation of foreign extracellular antigens; induces antibody production, and attracts immune cells to area of infection

12. 12 ANTIGEN PRESENTATION PATHWAYS Cross-presentation*  refers to the display on MHC class I of peptides from  extracellularly acquired  antigens

13. 13 CO-STIMULATION Signal 1: T Cell receptor-MHC Signal 2: CD28 – CD80 & CD86

14. 14 IMMUNE TOLERANCE

15. Unique case of tolerance of paternal allo -antigens in the Foetus – the Placenta creates an “immunologically privileged site” Evolutionary step * different from Transplantation Proposed theories: Induction of paternal antigens prior to actual conception Role of T-reg cells No MHC 1&2 expression in trophoblast tissues Increased Cytokine production which inhibits cell-mediated immunity 15 IMMUNE TOLERANCE IN PREGNANCY Failure of tolerance: Spontaneous miscarriage, Rh incompatibility, Pre-eclampsia

16. Gut, Respiratory lining & Skin Acquired tolerance starts at birth – role of breast milk Delicate balance between extent of colonization Sentinel surveillance by T cells – mucosal firewall Downregulation of MHC expression Tolerance improves development of Peyer’s patches, CD8 cells Recent changes: antibiotics, changes in diet- Higher incidence of Autoimmune, Allergic disorders 16 IMMUNE TOLERANCE OF THE NORMAL MICROBIOME

17. 17 HLA & ORGAN TRANSPLANT Perils of a normally functioning HLA!

18. 18 HLA antibodies* - alloimmunization: induced by pregnancy, multiple transfusions or prior transplantation Independent of ABO compatibility Role of Bg antigens : (Bennet & Goodspeed Bg a  is analogous with HLA-B7, Bg b  analogous with HLA-B17 ; Bg c  with HLA-A28, cross-reactivity ) Platelet refractoriness* TA-GVHD – Transfusion associated Graft-versus-host-disease Role of irradiation of blood products TRALI – Transfusion related acute lung injury Scope for Matching in long-term / repeated transfusion requiring cases? – Feasibility HLA AND TRANSFUSIONS

19. 19 HLA disparity between hematopoietic stem cell (HSC) donor and recipient – graft failure Antigen match* - High-resolution (HR) matching at 4 loci (8 alleles), that is, HLA-A, -B, -C, and -DRB1 - (8/8) or expanded to 10/10 (includes HLA-DQB1) alleles. Perfect HLA match between HSCT donor and recipient - crucial, but often not available. The HSCT from the alternative mismatched donor with one allele/antigen mismatch (9/10) can be as beneficial as a HSCT from a fully matched donor, especially in younger patients. Permissiveness depends not only on the potential adverse effect of the HLA mismatches, but also on the urgency of the transplantation, the desirable GVL effect and the potential efficacy of the alternative therapy available for the patient. HLA AND BONE MARROW TRANSPLANT

20. 20 Direct allorecognition : Acute rejection : Donor DC present donor peptide mounted on donor MHC molecules to recipient’s T cells (passenger leukocytes) Indirect allorecognition : Chronic rejection: Recipient’s DC acquire allogeneic donor antigens and present donor these MHC-derived peptides to recipient’s T cells. HLA antibodies: Hyperacute rejection CDC cross-match: Because T cells express class I antigens and B cells express both class I and II antigens, the interpretation of T cell together with B cell cross-match will help to establish whether class I and/or II anti-HLA antibodies are present. A positive B cell CDC crossmatch invariably accompanies a positive T cell CDC cross-match but this may reflect either anti-HLA antibodies to class I antigens and/or multiple antibodies to class I and/or II antigens. However, a positive B cell CDC cross-match may occur in the absence of a positive T cell CDC cross-match and suggest the presence of class II antigens or low levels class I antigens. The presence of a positive T cell CDC cross-match is an absolute contraindication for transplantation whereas a positive B cell cross-match is a relative contraindication because of the uncertainty regarding the clinical significance and the possibility of false-positive result HLA AND KIDNEY TRANSPLANT

