Kamla-Raj 2004 I nt JHum Genet, 4(2): 91-103 (2004)The Human Leukocyte Antigen (HLA) SystemU. ShankarkumarHLA Department, I nstitute of I mmunohaematology, 13th Floor, NMS, Bldg, K.E.M. HospitalCampus, Parel, Mumbai 400 012, Maharastra, I ndiaEmail: Shankarkumar16@hotmail.comKEYWORDSImmunogenetics;HLA;MHC;molecularbiologyABSTRACTThediscoveryofMajorHistocompatibilityComplex(MHC)anditsinvolvementingraftrejection,immuneresponseandthegeneticbasisofdiseaseassociationsleadtothebirthofthisnewfieldofsciencecalledImmunogenetics.Thisfieldisimportantnotonlyinbasicbiomedicalresearchbutalsoinclinicalmedicine.ThegrowthofthisfieldwasfurthersubstantiatedbythevariousInternationalHistocompatibilityWorkshops.SolutionstoidiopathicsyndromesandautoimmunediseasescomefromthefieldofImmunology,ImmunogeneticsandMolecularbiology.Instudyingthegeneticbasisofdiseasesusceptibilityinhumanbeings,onehastohaveadifferentapproachbecausepredictiveexperimentalmatingcannotbeachievedinhumansasinanimalmodels;moreoverthegenerationtimeislonger.Oneapproachisthroughastudyofrandomlyselectedpatientsandcomparingtheirresultswiththoseofcontrolsandtheotheristhroughastudyofaffectedfamilies(nuclearorextended)andlookingforthemodeofinheritanceofthediseasewithrelationtogeneticmarkers.Nonethelessthesamplingstratification,sampleheterogeneity,diseaseheterogeneity,andageatonset,epidemiologicalconditionsandothersocio-biologicalfactorslimitthesestudies.Essentiallyattemptsaretobemadetocontroltheseparametersandachieveameaningfulconclusion.Thecurrentconceptsofantigenpresentationtoimmunocompetentcellsindicatethatantigenprocessingtakesplaceintheacidenvironmentoftheendosomesofantigenpresentingcells.Proteolyticdegradationoftheantigenicproteinsresultsinpeptidesofdifferentfragments,whicharesubsequentlypresentedbyMHCmoleculesaftertheyareboundtopeptidebindingsiteoftheMHCmoleculeswhichcanbindavarietyofpeptideshavingincommon,apeptidebackbone.Theseimmunogenicpeptides(heldsnuglyinthegroovebyhydrogenbondsbetweentheMHCproteinandthepeptidebackbone)onantigenpresentingcells,withtheMHCmoleculeandT-cellreceptoronT-cellsformastabletrimolecularcomplex.ThusMHCmoleculesandalphabetaT-cellreceptorsorgammadeltaT-cellreceptorplayamajorroleinthesubsequentimmuneresponse.ThepolymorphismoftheMHCmoleculesandT-cellreceptormayplayanimportantroleintheantigenrecognitionprocess.ItispossiblethatafewofthemanyallelesofagivenHLAlocusmayprovideamorestabletrimolecularcomplexthanothers,thusresultinginahighresponderstatusofanindividualthantherestofthealleles.ThisfieldofHLAsystemhasbeenevolvingveryfastandthereforeanupdatedHLAsystemreviewhasbeenprovided.INTRODUCTIONThe term HLA refers to the Human LeucocyteAntigenSystem,whichiscontrolledbygeneson the short arm of chromosome six. The HLAloci are part of the genetic region known as theMajor Histocompatibility Complex (MHC) (Hughet al. 1984). The MHC has genes (including HLA)whichareintegraltonormalfunctionoftheimmune response. The essential role of the HLAantigensliesinthecontrolofself-recognitionandthusdefenseagainstmicroorganisms.TheHLAloci,byvirtueoftheirextremepoly-morphismensurethatfewindividualsareidentical and thus the population at large is wellequippedtodealwithattack (McDevitt1985).Because some HLA antigens are recognized onallofthetissuesofthebody(ratherthanjustblood cells), the identification of HLA antigensisdescribedasTissueTypingorHLATyping.HISTORYThe early development of HLA typing sprangfromattemptsbyredcellserologiststodefineantigensonleucocytesusingtheirestablishedagglutination methods. These methods, however,were plagued with technical problems and a lackof appreciation for the extreme polymorphism ofthe system. Although Jean Dausset reported thefirst HLA antigen, MAC (HLA-A2,A28) in 1958,the poor reproducibility of leuco-agglutinationwas hindering progress. It was five years laterthat the first glimpse of the polymorphic natureof the HLA system appeared (Terasaki 1990). Thedefinition of the 4a/4b series by Jan van Rood in1963 and the definition of LA1, LA2 and LA3(HLA-A1, HLA-A2, HLA-A3) by Rose Payne andWalterBodmerin1964indicatedaneedforInternational Standardization and thus was borna series of International Workshops, starting in1964 (Glen 1991).U.SHANKARKUMAR92A summary of the events occurring at theseworkshops provides a chronicle of the milestonesof achievement in HLA research (Roitt et al. 1998):1964*AcceptanceofCytotoxicityoveragglutination1965 * Allelism of HLA antigens proposed1967 * Segregation of Alleles demonstrated infamilies1970*singlelocusnowtwo-HLA-A,HLA-B1972*60worldpopulationstypedby75laboratories1975 * Third locus, HLA-C, demonstrated1977*HLA-DdefinedbyHomozygousTyping