Major Histocompatibility complex OR MHC

MHC The principal function of T cells are : The principal function of T cells are : Defense against intracellular microbes Activation of other cells(B cells,Macrophages ) peptide recognition by T and B cells is different. peptide recognition by T and B cells is different. B cells recognize soluble as well as cell associated Ags In contrast,T cells recognize peptide which is displaying only by APC in associated with MHC proteins.

MHC MHC genes are the most polymorphic genes MHC genes are the most polymorphic genes MHC genes are codominantly expressed in each individual MHC genes are codominantly expressed in each individual There are two main type of MHC:class I & II There are two main type of MHC:class I & II The physiologic function of MHC molecules is presentation of peptides to T cells,control of immune responsiveness to all proteins and graft rejection The physiologic function of MHC molecules is presentation of peptides to T cells,control of immune responsiveness to all proteins and graft rejection MHC-I present cytosolic peptides to T cytotoxic cells and MHC-II present endocytosed peptides to Th cells MHC-I present cytosolic peptides to T cytotoxic cells and MHC-II present endocytosed peptides to Th cells

MHC genes

HLA-G ( a role in Ag recog.by NK cells) HLA-G ( a role in Ag recog.by NK cells) HLA-H (involved in iron metabolism HLA-H (involved in iron metabolism HLA-DM (peptide binding to class II) HLA-DM (peptide binding to class II)

Class III C4,B,C2 C4,B,C2 TNF,LT TNF,LT joined to class II joined to class II Proteasome genes,TAP,DM

MHC –I structure

MHC-II structure

Conformational structure MHC-I MHC-II

Peptide location

Characteristics of peptide-MHC interaction Each class – I or II has a single peptide binding cleft that are accommodate many different peptides Each class – I or II has a single peptide binding cleft that are accommodate many different peptides Slow on-rate and very slow off-rate Slow on-rate and very slow off-rate The MHC molecules of an individual don ’ t discriminate between self and non self The MHC molecules of an individual don ’ t discriminate between self and non self

T cell receptor and MHC interaction

Polymorphism of class II HLA-DPA 12 HLA-DPA 12 HLA-DPB 88 HLA-DPB 88 HLA-DQA 17 HLA-DQA 17 HLA-DQB 42 HLA-DQB 42 HLA-DR >400 HLA-DR >400

Polymorphism of class I HLA-A >280 HLA-A >280 HLA-B >500 HLA-B >500 HLA-C >130 HLA-C >130

MHC genes and graft rejection

HLA expression

Peptide presentation by MHC-II

Peptide presentation by MHC-I

MHC –I and peptide presentation

Features of peptide binding to MHC

HLA and diseases

Testing of DNA sequences permits detection of many more subtypes or "splits" of HLA antigens or alleles. Testing of DNA sequences permits detection of many more subtypes or "splits" of HLA antigens or alleles. In serological typing, some antigens are difficult to identify and may even mask the presence of others. DNA typing can routinely define antigens at the allele level, assuring no ambiguity in interpretations. In serological typing, some antigens are difficult to identify and may even mask the presence of others. DNA typing can routinely define antigens at the allele level, assuring no ambiguity in interpretations. DNA typing does not require live blood cells from the patient, permitting more flexible sample requirements. Thus, LabCorp can perform DNA- based HLA typing on: DNA typing does not require live blood cells from the patient, permitting more flexible sample requirements. Thus, LabCorp can perform DNA- based HLA typing on:

More interesting facts Erythrocytes will adsorb some Class I antigens viz. Bg blood group system (B7,A28, B57….) Erythrocytes will adsorb some Class I antigens viz. Bg blood group system (B7,A28, B57….) HLA B most polymorphic system and studies have shown is most significant followed by A and then C HLA B most polymorphic system and studies have shown is most significant followed by A and then C 45Kd glycoprotein comprising three heavy chain domains, non-covalently associated 45Kd glycoprotein comprising three heavy chain domains, non-covalently associated

TYPING METHODS SEROLOGY used to be the ‘gold’ standard. Now being superceded by molecular techniques as they become more robust and time efficient SEROLOGY used to be the ‘gold’ standard. Now being superceded by molecular techniques as they become more robust and time efficient CELLULAR rarely used now. Orginally used for Class II typing CELLULAR rarely used now. Orginally used for Class II typing MOLECULAR fast becoming the method of choice. Many laboratories test of choice. MOLECULAR fast becoming the method of choice. Many laboratories test of choice.

SEROLOGY Complement Dependent Cytotoxicity (CDC) Complement Dependent Cytotoxicity (CDC) Viable peripheral blood lymphocytes are obtained by discontinous density gradient centrifugation using Ficoll / Tryosil or Ficoll / Sodium Metrizoate at a density of at 19º - 22ºC. Viable peripheral blood lymphocytes are obtained by discontinous density gradient centrifugation using Ficoll / Tryosil or Ficoll / Sodium Metrizoate at a density of at 19º - 22ºC. Microlymphocytotoxic test: 3 stages Microlymphocytotoxic test: 3 stages

Microlymphocyototoxic test 1.Viable lymphocytes are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound. 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. 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.

Microlymphocyototoxic test 2 3.Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Eosin have been used in the past. 3.Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Eosin have been used in the past. If the reaction has taken place the EB enters the cell and binds to the DNA. If the reaction has taken place the EB enters the cell and binds to the DNA. For ease double staining is normally used. We use a cocktail of Ethidium Bromide and Acridine Orange, quenched using Bovine Haemoglobin to allow simultaneous visualisation of both living and dead cells. For ease double staining is normally used. We use a cocktail of Ethidium Bromide and Acridine Orange, quenched using Bovine Haemoglobin to allow simultaneous visualisation of both living and dead cells.

Microlymphocytotoxicity test 3 Test is left for 10 minutes and then read using an inverted fluorescient microscope. Test is left for 10 minutes and then read using an inverted fluorescient microscope. A mixture of T and B lymphocytes can be used for HLA Class I typing. 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) 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. This can be achieved using a number of methods.

Easily performed does not require expensive equipment. Easily performed does not require expensive equipment. Takes around three hours to perform Takes around three hours to perform Low level resolution, with good antisera reliable results Low level resolution, with good antisera reliable results Requires large volumes of blood Requires large volumes of blood Requires viable lymphocytes Requires viable lymphocytes Difficult to find good antisera for rarer antigens in different populations Difficult to find good antisera for rarer antigens in different populations

molecular DNA extraction from the nucleated cells following cell lysis and protein digestion. DNA extraction from the nucleated cells following cell lysis and protein digestion. polymerase chain reaction (PCR) polymerase chain reaction (PCR)

Molecular Methods 4 Electrophoresis is used following amplification. PCR product is run out on an agarose gel containing ethidium bromide. Each product moves according to its size and is compared to a molecular weight marker. Electrophoresis is used following amplification. PCR product is run out on an agarose gel containing ethidium bromide. Each product moves according to its size and is compared to a molecular weight marker. Interpretation: every tube should produce an identical sized product as internal control and either a specific band or not dependent on whether the allele(s) is/are present or not. Interpretation: every tube should produce an identical sized product as internal control and either a specific band or not dependent on whether the allele(s) is/are present or not. Results are visualised using 312nm UV transillumination and recorded either by video imaging or polaroid photograghy. Results are visualised using 312nm UV transillumination and recorded either by video imaging or polaroid photograghy.