21. 21 HLA AND LIVER TRANSPLANT HLA matching - Rarely undertaken for liver transplantation so far Measures of outcome of liver Tx: Age, extent of transplantation, condition of patient around Tx and available waiting time Initial years – logistic challenges- The scarcity of donor organs Poor condition of the patient at referral for transplantation Challenges in maintaining graft viability - cold ischemic time limited to less than 12 hours Early results failed to show any benefit from HLA matching and cases of acceptable outcomes even in non-matched events Liver an Immunologically privileged site? Greater expression of MHC -2 as compared to MHC-1 in the liver Complex tolerogenic mechanisms in the liver – largely due to its structural architecture – large surface area, extensive sinusoidal network Role of IL-10 and PDL-1 – downregulate T cell activity and increased levels of these found in the liver However, long-term rejection secondary to MHC-2 now being recognized – eventually HLA typing will be preferred wherever feasibility allows

22. 22 HLA AND HEART TRANSPLANT HLA matching – presently not done, infeasible – future possibilities being considered All logistic challenges similar to liver transplant – much more enhanced for heart transplant – sicker patient, extremely narrow cold ischemic time and critical transport Advent of external support devices now changing the paradigm of heart transplant in triaging patients for whom transplant should be actually done : LVAD – Left Ventricular Assist Device, Total artificial heart – Syncardia

23. 23 HLA IN DISEASE

24. 24 MHA class 1 deficiency : Failure of TAP-1 molecule in transport of peptide CD8+ deficiency – recurrent viral infections CD4+ & antibody normal MHA class 2 deficiency – Bare Lymphocyte syndrome Transcription defect No presentation by APCs hence no CD4+ activation & Antibody production SCID-like presentation Fatal in early life HLA DEFICIENCY

25. 25 HLA ASSOCIATIONS – AUTO-IMMUNE DISEASES

26. 26 Approximately 90% of people with AS express the HLA-B27 genotype, But only 1–2% of individuals with the HLA-B27 genotype develop the disease. However, in symptomatic patients - negative predictive value 98% Possible theories: Abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells K lebsiella pneumonia (KP) infection and HLA-B 27 have been shown to be strongly associated with ankylosing spondylitis (AS) – molecular mimicry* HLA & ANKYLOSING SPONDYLITIS https://www.ncbi.nlm.nih.gov/pubmed/1298592 [Role of enteric Klebsiella pneumonia infection and HLA-B27 in ankylosing spondylitis] Huang F 1 ,  Cai XH ,  Shi GY . Armstrong RD, Panayi GS, Welsh KI. Histocompatibility antigens in psoriasis, psoriatic arthropathy, and ankylosing spondylitis.  Annals of the Rheumatic Diseases . 1983;42(2):142-146.

27. 27 Children with DR-DQ haplotypes DRB1 *03:01-DQA1*05:01- DQB1 *02:01 (abbreviated “DR3 ”) and  DRB1 *04:01/02/04/05/08-DQA1*03:01- DQB1 *03:02/04 (or DQB1*02; abbreviated “ DR4 ”) have a risk of approximately 1 in 20 for a diagnosis of T1D by the age of 15 years. If the child has the high-risk genotype and has a sibling who has T1D, the risk is even higher (~ 55%) . Some haplotypes confer strong protection from disease, such as DRB1*15:01-DQA1*01:02-DQB1*06:02 (abbreviated “ DR2 ”; OR = 0.03; 95% CI, 0.01–0.07) ROLE OF SCREENING*   HLA & TYPE-1 DIABETES MELLITUS Noble JA, Valdes AM. Genetics of the HLA Region in the Prediction of Type 1 Diabetes.  Current diabetes reports . 2011;11(6):533-542. doi:10.1007/s11892-011-0223-x.

28. 28 Initially RA was reported to be associated with DRB4. Later – shared epitopes hypothesis discovered: RA-associated DRB1 alleles also encode a common sequence of amino acids corresponding to residues 67-74 (shared epitopes – SE) . Several SE-positive (SE+) DRB1 alleles associated with RA include DRB1 * 0401 ,  * 0404,   * 0405 and * 0408 as well as the DRB1 * 0101 ,  * 1402, and * 1001 alleles. Relative risk estimates for most common 3 alleles: 6 for the DRB1 * 0401 allele 5 for the DRB1 * 0404 allele 1 for the DRB1 * 0101 allele Prevailing hypothesis is that the RA-associated HLA-DR molecules present self-antigens to auto-aggressive T cells, which subsequently induce an inflammatory response that leads to the development of arthritis.  HLA & RHEUMATOID ARTHRITIS Fugger L, Svejgaard A. Association of MHC and rheumatoid arthritis: HLA-DR4 and rheumatoid  arthritis - studies in mice and men.  Arthritis Research . 2000;2(3):208-211. doi:10.1186/ar89.