Cells1977*Theserum-detected,D-related,HLA-DR defined1984*HLAandDiseaseassociationsexplored1984* Studies of gene structure1984*WorldwideRenalTransplantationdatabases1984* Definition of MB (later to be HLA-DQ)1987*DNAtechniqueswithserological,biochemical and cellular methods1987 * Definition of HLA-DP and HLA-DQ1992 * Use of Polymerase Chain Reaction -e.g. for SSOP1996 * Molecular definition of HLA-Class I1996 * Roles of HLA-G, E, DM, Tap & LMPsbetterunderstood2002 * Molecular characterization of HLAalleles and Non HLA genes2003*NomenclatureofKIRgenesbetterdefinedTHEHLAANTIGENSBasedonthestructureoftheantigensproduced and their function, there are two classesof HLA antigens, termed accordingly, HLA ClassI and Class II.. The overall size of the MHC isapproximately 3.5 million base pairs (Fig.1). Withinthis the HLA Class I genes and the HLA Class IIgenes each spread over approximately one thirdof this length. The remaining section, sometimesknown as Class III, contains loci responsible forcomplement,hormones,intracellularpeptideprocessing and other developmental characteris-Fig.1.TheHLAgenecomplexTHEHUMANLEUKOCYTEANTIGEN(HLA)SYSTEM93tics (Sanfilippo and Amos1986). Thus the ClassIIIregionisnotactuallyapartoftheHLAcomplex, but is located within the HLA region,because its components are either related to thefunctions of HLA antigens or are under similarcontrol mechanisms to the HLA genes.HLA Class I AntigensThe cell surface glycopeptide antigens of theHLA-A, -B and -C series are called HLA Class Iantigens(Roittetal.1998).Alistingofthecurrently recognized HLA Class I antigens areexpressed on the surface of most nucleated cellsofthebody.Additionally,theyarefoundinsolubleforminplasmaandareadsorbedontothe surface of platelets. Erythrocytesalso adsorbHLAClassIantigenstovaryingdegreesdepending on the specificity (e.g. HLA-B7, A28and B57 are recognizable on erythrocytes as socalledBgantigens).ImmunologicalstudiesindicatethatHLA-B(whichisalsothemostpolymorphic) is the most significant HLA Class Ilocus,followedbyHLA-AandthenHLA-C.ThereareotherHLAClassIloci(e.g.HLA-E,F,G,H,J,K and L), but most of these may not beimportant as loci for peptide presenters.TheHLAClassIantigenscomprisea45Kilodalton (Kd) glycopeptide heavy chain withthreedomains,whichisnon-covalentlyassociated with b - 2 microglobulin, which playsan important role in the structural support of theheavychain.TheHLAClassImoleculeisassembled inside the cell and ultimately sits onthe cell surface with a section inserted into thelipid bilayer of the cell membrane and has a shortcytoplasmic tail (Fig. 2).The general structure of HLA Class I, HLAClass II and IgM molecules show such similarityof subunits, that a common link between HLAand immunoglobulins, back to some primordialcellsurfacereceptorislikely.Thefull3-dimensionalstructureofHLA-AClassImoleculeshasbeendeterminedfromX-raycrystallography(BrowningandMcMichael1996). This has demonstrated that the moleculehas a cleft on its outermost surface, which holdsapeptide(Fig.3).Infact,ifacellbecomesFig.2.BiochemicalstructureoftheHLAClassIandClassIImolecule.Fig.3.TopviewoftheHLAmoleculedepictedbyX-raycrystallography.infected with a virus, the virally induced proteinswithinthecellarebrokendownintosmallpeptidesandthesearethepeptideswhicharethen inserted into this cleft during the synthesisof HLA Class I molecules. The role of HLA ClassImoleculesistotakethesevirallyinducedpeptides to the surface of the cell and by linkingto the T-Cell receptor of a Cytotoxic (CD8) T Cell,demonstrate the presence of this virus. The CD8T Cell will now be educated and it will be abletoinitiatetheprocessofkillingcellswhichsubsequentlyhasthatsameviralprotein/HLAClass I molecule on its surface. This role of HLAClass I, in identifying cells, which are changed(e.g. virally infected), is the reason why they needtobepresentonallcells(BrowningandMcMichael 1996).HLA Class II Antigens The cell surface glycopeptide antigens ofthe HLA-DP, -DQ and -DR loci are termed HLAClass II (Sanfilippo and Amos 1986). The tissuedistribution of HLA Class II antigens is confinedU.SHANKARKUMAR94totheimmunecompetentcells,includingB-lymphocytes,macrophages,andendothelialcellsandactivatedT-lymphocytes.Theexpression of HLA Class II, on cells, which wouldnotnormallyexpressthem,isstimulatedbycytokines like interferon g and in a transplant,thisisassociatedwithacutegraftdestruction.HLAClassIImoleculesconsistoftwochainseach encoded by genes in the HLA Complexon Chromosome 6 (Fig. 2). The T Cells, whichlink up to the HLA Class II molecules, are Helper(CD4)Tcells.Thustheeducationprocesswhich occurs from HLA Class II