Major Histocompatibility complex OR MHC MHC The principal function of T cells are : The principal function of T cells are : Defense against intracellular.

Major Histocompatibility Major Histocompatibility complexcomplex

OROR

MHCMHC

MHCMHC

The principal function of T cells are :The principal function of T cells are :Defense against intracellular microbesDefense against intracellular microbesActivation of other cells(B cells ,Macrophages )Activation of other cells(B cells ,Macrophages )

peptide recognition by T and B cells peptide recognition by T and B cells is different.is different.

B cells recognize soluble as well as cell associated AgsB cells recognize soluble as well as cell associated AgsIn contrast ,T cells recognize peptide which is displaying only In contrast ,T cells recognize peptide which is displaying only

by APC in associated with MHC proteins.by APC in associated with MHC proteins.

MHCMHC MHC genes are the most polymorphic genesMHC genes are the most polymorphic genes MHC genes are codominantly expressed in MHC genes are codominantly expressed in

each individualeach individual There are two main type of MHC:class I & IIThere are two main type of MHC:class I & II The physiologic function of MHC molecules The physiologic function of MHC molecules

is presentation of peptides to T cells ,control is presentation of peptides to T cells ,control of immune responsiveness to all proteins of immune responsiveness to all proteins and graft rejectionand graft rejection

MHC-I present cytosolic peptides to T MHC-I present cytosolic peptides to T cytotoxic cells and MHC-II present cytotoxic cells and MHC-II present endocytosed peptides to Thendocytosed peptides to Th cellscells

MHC genesMHC genes

HLA-G ( a role in Ag recog.by NK HLA-G ( a role in Ag recog.by NK cells)cells)

HLA-H (involved in iron metabolismHLA-H (involved in iron metabolism HLA-DM (peptide binding to class II)HLA-DM (peptide binding to class II)

Class IIIClass III C4,B,C2C4,B,C2 TNF,LTTNF,LT

joined to class IIjoined to class II

Proteasome genes,TAP,DMProteasome genes,TAP,DM

MHC –I structureMHC –I structure

MHC-II structureMHC-II structure

Conformational structureConformational structure

MHC-IMHC-II

Peptide locationPeptide location

Characteristics of peptide-MHC interactionCharacteristics of peptide-MHC interaction

Each class Each class ––I or II has a single I or II has a single peptide binding cleft that are peptide binding cleft that are accommodate many different accommodate many different peptidespeptides

Slow on-rate and very slow off-rateSlow on-rate and very slow off-rate The MHC molecules of an individual The MHC molecules of an individual

dondon’’t discriminate between self and t discriminate between self and non selfnon self

T cell receptor and MHC T cell receptor and MHC interactioninteraction

Polymorphism of class IIPolymorphism of class II

HLA-DPA 12HLA-DPA 12 HLA-DPB 88HLA-DPB 88 HLA-DQA 17HLA-DQA 17 HLA-DQB 42HLA-DQB 42 HLA-DR >400HLA-DR >400

Polymorphism of class IPolymorphism of class I

HLA-A >280HLA-A >280 HLA-B >500HLA-B >500 HLA-C >130HLA-C >130

MHC genes and graft rejectionMHC genes and graft rejection

HLA expressionHLA expression

Peptide presentation by MHC-IIPeptide presentation by MHC-II

Peptide presentation by MHC-IPeptide presentation by MHC-I

MHC 1.exe

MHC –I and peptide presentationMHC –I and peptide presentation

MHC 1.exe

Features of peptide binding to MHCFeatures of peptide binding to MHC

HLA and diseasesHLA and diseases

Testing of DNA sequences permits Testing of DNA sequences permits detection of many more subtypes or detection of many more subtypes or "splits" of HLA antigens or alleles. "splits" of HLA antigens or alleles.

In serological typing, some antigens are In serological typing, some antigens are difficult to identify and may even mask the difficult to identify and may even mask the presence of others. DNA typing can presence of others. DNA typing can routinely define antigens at the allele level, routinely define antigens at the allele level, assuring no ambiguity in interpretations. assuring no ambiguity in interpretations.

DNA typing does not require live blood DNA typing does not require live blood cells from the patient, permitting more cells from the patient, permitting more flexible sample requirements. Thus, flexible sample requirements. Thus, LabCorp can perform DNA-based HLA LabCorp can perform DNA-based HLA typing on: typing on:

More interesting factsMore interesting facts

Erythrocytes will adsorb some Class I Erythrocytes will adsorb some Class I antigens viz. Bg blood group system antigens viz. Bg blood group system (B7,A28, B57….)(B7,A28, B57….)