29. 29 Patients with psoriasis had a significantly raised frequency of HLA-Cw6. Those with arthritis in addition to their psoriasis also had raised frequencies of HLA-B27 and DR7.   In patients with psoriasis only, the presence of Cw6 was associated with a significantly earlier age of onset. Woodrow JC, Ilchysyn A. HLA antigens in psoriasis and psoriatic arthritis.  Journal of Medical Genetics . 1985;22(6):492-495. HLA & PSORIASIS

30. 30 More than 95% of celiac patients share the major histocompatibility complex II class human leukocyte antigen (HLA) DQ2 or DQ8 haplotype; Patients negative for both types are unlikely to suffer from celiac disease - Very high Negative predictive value- almost 100% – independent of dietary gluten content The positive predictive value of HLA DQ2/DQ8 is however limited as they can be present in approximately 40% of the general population, most of whom will never develop CD  Am J Gastroenterol.  2002 Mar;97(3):695-9. HLA-DQ typing in the diagnosis of celiac disease. Kaukinen K 1 ,  Partanen J ,  Mäki M ,  Collin P . Pallav K, Kabbani T, Tariq S, Vanga R, Kelly CP, Leffler DA. Clinical utility of celiac disease associated HLA testing.  Digestive diseases and sciences . 2014;59(9):2199-2206. doi:10.1007/s10620-014-3143-1. HLA & CELIAC DISEASE

31. 31  HLA-DR3 was related to the presence of anti-Ro/SSA or anti-La/SSB, or both. HLA-DR3, DR9, DR15 were potent risk factors for SLE. HLA-DR4, DR11 and DR14 were identified as protective factors for SLE. Smolen JS, Klippel JH, Penner E, et al. HLA-DR antigens in systemic lupus erythematosus: association with specificity of autoantibody responses to nuclear antigens.  Annals of the Rheumatic Diseases . 1987;46(6):457-462. Value of HLA-DR genotype in systemic lupus erythematosus and lupus nephritis: a meta-analysis. Niu Z 1 ,  Zhang P ,  Tong Y . HLA & SYSTEMIC LUPUS ERYTHEMATOSUS

32. 32 HLA - DRB1*15:01   has the strongest effect with an average odds ratio of 3.08 towards susceptibility. Several others under study Hollenbach JA, Oksenberg JR. The Immunogenetics of Multiple Sclerosis: A Comprehensive Review.  Journal of autoimmunity . 2015;64:13-25. doi:10.1016/j.jaut.2015.06.010. HLA & MULTIPLE SCLEROSIS

33. 33 Early studies showed association of HLA-DR3 with Graves’ disease (GD) - amino acid substitution at position 74 of the DR beta 1 chain of HLA-DR3 (DRb1-Arg74),with increased susceptibility to Graves’ disease Increasing evidence for a genetic interaction between thyroglobulin variants and DRb1-Arg74 in conferring risk for GD.  POST-PARTUM THYROID DYSFUNCTION - PPTD Compared with a local control population ( n  = 600), and using multiplex analysis, there was a significant increase in the combinations HLA B8, DR3 and HLA Al, B8, DR3 from 22.5% to 40.0% ( P <0.02) and from 18.6% to 35.6% ( P <0.01) respectively in the women who developed PPTD.  Jacobson EM, Huber A, Tomer Y. THE HLA GENE COMPLEX IN THYROID AUTOIMMUNITY: FROM EPIDEMIOLOGY TO ETIOLOGY.  Journal of autoimmunity . 2008;30(1-2):58-62. doi:10.1016/j.jaut.2007.11.010. HLA & GRAVES DISEASE