presentation,involvesthehelper-functionofsettingupageneralimmunereactionwhichwillinvolvecytokines, cellular and humoral defense againstthe bacterial (or other) invasion. This role of HLAClass II, in initiating a general immune response,is the reason why they need only be present onimmunologically active cells (B lymphocytes,macrophages,etc.)andnotonalltissues(Browning and Mc Michael 1996).The HLANomenclatureThere are a number of ways that you may seean HLA antigen is written. For example, you maysee HLA-DR3, HLA-DR17, HLA-DRB1*03 orHLA-DRB1*0301. These could all refer to thesame antigen! What do they mean?Firstly, asyou know, HLA is the name for the gene clusterwhichtendstobeinheriteden-bloconhumanchromosome number 6. These HLA antigens areresponsibleforthepresentationofforeignpeptides(antigens)totheimmunecompetentcells of the immune system. H.L.A. stands forHuman Leucocyte Antigen - a name that hasbeen kept more as a tribute to history than actualfunction.The second part e.g. DR - is the name of thespecific locus. There are 6 loci (Fig. 1) to whichpeople normally refer. These are A, B, C, DR, DQandDP.TheHLA-A,BandClociproducemolecules(antigens)thatnormallypresentpeptides of viral origin and are expressed on allnucleated cells. The HLA-A, B, C antigens aretermed Class I. The HLA-DR, DQ and DP lociproduce antigens that normally present peptideswhich have been broken down from bacterial orotherproteinsthathavebeenengulfedbythecell in a process of immune surveillance. Theyare only expressed on cells actively involved intheimmuneresponse,e.g.Blymphocytesmonocytes and activated T lymphocytes.The HLA-DR, DQ and DP antigens are termedClass II. There are other Class I loci besides A, Band C and there are other Class II loci besidesDR,DQandDP.However,theselociarenotnormally tested for and their significance is notentirely clear. The third part - the number, e.g. 3,17, 03, 0301, refers to the actual antigen at thelocus. For example, the DNA in the gene regionthatwecalltheHLA-DRlocustendstobedifferent from person to person. This differencewill result in a different type of HLA-DR molecule.These different types of HLA-DR molecules aregiven names, such as DR17. Actually, HLA-DR17is the old way of writing this antigen - based onusingantibodiesthatreacttotheantigensonthe cells. Now we can look directly at the DNAand therefore the accuracy is ofmuch greaterclarification. The problem is that now we can seea lot more variation between the different antigensand so we need a different way of writing them!So, when we look at the antigens above: HLA-DR3 is the broadest description of the antigen. Itis the name for a specific group of antigens. TheDR3 group can be divided into HLA DR17 andHLA-DR18 by using antibodies (serology). Whenwe look at this antigen at the DNA level we calltheDRlocusDRB1(becausethereareotherstermed A and B2,B3,etc) and the antigen 03 (forthe general antigen) and 01 for the specific variantof the 03. So, HLA-DR17 is now called HLA-DRB1*0301.This is similar for other antigens inthe system, at either HLA Class II or Class I. e.g.HLA-B60 (HLA-B*4001 molecularly).How Many HLA Loci are There? The currently recognized loci are given below(Marsh et al. 2002). Notice that HLA-DRB1 is thenormal DR locus and the old DRw52 and DRw53are DRB3 and DRB4, respectively:The Common HLA Antigens and TheirMolecular DiversityThenumberofallelesthatcannowberecognized by molecular techniques is huge andis being increased rapidly. The tables 1 and 2 willhelp to demonstrate the antigens with the greatestdiversityandtoshowthemostfrequentmolecular variants.THEHUMANLEUKOCYTEANTIGEN(HLA)SYSTEM95GENETICSOFHLARoutine Tissue Typing identifies the allelesat the three HLA Class I loci (HLA-A, -B, and -C)andthethreeclassIIloci(HLA-DR,-DPand-DQ). Thus, as each chromosome is found twice(diploid) in each individual, a normal tissue typeof an individual will involve 12 HLA antigens(Sullivan and Amos 1986). These 12 antigens areinheritedco-dominantly-thatistosay,all12antigensarerecognizedbycurrenttypingmethods and the presence of one does not affectourabilitytotypefortheothers.ThereareanumberofgeneticcharacteristicsofHLAantigens, they are:PolymorphismThepolymorphismattherecognizedHLAloci is extreme. As the role of HLA molecules isto present peptides from invasive organisms, itislikelythatthisextremepolymorphismhasHLAClass TheHLA-loci(genes) Routinelytyped?Class I HLA-A YESClass I HLA-B YESClass I HLA-C YESClass I HLA-E -Class I HLA-F -Class I HLA-G -Class I HLA-H -Class I HLA-J -Class I HLA-K -Class I HLA-L -ClassII HLA-DRA -ClassII HLA-DRB1 YESClassII HLA-DRB2 -ClassII HLA-DRB3 YESClassII HLA-DRB4 YESClassII