HLA B most polymorphic system and HLA B most polymorphic system and studies have shown is most studies have shown is most significant followed by A and then Csignificant followed by A and then C

45Kd glycoprotein comprising three 45Kd glycoprotein comprising three heavy chain domains, non-covalently heavy chain domains, non-covalently associatedassociated

TYPING METHODSTYPING METHODS

SEROLOGY used to be the ‘gold’ SEROLOGY used to be the ‘gold’ standard. Now being superceded by standard. Now being superceded by molecular techniques as they become molecular techniques as they become more robust and time efficientmore robust and time efficient

CELLULAR rarely used now. Orginally CELLULAR rarely used now. Orginally used for Class II typingused for Class II typing

MOLECULAR fast becoming the MOLECULAR fast becoming the method of choice. Many laboratories method of choice. Many laboratories test of choice.test of choice.

SEROLOGYSEROLOGY Complement Dependent Cytotoxicity Complement Dependent Cytotoxicity

(CDC)(CDC) Viable peripheral blood lymphocytes Viable peripheral blood lymphocytes

are obtained by discontinous density are obtained by discontinous density gradient centrifugation using Ficoll / gradient centrifugation using Ficoll / Tryosil or Ficoll / Sodium Metrizoate Tryosil or Ficoll / Sodium Metrizoate at a density of 1.077 at 19º - 22ºC.at a density of 1.077 at 19º - 22ºC.

Microlymphocytotoxic test: 3 stagesMicrolymphocytotoxic test: 3 stages

Microlymphocyototoxic testMicrolymphocyototoxic test 1.Viable lymphocytes are incubated 1.Viable lymphocytes are incubated

with HLA specific antibodies. If the with HLA specific antibodies. If the specific antigen is present on the cell specific antigen is present on the cell the antibody is bound.the antibody is bound.

2.Rabbit serum as a source of 2.Rabbit serum as a source of complement is added, incubate. If complement is added, incubate. If antibody is bound to the HLA antigen antibody is bound to the HLA antigen on the cell surface it activates the on the cell surface it activates the complement which damages the cell complement which damages the cell membrane making it permeable to membrane making it permeable to vital stains. vital stains.

Microlymphocyototoxic test 2Microlymphocyototoxic test 2 3.Results are visualised by adding dye 3.Results are visualised by adding dye

usually a fluorochrome eg Ethidium usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Bromide although both Trypan Blue and Eosin have been used in the past.Eosin have been used in the past.

If the reaction has taken place the EB If the reaction has taken place the EB enters the cell and binds to the DNA.enters the cell and binds to the DNA.

For ease double staining is normally used. For ease double staining is normally used. We use a cocktail of Ethidium Bromide and We use a cocktail of Ethidium Bromide and Acridine Orange, quenched using Bovine Acridine Orange, quenched using Bovine Haemoglobin to allow simultaneous Haemoglobin to allow simultaneous visualisation of both living and dead cells. visualisation of both living and dead cells.

Microlymphocytotoxicity test 3Microlymphocytotoxicity test 3 Test is left for 10 minutes and then read Test is left for 10 minutes and then read

using an inverted fluorescient using an inverted fluorescient microscope.microscope.

A mixture of T and B lymphocytes can be A mixture of T and B lymphocytes can be used for HLA Class I typing. used for HLA Class I typing.

B lymphocytes are required for HLA B lymphocytes are required for HLA Class II typing by serology. (Normal Class II typing by serology. (Normal population 85-90% T and 10-15% B cells)population 85-90% T and 10-15% B cells)

This can be achieved using a number of This can be achieved using a number of methods.methods.

Easily performed does not require Easily performed does not require

expensive equipment.expensive equipment. Takes around three hours to performTakes around three hours to perform Low level resolution, with good antisera Low level resolution, with good antisera

reliable resultsreliable results Requires large volumes of bloodRequires large volumes of blood Requires viable lymphocytesRequires viable lymphocytes Difficult to find good antisera for rarer Difficult to find good antisera for rarer

antigens in different populationsantigens in different populations

molecularmolecular

DNA extraction from the nucleated DNA extraction from the nucleated cells following cell lysis and protein cells following cell lysis and protein digestion.digestion.

polymerase chain reaction (PCR)polymerase chain reaction (PCR)

Molecular Methods 4Molecular Methods 4 Electrophoresis is used following Electrophoresis is used following

amplification. PCR product is run out on an amplification. PCR product is run out on an agarose gel containing ethidium bromide. agarose gel containing ethidium bromide. Each product moves according to its size and Each product moves according to its size and is compared to a molecular weight marker. is compared to a molecular weight marker.

Interpretation: every tube should produce an Interpretation: every tube should produce an identical sized product as internal control identical sized product as internal control and either a specific band or not dependent and either a specific band or not dependent on whether the allele(s) is/are present or on whether the allele(s) is/are present or not.not.

Results are visualised using 312nm UV Results are visualised using 312nm UV transillumination and recorded either by transillumination and recorded either by video imaging or polaroid photograghy.video imaging or polaroid photograghy.