34. 34 DR2, DR9, and DRB1*0103 were positively associated with ulcerative colitis, and a negative association was found for DR4 and ulcerative colitis. For Crohn's disease a positive association was found with DR7, DRB3*0301, and DQ4 and a negative association with DR2 and DR3. Stokkers P, Reitsma P, Tytgat G, van Deventer SJH. HLA-DR and -DQ phenotypes in inflammatory bowel disease: a meta-analysis.  Gut . 1999;45(3):395-401. HLA & INFLAMMATORY BOWEL DISEASE

35. 35 Tumour escape* Downregulation of MHC -1 expression linked with progression of cancers, whereas preserved MHC associated with better outcomes Upregulation of IL-10 and PDL-1 leading to immunosuppression MECHANISMS: T-cell antigen recognition failure - antigen loss/mutation Immunosuppressive proteins Dhanik A, R. Kirshner J, MacDonald D, et al. In-silico discovery of cancer-specific peptide-HLA complexes for targeted therapy.  BMC Bioinformatics . 2016;17:286. doi:10.1186/s12859-016-1150-2. HLA & CANCER

36. 36 Dendritic cell therapy: Involves the extraction of mononuclear (CD14+) cells from the patient's own blood, transformation of these cells into cancer-fighting cells and giving back to the patient. Sipuleucel -T ( Provenge ): Approved for metastatic castration-resistant prostate cancer - fusion protein PA2024 Newer research: Role of targeting specific MHC-1 peptides derived from tumour cells in targeted immunotherapy for cancer HLA & CANCER IMMUNO-THERAPY

37. 37 HLA-DRB1 in allergic asthma, HLA-DQB1 in occupational asthma and HLA-DPB1 in aspirin-sensitive asthma. West Bengal study Confirmed significantly higher frequency of HLA-DRB1*03 in asthmatics - implicated in the susceptibility to asthma in the pediatric population. Role of Biological – Immunotherapy* HLA and asthma phenotypes/endotypes: a review. Kontakioti E 1 ,  Domvri K 1 ,  Papakosta D 2 ,  Daniilidis M 3 . Tissue Antigens.  2014 Sep;84(3):316-20. doi : 10.1111/tan.12403. Epub 2014 Jun 25 A study of the association of childhood asthma with HLA alleles in the population of Siliguri, West Bengal, India. Lama M 1 ,  Chatterjee M ,  Chaudhuri TK . HLA & ASTHMA

38. 38 HLA & AUTOIMMUNE HEPATITIS AIH-1 is associated with the presence of DRB1*0301, DRB3*0101 and DRB1*0401 alleles, AIH-2 is associated with DRB1*0301 or DRB1*07.

39. 39 HLA ASSOCIATIONS – INFECTIOUS DISEASES

40. 40 Resistance conferred factors:   B*53:01 ,  DQB1 *05:01 ,  DRB1 *01:01 , and   DRB1 *13:02 ; Increased Susceptibility factors:   A*30 :01 ,  A*33 :01 ,  DPB1*17:01 , and  DRB1 *04:01 .  CP/CSP : the circumsporozoite surface protein (CSP) and MSP: Merozoite surface proteins (MSPs) MSP-1, MSP-3, and MSP-9 - important vaccine candidates Role of TNF – Cerebral malaria susceptibility – in review – only proven role in blocking iron absorption Garamszegi LZ. Global distribution of malaria-resistant MHC-HLA alleles: the number and frequencies of alleles and malaria risk.  Malaria Journal . 2014;13:349. doi:10.1186/1475-2875-13-349. Lima-Junior J da C, Pratt-Riccio LR. Major Histocompatibility Complex and Malaria: Focus on  Plasmodium vivax  Infection.  Frontiers in Immunology . 2016;7:13. doi:10.3389/fimmu.2016.00013. HLA & MALARIA

41. 41 HLA class I Cw *6 and HLA class II DRB *10 alleles promote viral clearance group The HLA class I B *38 allele : more prone for chronic hepatitis C HLA DRB1 *11/*12 alleles and DQB1 *0301 are associated with HBV persistence but with HCV clearance worldwide. Consistent association of DRB1*03 and *07 is observed with HCV susceptibility and non-responsiveness to HBV vaccination across the population. HLA DR13 is protective for vertical HBV and HCV transmission in Chinese and Italian neonates, alleles under study Ocal S, Selcuk H, Korkmaz M, Altun R, Yildirim AE, Akbas E. Effect of HLA on Hepatitis C Virus Clearance and Persistence in Anti-HCV-positive End-stage Renal Disease Patients.  Saudi Journal of Gastroenterology : Official Journal of the Saudi Gastroenterology Association . 2014;20(3):175-181. doi:10.4103/1319-3767.133007. Singh R, Kaul R, Kaul A, Khan K. A comparative review of HLA associations with hepatitis B and C viral infections across global populations.  World Journal of Gastroenterology : WJG . 2007;13(12):1770-1787. doi:10.3748/wjg.v13.i12.1770. HLA & HEPATITIS B & C