HLA-DRB5 YESClassII HLA-DRB6 -ClassII HLA-DRB7 -ClassII HLA-DRB8 -ClassII HLA-DRB9 -ClassII HLA-DQA1 -ClassII HLA-DQB1 YESClassII HLA-DQA2 -ClassII HLA-DQB2 -ClassII HLA-DQB3 -ClassII HLA-DOB -ClassII HLA-DMA -ClassII HLA-DMB -ClassII HLA-DNA -ClassII HLA-DPA1 -ClassII HLA-DPB1 notroutineClassII HLA-DPA2 -ClassII HLA-DPB2 -Table1: ThecurrentlyrecognizedHLAlocigenesandthosethatarebeingroutinelytypedarepresented.Table2: ThecommonHLAantigensandtheirmo-leculartypesexpressedmorefrequentlyamongtheHLA-A,HLA-B,HLA-C,HLA-DRlociarepresented.HLAAA1 9 A*0101A2 58 A*0201,A*0202A3 9 A*0301A11 13 A*1101A23 A9 9 A*2301A24 A9 36 A*2402A25 A10 4 A*2501A26 A10 18 A*2601A29 A19 6 A*2901,A*2902A30 A19 12A31 A19 8 A*3101A32 A19 7 A*3201A33 A19 6 A*3301A34 A10 4 A*3401A36 3 A*3601A43 1 A*4301A66 A10 4 A*6601A68 A28 22 A*6801A69 A28 1 A*6901A74 8 A*7401A80 1 A*8001HLABB7 31 B*0702B8 16 B*0801B13 10 B*1301B14 6 B*1401(64),B*1402(65)B15 73 B*1501B18 18 B*1801,B*1802B27 24 B*2701,B*2702B35 44 B*3501,B*3502B37 5 B*3701B38 B16 8 B*3801B39 B16 26 B*3901B40 44 B*4001B41 6 B*4101B42 4 B*4201B44 B12 32 B*4402B45 B12 6 B*4501B46 2 B*4601B47 4 B*4701B48 7 B*4801B49 B21 3 B*4901B50 B21 3 B*5001B51 B5 29 B*5101B52 B5 4 B*5201B53 B5 9 B*5301B54 B22 2 B*5401B55 B22 12 B*5501,B*5502B56 B22 8 B*5601B57 B17 9 B*5701B58 B17 6 B*5801B59 1 B*5901B67 2 B*6701B73 1 B*7301B78 5 B*1517B81 1 B*8101HLAAntigensBroadGroupNo.ofmoleculartypes*MostcommonallelesU.SHANKARKUMAR96evolved as a mechanism for coping with all ofthe different peptides that will be encountered.That is to say, each HLA molecule differs slightlyfrom each other in its amino acid sequence - thisis what we see as different HLA antigens. Thisdifferencecausesaslightlydifferent3-dimen-sional structure in the peptide binding cleft. Sincedifferentpeptideshavedifferentshapesandchargecharacteristics,itisimportantthatthehuman race has a large array of different HLAantigens,eachwithdifferentshapedpeptidebindingareas(clefts)tocopewithallofthesepeptides.Howeverthatisnotall,asthepoly-morphismispopulationspecific.ThefrequentHLA antigens in different populations are clearlydifferent. For example, HLA-A34, which is presentin 78% of Australian Aborigines, has a frequencyoflessthan1%inbothAustralianCaucasoidand Chinese. Several workers have reported HLAstudiesfromvariouspopulationsofWorld(Imanishi et al. 1992; Clayton and Lonjou 1997)and India (Shankarkumar et al. 1999, 2000, 2001,2002, 2003; Mehra et al. 1984; Pitchappan et al.1984).ThusHLAantigensareofgreatsignificanceinanthropologicalstudies.PopulationswithverysimilarHLAantigenfrequenciesareclearlyderivedfromcommonstock.Conversely,fromthepointofviewoftransplantation, which will be discussed later, itisverydifficulttomatchHLAtypesbetweenpopulations.Inheritance of HLAThe normal way to present a tissue type is tolist the HLA antigens as they have been detected.Thereisnoattempttoshowwhichparenthaspassed on which antigen. This way of presentingtheHLAtypeisreferredtoasaPhenotype(Thomas et al. 1998). HLA PHENOTYPE example:HLA - A1, A3; B7, B8; Cw2, Cw4; DR15, DR4,When family data is available, it is possible toassign one each of the antigens at each locus toaspecificgroupingknownasahaplotype.Anhaplotype is the set of HLA antigens inheritedfrom one parent (Fig. 4). For example, the motherof the person whose HLA type is given abovemay be typed as HLA-A3, A69; B7, B45; Cw4,Cw9; DR15, DR17; Now it is evident that the A3,B7, Cw4 and DR15 were all passed on from themother to the child above. This group of antigensis a haplotype.B82 2 B*8201B83 1 B*8301HLACCw1 6 Cw*0101Cw2 5 Cw*0202Cw3 15 Cw*0303Cw4 10 Cw*0401Cw5 5 Cw*0501Cw6 7 Cw*0602Cw7 16 Cw*0701,Cw*0702Cw8 9 Cw*0802Cw12 8 Cw*1203Cw14 5 Cw*1401Cw15 11 Cw*1502Cw16 3 Cw*1601Cw17 3 Cw*1701Cw18 2 Cw*1801HLADRDR1 8 DRB1*0101,0103DR15 DR2 13 DRB1*1501,1502DR16 DR2 8 DRB1*1601,1602DR3 23 DRB1*0301DR4 44 DRB1*0401,0404DR11 DR5 43 DRB1*1101DR12 DR5 8 DRB1*1201DR13 DR6 52 DRB1*1301,1302DR14 DR6 43 DRB1*1401,1402DR7 6 DRB1*0701DR8 24 DRB1*0801,0802,0803DR9 2 DRB1*0901DR10 2 DRB1*1001Fig.4.ThesegregationofHLAantigensinafamily.