W7L8 - Human Leucocyte Antigen (HLA) Flashcards
HLA
Cell surface proteins encoded by genes within the MHC
Called HLA antigens (MHC antigens) in context of transplantation
Inherited co-dominantly
Minor Histocompatibility Antigens (mHAgs)
Other surface proteins can act as antigens in transplantation
mH antigens - non HLA antigens associated with graft rejection
Complete matching for HLA often lead to graft rejection (requirement for immunosuppressive therapy)
MHC Class 1 and 2
MHC divided into classes related to structure, function and location within MHC
Three independent genes in class 1
- HLA-A, HLA-B, HLA-C (also E, F, G, H, J, K, L)
Three sets of genes in class 2
- HLA-DR, HLA-DQ, HLA-DP (also DM and DO, not expressed on surface)
Class III genes made of a variety of genes most related to immune function
- complement, tumour necrosis factor (TNF)
- LMP and TAP found within class 2 region but are class 3
MHC Class 1
Single gene coding for transmembrane glycoprotein (α or heavy chain) forming three globular domains
- α1, α2, and α3
Linked to β2-microglobulin
Sequence differences between alleles are limited to α1 and α2
The α1 and α2 domains form a peptide binding groove
- 8-9 amino acids peptides
Variation in α1 and α2 domains determine peptide binding and antigenicity
MHC Class 2
Consists of two transmembrane glycoproteins coded for by separate genes
- α and β
Form four domains:
α1 and β1
- most distant from cell membrane, forms binding groove, polymorphic region
- binds 12-17 amino acid peptides
α2 and β2
- Nearest to cell membrane, less variation, bind to CD4 cells
What is HLA typing?
Test used to identify certain individual variations in a person’s immune system
HLA Inheritance Example
Since HLA is inherited co-dominantly, if mother has A1 and A23 and father has A2 and A3, then there are 4 different haplotypes
- A1 and A2
- A1 and A3
- A23 and A2
- A23 and A3
Linkage Disequilibrium
Mendelian genetics suggests that the frequency of one locus is not influenced by another
So what is inherited at HLA-A should be independent of what is inherited at HLA-DR
But this is not true for HLA
Some HLA antigens are found in higher frequencies with other HLA antigens
HLA Types
HLA antigens extremely polymorphic
- many variations of same proteins
Expressed on all cells (class I) so HLA typing is referred to as “tissue typing”
3 class I (A, B, C) and 3 class II (DR, DQ, DP)
- total of 12 antigens per person
Many allelic variation of HLA antigens
In transplantation these are recognised by recipient as being foreign
HLA Nomenclature
Starts with HLA
Then locus - name of specific locus (e.g. DR)
- for molecular typing of class 2 the region is added (e.g. DRB1)
Number - refers to allele within locus
- HLA-DR3 = HLA-DR03
- HLA-DR17 = HLA-DR0301
- HLA-DR18 = HLA-DR0302
For class I (only one gene, region not added): HLA-A1 (serology), HLA-A0101 to 0109
Transplantation
Graft - a piece of living tissue that is transplanted surgically
Recipient has an immune system that will recognise graft as foreign
- self vs non-self
- graft rejection
Most rejection is due to differences in HLA (some mHAg) due to large numbers of possible alleles
Types of Transplantations
Autograft (autologous graft)
- transplant of tissue from one area to another area of same individual
- e.g. skin graft
Isograft (syngeneic graft)
- transplant from one individual to another who is genetically identical (syngeneic)
- e.g. kidney transplant between identical twins
Allograft
- most common
- transplantation between individuals of the same species
- MHC polymorphism will lead to probable rejection (immunosuppressive therapy required)
Xenograft
- transplantation across species
Allograft Rejection - Hyperacute Rejection
Occurs few minutes or hours of transplantation
There are pre-existing humoral antibodies
Blood group incompatibility (ABO)
Pre-sensitisation to class I MHC (blood transfusion, previous transplant, pregnancy)
Symptoms:
- complement activation
- thrombosis
- swelling
- interstitial haemorrhage
- fibrinoid necrosis
Cell mediated immunity not involved
Allograft Rejection - Acute Rejection
Occurs few days after transplant (complete loss 10-14 days)
Predominately mediated by T cells
- direct recognition of alloantigens on donor cells by recipient
Antibody binding of graft cells
- antibody mediated cellular cytotoxicity
- antibody binding blood vessel of graft with complement activation
HLA mismatch, insufficient immunosuppression
Allograft Rejection - Chronic Rejection
Some month of years after transplantation
Both antibody and cell mediated immunity
Rate, extent and mechanism may vary with tissue/organ
Immune injury already taken place so immunosuppressive therapy of no use
Chronic Rejection Examples with Different Tissues
Bone marrow and skin sensitive to rejection
Heart, kidney and liver less sensitive
For a kidney tx:
- slow, progressive renal failure
- cell-mediated inflammatory lesions of blood vessels
- thickening of glomerular basement membranes
Tissue Typing for Transplantation
HLA phenotype both donor and recipient must be determined
For transplantation
- HLA – A, B, DR (6 antigens)
- match as many as possible, DR locus matching most beneficial followed by B locus, then A locus
- most antigenic, some evidence that DQ and DP are important
Serology Based Typing
Based on antibody-antigen reactions
T cells used for class I (HLA - A, B, C)
B cells used for class II (HLA – DR, DQ)
Detects what is expressed on cell surface
Complement Dependent Cytotoxicity (CDC)
Serum containing known specific anti-HLA antibodies in tray wells
Lymphocytes added to serum
- react with specific antibody if express specific HLA type
Rabbit complement added
- lymphocytes with Ag-Ab complex killed (enlarge)
Detect killing by addition of eosin dye, ethidium bromide
- killed cells larger and stained
- live cells smaller and retractile
HLA Typing by DNA
Detects what is coded at molecular level (not what is expressed on cell surface)
DNA region of interest - generally correspond to regions on cell surface
Done by PCR
- sequence specific primers (SSP)
- sequence specific oligonucleotides (SSO)
- DNA sequencing
Sequence Specific Oligonucleotides
A single PCR amplifying all alleles
Hybridisation probes placed onto nitrocellulose membrane (dot blot)
PCR product reacted with nitrocellulose membrane
PCR product only reacts with probes specific for the HLA type
HLA Typing by DNA Sequencing
PCR relevant region (e.g. exon 2, DRB1 - highly polymorphic)
Use of primers
- for DR single primer 3’ end and eight primers at 5’ end
- amplify all known alleles of DR in single PCR tube
Sequence using single 3’ primer
DNA sequence compared to data base