42. 42 Indian study : HLA-DRB1 *1501 and DQB1*0601 predisposed to sputum positive pulmonary tuberculosis, and DPB1*04 was preventive and epistatic to this risk. Amazon study :  The  HLA-DRB1*04  was prevalent in patients with pulmonary TB OR = 2.94 The subtype  HLA-DRB1*04 : 11 : 01 OR = 2.23; 95% was associated with susceptibility to pulmonary TB while  DRB1*04 : 07 : 01  OR = 0.02; to protection.  All three subtypes - potential immunogenetic markers HLA & TUBERCULOSIS Alleles of  HLA-DRB1*04  Associated with Pulmonary Tuberculosis in Amazon Brazilian Population Dhêmerson Souza de Lima,  Mauricio Morishi Ogusku ,  Maisa Porto dos Santos,  Cláudia Maria de Melo Silva,  Vanessa Alves de Almeida,  … Tuber Lung Dis.  1999;79(5):309-17. Associations of HLA-DRB1, DQB1 and DPB1 alleles with pulmonary tuberculosis in south India. Ravikumar M 1 ,  Dheenadhayalan V ,  Rajaram K ,  Lakshmi SS ,  Kumaran PP ,  Paramasivan CN ,  Balakrishnan K ,  Pitchappan RM .

43. 43 MUMBAI STUDY Increase in HLA A2, A11, B40 and Cw7, while a decrease of A28, B12, B15 and Cw3 Haplotypes A*1102-B*4006-Cw*1502; A*0203-B*4016-Cw*0703; A*11-B*40 increased in multi-bacillary NORTH INDIA STUDY Much stronger association of DRB1*1501 with the multibacillary form than with the TT type of leprosy - possible role in the differential immune response to M. leprae antigens. HLA & LEPROSY Lepr Rev.  2004 Mar;75(1):79-85. HLA associations in leprosy patients from Mumbai, India. Shankarkumar U 1 Tissue Antigens.  1993 Sep;42(3):133-7. Study of HLA class II alleles by PCR oligotyping in leprosy patients from north India. Rani R 1 ,  Fernandez-Vina MA ,  Zaheer SA ,  Beena KR ,  Stastny P .

44. 44 HLA-B*57 and HLA-B*27 are associated with improved HIV control Association of HLA-B*35 - with worse outcome CTL escape: Loss of T-cell reactivity may not be directly linked to HIV-specific CD4 +  T-cell responses but that increased viremia after CTL escape may influence CD4 +  T-helper responses. Goulder PJR, Walker BD. HIV and HLA Class I: an evolving relationship.  Immunity . 2012;37(3):426-440. doi:10.1016/j.immuni.2012.09.005 CTL escape and increased viremia irrespective of HIV-specific CD4+ T-helper responses in two HIV-infected individuals Mark J. Geels a,1, Christine A. Jansen b,d,1, Elly Baan a , Iris M. De Cuyper b , Gijs J.M. van Schijndel b , Hanneke Schuitemaker b , Jaap Goudsmit c , Georgios Pollakis a , Frank Miedema b,d , William A. Paxton a , Debbie van Baarle b,d ,* . HLA & HIV-AIDS

45. 45 HLA TYPING

46. 46 HLA typing along with ABO (blood type) grouping - used to evaluate tissue compatibility between a donor and a potential transplant recipient. Serological typing methods or molecular typing methods: Serologic HLA typing methods detect the epitopes of the HLA molecules, whereas the molecular methods detect the nucleotide sequences. HLA typing that defines groups of alleles is referred to as low-resolution or generic typing (HLA- DRBl-04). HLA Typing that resolve all known alleles is referred to as high-resolution HLA typing (HLA- DR BI x401). HLA Typing that resolves beyond serologic specificities but that does not achieve allele level is referred to as intermediate -resolution typing (For example, HLA- DRBl-0401/09/13/16/21/26/33). Serological: Complement Dependent Cyto -toxicity (phenotyping) HLA antibody screening Molecular: (genotyping) PCR amplification & sequencing PCR-RFLP PCR-SSO PCR-SSP PCR-SBT HLA TYPING