*Thenumberofvariantsisapproximate,astherewillbemorereportedregularlyTable2:Contd...HLAAntigensBroadGroupNo.ofmoleculartypes*MostcommonallelesTHEHUMANLEUKOCYTEANTIGEN(HLA)SYSTEM97Intheabsenceofgeneticcrossingover,2siblingswhoinheritthesametwoHLAchromosomes (haplotypes) from their parents willbe HLA identical. There is a one in four chancethat this will occur and therefore in any familywith more than four children at least two of themwill be HLA identical. This is because there areonly two possible haplotypes in each parent.Linkage DisequilibriumBasicMendeliangeneticsstatesthatthefrequencyofallelesatonelocusdoesnotinfluence the frequency of alleles at another locus(Law of independent segregation). However inHLA genetics this is not true. There are a numberof examples from within the HLA system of allelesat different loci occurring together at very muchhigher frequencies than would be expected fromtheir respective gene frequencies. This is termedlinkage disequilibrium. The most extreme exampleis in Caucasians where the HLA-A1, B8, DR3(DRB1*0301), DQ2 (DQB1*0201) haplotype is soconservedthateventheallelesatthecomplement genes (Class III) can be predictedwithgreataccuracy.Similarhaplotypesareobservedinselectedcastegroupsandtribalgroups of India (Shankarkumar et al. 1999). Also,atHLAClassII,thisphenomenonissopronounced,thatthepresenceofspecificHLA-DRallelescanbeusedtopredicttheHLA-DQ allele with a high degree of accuracybefore testing. Because of linkage disequilibrium,a certain combination of HLA Class I antigen,HLA Class II antigen and Class III products willbe inherited together more frequently than wouldnormallybeexpected.Itispossiblethatthesesets of alleles may be advantageous in someimmunological sense, so that they have a positiveselectiveadvantage.Cross-ReactivityCross-reactivity is the phenomenon wherebyoneantibodyreactswithseveraldifferentantigens,usuallyattheonelocus(asopposedtoamixtureofantibodiesintheoneserum)(Shankarkumar et al. 1998). This is not a surprisingevent as it has been demonstrated that differentHLA antigens share exactly the same amino acidsequence for most of their molecular structure.Antibodiesbindtospecificsitesonthesemolecules and it would be expected that manydifferent antigens would share a site (or epitope)forwhichaspecificantibodywillbind.Thuscross-reactivityisthesharingofepitopesbetweenantigens. The term CREG is often used to describeCross Reacting Groups of antigens. It is usefulto think in terms of CREGs when screening seraforantibodies,asmostserafoundaremulti-specificanditisraretofindoperationallymonospecificsera.Therarityofmonospecificsera means that most serological tissue typing isdoneusingseradetectingmorethanonespecificityandatypingisdeducedbysubtraction. For example, a cell may react with aserum containing antibodies to HLA-A25, A26,and A34 and be negative for pure A26 and pureA25antisera.Inthiscase,HLA-A34canbeassigned, even in the absence of pure HLA-A34antiseraMETHODSOFTESTING FORHLAANTIGENSLymphocytotoxicity (Serological Testing)In the lymphocytotoxicity test (Terasaki andMcClleland 1964), lymphocytes are added to sera,which may or may not have antibodies directedtoHLAantigens.Iftheserumcontainsanantibody specific to an HLA (Class I or Class II)antigenonthelymphocytes,theantibodywillbind to this HLA antigen. Complement is thenadded. The complement binds only to positivecells (i.e. where the antibody has bound) and indoingso,causesmembranedamage.Thedamaged cells are not completely lysed but suffersufficient membrane damage to allow uptake ofvitalstainssuchaseosinorfluorescentstainssuchasEthidiumBromide.Microscopicidentification of the stained cells, indicates thepresence of a specific HLA antibody. The cellsusedforthetestarelymphocytesbecauseoftheir excellent expression of HLA antigens andease of isolation compared to most other tissue.The most important use of this test is to detectspecificdonor-reactiveantibodiespresentinapotential recipient prior to transplantation.Historically, this test has long been used totype for HLA Class I and Class II antigens, usingantiseraofknownspecificity.However,theproblems of cross-reactivity and non-availabilityof certain antibodies has led to the introductionofDNAbasedmethods.Currently,manyU.SHANKARKUMAR98laboratories have changed to molecular geneticmethods for HLA Class typing.MixedLymphocyteCulture (MLC)When lymphocytes from two individuals arecultured together, each cell population is able torecognize the foreign HLA class II antigens oftheother.Asaresponsetothesedifferences,the lymphocytes transform into blast cells, withassociatedDNAsynthesis.Radio-labelledthymidine, added to the culture, will be used inthisDNAsynthesis.Therefore,radioactiveuptake is a measure of DNA synthesis and thedifference between the HLA Class II types of thetwopeople.Thistechniquecanberefinedbytreatingthelymphocytesfromoneoftheindividuals to prevent cell division, for exampleby irradiation. It is thus possible to measure theresponse of T lymphocytes from one individualtoarangeofforeignlymphocytes.Ithasthusproved possible by using the mixed lymphocyteculture (MLC) test to use T lymphocytes to definewhatwerepreviouslycalledHLA-Dantigens.The HLA-D defined in this way is actually acombination of HLA-DR,DQ and -DP.An important use of the MLC is in its use asa cellular crossmatch prior to transplantation -especiallybonemarrow.Bytestingthepros-pective donor and recipient, an in-vitro transplantmodelisestablishedwhichisanextremelysignificantindicatorofpossiblerejectionorGraft-Versus-Host reaction.MolecularGeneticTechniquesRFLP (Restriction Fragment LengthPolymorphism) Restriction Fragment Length Polymorphism(RFLP) methods (Dupont 1989) rely on the abilityofcertainenzymestorecognizeexactDNAnucleotide sequences and to cut the DNA at eachof these points. Thus the frequency of a particularsequencewilldeterminethelengthsofDNAproduced by cutting with a particularenzyme. The DNA for one HLA (Class II) antigen,e.g.DR15,willhavetheseparticularenzymecutting sites (or restriction sites) at differentpositions to another antigen, e.g. DR17. So thelengthsofDNAseenwhenDR15iscutbyaparticular enzyme, are characteristic of DR15 anddifferent to the sizes of the fragments seen whenDR17 is cut by the same enzyme (Bidwell 1988).Polymerase Chain ReactionThePolymeraseChainReaction(PCR)(Michael et al. 1995) is a recently developed andrevolutionary new system for investigating theDNA nucleotide sequence of a particular regionof interest in any individual. Very small amountsofDNAcanbeusedasastartingpoint,suchthat it is theoretically possible to tissue type usingasinglehairroot.SequencingDNAhasbeentransformed from a long and laborious exerciseto a technique that is essentially automatable inthe not too distant future. The first step in this technique is to obtainDNA from the nuclei of an individual. The doublestrandedDNAisthendenaturedbyheatintosinglestrandedDNA.Oligonucleotideprimersequences are then chosen to flank a region ofinterest. The oligo- nucleotide primer is a shortsegmentofcomplementaryDNA,whichwillassociate with the single stranded DNA to act asastartingpointforreconstructionofdoublestranded DNA at that site. If the oligonucleotide is chosen to be closeto a region of special interest like a hypervariableregion of HLA-DRB then the part of the DNA,and only that part, will become double strandedDNA,whenDNApolymeraseanddeoxy-ribonucleotide triphosphates are added. From onecopyofDNAitisthuspossibletomaketwo.Those two copies can then, in turn, be denatured,reassociate with primers and produce four copies.Thiscyclecanthenberepeateduntilthereissufficient copies of the selected portion of DNAto isolate on a gel and then sequence or type.There are a number of PCR based methods inuse. For example:Sequence Specific Priming (SSP)In this test, the oligonucleotide primers usedto start the PCR have sequences complimentaryto known sequences which are characteristic tocertain HLA specificities. The primers, which arespecific to HLA-DR15, for example, will not beable to instigate the PCR for HLA-DR17. Typingis done by using a set of different PCRs, eachwith primers specific for different HLA antigens.Sequence Specific Oligonucleotide (SSO)TypingBy this method, the DNA for a whole regionTHEHUMANLEUKOCYTEANTIGEN(HLA)SYSTEM99(e.g. the HLA DR gene region) is amplified in thePCR. The amplified DNA is then tested by addinglabeled (e.g. Radioactive) oligonucleotide probes,which are complementary for DNA sequences,characteristicforcertainHLAantigens.Theseprobeswillthentypeforthepresenceofspecific DNA sequences of HLA genes (Fig. 5).CLINICALRELEVANCEOFTHEHLASYSTEMDespitethetemptationtothinkofthemastransplantation antigens, HLA antigens are notpresent on tissues simply to confound transplantsurgeons. The most important function of MHCmoleculeisintheinductionandregulationofimmuneresponses.T-lymphocytesrecognizeforeignantigenincombinationwithHLAmolecules. In an immune response, foreign antigen isprocessed by and presented on the surface of acell (e.g.. macrophage). The presentation is madeby way of an HLA molecule. The HLA moleculehasasection,calleditsantigen(orpeptide)bindingcleft,inwhichithastheseantigensinserted. T-lymphocytes interact with the foreignantigen/HLA complex and are activated. Uponactivation, the T cells multiply and by the releaseofcytokines,areabletosetupanimmuneresponsethatwillrecognizeanddestroycellswiththissameforeignantigen/HLAcomplex,when next encountered. The exact mode of actionofHLAClassIandHLAClassIIantigensisdifferent in this process. HLA Class I molecules,by virtue of their presence on all nucleated cells,present antigens that are peptides produced byinvadingviruses.Thesearespecificallypresented to cytotoxic T cells (CD8) which willthen act directly to kill the virally infected cell.HLAClassIImolecules,haveanintracellularchaperone