47. 47 Isolation of Lymphocytes for HLA Typing: Peripheral venous blood is mixed with Ficoll - Hypaque and centrifuged, Layer containing peripheral blood mononuclear cells is aspirated, washed, and used Anti-CD2 or anti-CD3 mAbs coated magnetic beads are used to isolate T cells; and anti-CD19 mAbs coated magnetic beads are used to isolate B cells. HLA TYPING - PREPARATION

48. 48 CYTOTOXIC CROSS-MATCH - PRA Set of cell donors - randomly selected to be representative of a population (representative of the population of potential deceased donors) - 30 to 40 different donor lymphocytes. Similar to that of serologic typing except - here recipient serum is mixed with “cell donor” lymphocytes in individual wells along with complement and dye. Where the serum contains antibodies that bind to the cell surface with adequate density complement pathways are activated which results in cell death and uptake of the dye. The degree of cytotoxicity is expressed as percentage PRA (panel reactive antibody) - tool employed to approximate the risk of a given recipient of having a positive crossmatch  Limitations: PRA percent can be different numerically - depending on the cell panel used which are commercially produced and may not truly represent the population – HENCE FALSE POSITIVE – reducing the chances of a recipient getting a transplant just because the PRA was significant CDC –crossmatch with calculated PRA using specific antigens/antibodies preferred – cytometry or beads can be used

49. 49 Can be used to detect either HLA antigen or antibody based on the same concept of complement dependent killing – Mapping* Test lymphocytes/serum are incubated with known HLA antigen/antibodies (binding occurs if specific HLA antigens/antibodies are present.) Five µl complement (usually, rabbit serum) is added to each well and incubated for 60 minutes, (complement proteins cause death of lymphocytes that are coated with mAbs . In the absence of mAb binding with lymphocytes, the complement is not activated and the lymphocytes are not killed). The dye eosin Y, followed by formaldehyde (formaldehyde fixes the reaction) are added and viewed under an inverted/phase contrast microscope COMPLEMENT DEPENDENT CYTO-TOXICITY

50. 50

51. 51 ELISA method is easy, fast, and doesn’t require live lymphocytes as well as complement. Affinity-purified HLA antigens are coated on the walls of the microtiter plate. Recipient’s serum is added and incubated After washing, enzyme conjugated anti-human IgG is added and incubated After washing, substrate is added and incubated. i . Development of color in a particular well indicates the presence of IgG antibodies against the HLA antigen coated on that particular well. ii. Non-development of color in a particular well indicates the absence of antibodies to the particular HLA antigen coated on that well. ANTIBODY SCREENING - ELISA

52. 52 Flow cytometer cross matching : 30-250 times more sensitive than the visual serologic methods for the detection of IgG antibodies against HLA antigens on the surface of lymphocytes. Peripheral blood lymphocytes of the proposed donor are incubated with fluorescent labeled anti-CD3 mAbs , which bind to T cells Beads (micro-particles) coated with required antigens can also be used The labeled ani-CD3 bound T cells are separated from B cells in the flow cytometer and the separated, stained T cells are now incubated with the waiting recipient’s serum. Recipient’s serum antibodies against the donor T cell HLA antigens, if present, will bind to the T cells. After washing, a fluorochrome (that emits a color, other than green, say red color) labeled anti-human IgG is added and incubated. The fluorochrome labeled anti-human IgG will bind to the recipient’s HLA antibodies, which are bound to the donor T cells. The flow cytometer counts the number of T cells (red and green color) and T cells (green color) and creates a histogram. FLOW CYTOMETRY