network which prevents endogenouspeptidefrombeinginsertedintoitsantigenbindingcleft.Theyinsteadbindantigens(peptides) which are derived from outside of thecell(andhavebeenengulfed).Suchpeptideswould be from a bacterial infection. The HLAClassIImoleculepresentsthisexogenouspeptide to helper T cells (CD4) which then set upa generalized immune response to this bacterialinvasion. Thus it is apparent that MHC productsare an integral part of immunological health andtherefore it is no surprise to see a wide variety ofareasofclinicalandgeneticimplications.Thefollowing is a general overview of some of theimportant functional aspects of HLA antigens.HLA and TransfusionThe HLA Class I antigens are carried in highFig.5.TheSSOPhybridizedbandsoftheHLAgene5 10 15 20 25 30 35 40 45 50 555 10 15 20 25 30 35 40 45 50 55U.SHANKARKUMAR100concentrations by leucocytes and platelets, butonlyintraceamountsonerythrocytes.Eachtransfusionofeitherplateletsorleucocytestherefore carries a risk of immunizing the patient.Patients,withanintactimmunesystem,whorequiremultipletransfusionsofwholeblood,platelets or leucocyte concentrates will thereforeusuallydevelopantibodiestoHLAantigens(Rudmann 1995). This risk can be minimized bywashing or filtering the red cell preparations andbyreducingleucocytecontaminationasfaraspracticable.Inmulti-transfusedpatients,suchasthosewith leukemia, anti-HLA antibodies may lead totwoproblems.Firstly,thesepatientsbecomerefractorytoplatelettransfusions,whichtheydestroyrapidly,andsecondlynon-haemolytictransfusion reactions may occur in response toHLAantigens.Boththeseproblemscanbecircumvented with some difficulty. Family donors,especially HLA identical siblings, provide onesource of platelets, which may not be consumed.Itispossibletouseplateletorlymphocytecrossmatchingtechniquestoconfirmthesuitability of an individual donor, but there is alimittothefrequencywithwhichasingleindividual can provide platelets. An alternativesource, is to HLA phenotype a bank of potentialplateletvolunteersforuseintheappropriatepatients.Thedisadvantageofthisapproachisthat the polymorphism of each of the HLA classIallelicsystemsgivesrisetolowchancesoffinding HLA matched donors for patients withany tissue type other than an extremely commonone. The volunteer banks thus have to be largeto offer any chance of success.HLA and TransplantationRenal TransplantsHLAtypingwasappliedtokidneytransplantationverysoonafterthefirstHLAdeterminants were characterized (Terasaki 1992;Opelz 1985; Sanfilipo et al. 1984). The importanceofreducingmismatchedantigensindonorkidneys was immediately apparent with superiorsurvivalofgraftsfromHLAidenticalsiblingscompared to one haplotype matches or unrelateddonors.ItisapparentthattheeffectofHLAmatchingissignificant,evenwiththehighlyefficient immunosuppression used today. In renaltransplantation there are two major priorities thatreduce the (already low) chance of obtaining goodHLAmatching.ThesearetheneedforABOcompatibilityandtheneedforanegativeT-lymphocyte crossmatch (using cytotoxicity).Anti-HLA Class I antibodies present at the timeof transplant will cause hyperacute rejectionof the graft (i.e. when the T cell crossmatch ispositive).Liver TransplantationPatientsawaitinglivertransplantationcanseldom afford to wait for a well matched graft.Therefore, liver transplantation is more involvedwith problems such as physical size rather thanHLA. Also with the effects of Cyclosporin-A andtheactionoftheliveritselfasaformofimmunologicalsponge(tomopupimmunecomplexes) the effect of HLA matching is difficulttodetermine.ThelymphocytotoxicTcellcrossmatchisanimportantfactorinlivertransplantation. Transplants, which are, throughurgency,carriedoutdespiteapositiveTcellcrossmatch, have a significantly lower successrate.Heart TransplantsTherehasonlyrecentlybeensufficientaccumulated experience to show the effect of HLAantigens on cardiac allograft survival. It is nowclearthatHLA-DRantigensexertapowerfuleffect, analogous to that seen in renal transplanta-tion.However,theproblemofapplyingthatknowledge to clinical practice is more analogousto liver transplantation. Cardiac size match andavailability at the right time, are of more pressingimportance than matching HLA antigens.Corneal TransplantationThere is evidence that the cornea may surviveslightlybetteriftheHLAclassIantigensismatched (Mayer et al. 1983). In the low risk, firstcorneal graft recipient, the efforts to HLA matchandthehindrancetoroutinesofcornealprocurement and transplantation do not appearto warrant tissue typing. The situation is howeversomewhat different when the recipients corneahasbecomevascularisedfrompreviousinflammation or they have rejected one or moreprevious grafts. In those patients, matching forHLAclassIantigensdoesseemtobeworthwhile.THEHUMANLEUKOCYTEANTIGEN(HLA)SYSTEM101Bone Marrow Transplantation (BMT) orHaematopoitic Stem Cell Transplantation(HSCT) CompleteHLAmatchingofbonemarrowdonor and recipient is crucial to the success ofallogenic BMT (Shankarkumar and Undevia 1999;Ghosh 1999; Shankarkumar 2001).Incompatibilitymaynotonlyleadtorejectionbutalsotothegreaterproblemofgraft-versus-host-disease(GVHD)inwhichtheimmunologicallycompromisedrecipientisattackedbythegraftedbonemarrow.Mostbonemarrowtransplants involve HLA-identical siblings withthe HLA identity confirmed by family study andMLCorhighlydefinitivemoleculargenetictechniques. Failing an HLA identical sibling beingavailable, a close relative with very similar (e.g.one HLA antigen mismatch) may be considered.Howeversince60%to70%ofpotentialcandidates do not have a suitable family membertoactasadonor,therehasbeeninterestindevelopinglistsoftissuetypedvolunteerspreparedtodonatebonemarroworperipheralblood stem cells. There are now a number of suchRegistriesestablished,whichbyInternationalcollaboration now able to find an HLA-A, B, C,DR, and DQ matched donor, for a further 30% or40%ofcandidatesofCaucasianpatients.Thesuccessratesofthesetransplantswasinitiallydismal, but better conditioning of both the patientandbonemarrowarenowresultinginmuchbetter results.HLA and Paternity TestingThe vast polymorphism of the HLA systemmakesitamostvaluabletoolinthefieldofpaternitytesting(Bryant1988).Therearetwopossiblerolesofpaternitytestingdependinguponthesituation.Probabilityofexclusionofpaternity,andprobabilityofpaternityrequiredifferentmathematicalformulae.Excludingpaternity may in some cases be straightforward,for example when a putative father does not haveany of the HLA antigens that a child must haveinheritedfromitsfather,thisisfirstorderexclusion.Whenthefatherishomozygousorthe child appears to be homozygous it is possiblethat unidentified antigens explain the differences.The probability of paternity on the other handhastoconsider,evenwhentheHLAantigensarefullycompatiblewithpaternity,thatanalternate father may have possessed those sameantigens.TheprobabilityofexclusionfortheHLA test alone is 93% which means that out ofevery 100 falsely accused fathers 93 would beexcluded from paternity. If the alleged father doesexpress the requisite HLA tissue type, the matchbetweenthechildandallegedfatherisasignificantpieceofevidenceconsistentwithpaternity(Rudmann1995).Howeveritisuncommon due to the diverse nature of the HLAsystemthattherequisiteHLAtissuetypeisfound in the alleged fathers population group.Here one could analyze the haplotype frequencydata for HLA A, B and DR and would be veryprobative with a power of exclusion increasingto 99%. Most laboratories now combine red celland HLA testing of the mother, child and putativefather,togetherwithfurthergeneticanalysis,suchasDNAfingerprinting-wheredirectcomparison of DNA fragments yields excellentresults.HLA and Disease SusceptibilityIn the 1960s, it was discovered that the mouseMHC(calledH-2)controlledboththegeneticsusceptibilitytocertainleukemiasandtheimmune response to certain antigens. Since theninnumerable reports have been published aimedat discovering the role of the human MHC in thecontrol of responsiveness and disease suscepti-bility (Tiwari and Terasaki 1985).There are two general explanations for HLAand disease associations (McDevitt 1985).Firstly, there may be a linkage disequilibriumbetween alleles at a particular disease associatedlocus and the HLA antigen associated with thatdisease - this is so for HLA-A3 and IdiopathicHaemochromatosis.Anotherpossibleexplanationfortheseassociations is that the HLA antigen itself playsa role in disease, by a method similar to one ofthe following models: a) By being a poor presenter of a certain viral orbacterial antigenb) By providing a binding site on the surface ofthecellforadiseaseprovokingvirusorbacteriumc) By providing a transport piece for the virusto allow it to enter the celld) By having such a close molecular similarityto the pathogen, that the immune system failsto recognize the pathogen as foreign and soU.SHANKARKUMAR102fails to mount an immune response against it.It is most likely that all these mechanisms areinvolved,buttoavaryingextentindifferentdiseases(Trosby1997).Inmultiplesclerosis(Kankonkaretal.2003)andankylosingspondylitis(Shankarkumaretal.2002),cellmediated immunity is often depressed, not onlyinthepatientsbutalsointheirparentsandsiblings. 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