53. 53 The sample is added to a mixture of color-coded beads, pre-coated with analyte-specific capture antibodies. The antibodies bind to the analytes of interest. Biotinylated detection antibodies specific to the analytes of interest are added and form an antibody-antigen sandwich. Phycoerythrin (PE)-conjugated streptavidin is added. It binds to the biotinylated detection antibodies. Polystyrene beads are read on a dual-laser flow-based detection instrument, such as the Luminex ®  100 ™ , Luminex 200 ™  or Bio-Rad ® Bio-Plex ®  analyzer. One laser classifies the bead and determines the analyte that is being detected. The second laser determines the magnitude of the PE-derived signal, which is in direct proportion to the amount of analyte bound. ANTIBODY SCREENING - LUMINEX BEAD ASSAY

54. 54 MOLECULAR METHODS PCR-RFLP: Polymerase Chain Reaction – Restriction Fragment PCR amplified DNA digested with restriction enzyme to generate specific restriction pattern and alleles are identified according to pattern. Can distinguish polymorphisms associated with DR3, DR5 and DR6 haplotypes Long procedure and extensive handling of samples PCR-SSO Polymerase Chain Reaction – Sequence Specific Oligonucleotides Labelled sequence specific probes are hybridized to PCR amplified DNA and then detected. Most specific technique, results within 10 hours, easier handling of several samples in one run. Very precise requirements in terms of temperature or hybridization for example PCR-SSP Polymerase Chain Reaction – Sequence Specific Primer Primers are designed with specificity-dependent nucleotide on the 3’ end. Faster than PCR-RFLP and SSO, slightly cheaper Some equivocal results of typing of certain types of alleles observed PCR-SBT Polymerase Chain Reaction – Sequence Based Testing DNA amplified using PCR specific for site of interest, products purified and then sequenced, most specific and reliable Very expensive equipment

55. 55 HLA NEWER ASSOCIATIONS & CONCEPTS

56. 56 Sleep disorder associated with HLA DR15 (DR2) and DQB1*0602. DQB1*0602 frequency was strikingly higher in patients with cataplexy versus patients without cataplexy (76.1% in 421 patients versus 40.9% in 88 patients). Positivity was highest in patients with severe cataplexy (94.8%) and progressively decreased to 54.2% in patients with the mildest cataplexy. Sleep.  1997 Nov;20(11):1012-20. HLA DQB1*0602 is associated with cataplexy in 509 narcoleptic patients. Mignot E 1 ,  Hayduk R ,  Black J ,  Grumet FC ,  Guilleminault C . HLA & NARCOLEPSY

57. 57 A significant association between C282Y and the HLA-A3/B7 haplotype was found Pacho A, Mancebo E, del Rey MJ, et al. HLA haplotypes associated with hemochromatosis mutations in the Spanish population.  BMC Medical Genetics . 2004;5:25. doi:10.1186/1471-2350-5-25. HLA & HEMOCHROMATOSIS

58. 58 HLA STRIPPED PLATELETS Platelet transfusion refractoriness is typically diagnosed using the  corrected count increment , : Pre-transfusion platelet count Post-transfusion platelet count: (END OF 1 HOUR) Body surface area Number of platelets transfused (@ 3.0 x10 11  platelets/unit) CCI = (Post-transfusion platelet count - Pre-transfusion platelet count)(BSA) ÷ (number of platelets transfused). A CCI > 7500 - sufficient post-transfusion increment, CCI < 7500 : Diagnostic of platelet refractoriness. HLA STRIPPED PLATELETS : Standard platelets of HLA class I using a brief incubation at low pH. Such HLA‐stripping results in a reduction in HLA class I expression at the surface, followed by loss of reactivity to HLA class I‐specific antibodies.

59. 59 SAVIOR SIBLING The creation of saviour siblings using fertility treatment is now possible in the United Kingdom as a result of the decision in Quintavalle v Human Fertilisation and Embryology Authority: - A licence …cannot authorise the testing of an embryo, except for one or more of the following purposes: (d) in a case where a person (‘the sibling’) who is the child of the persons whose gametes are used to bring about the creation of the embryo (or of either of those persons) suffers from a serious medical condition which could be treated by umbilical cord blood stem cells, bone marrow or other tissue of any resulting child, establishing whether the tissue of any resulting child would be compatible with that of the sibling. Britain's only saviour sibling twins At the age of two, little do they know it but Amy and Anthony Maguire are Britain's only ' saviour sibling' twins, created to be donors for their sick older brother.

60. 60 Thank you