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HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude)
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HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

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Page 1: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA – Basics

(or how to recognize “self”)

Directors’ Affairs Committee plus others

(with our gratitude)

Page 2: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

Overview

• Some Definitions

– MHC and HLA

– Class I and Class II

– Nomenclature

• Class II extended haplotypes

• HLA Typing Technology

• HLA Immunogenicity

• Solid Phase Antibody detection methods

• HLA and Disease Associations

• HLA and Pharmacogenetics

• Review

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Page 3: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

Polymorphism slide-new alleles each year

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Human Leukocyte Antigens

Page 4: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

Major Histocompatibility Complex (MHC)

• MHC in humans is named “Human Leukocyte Antigens”

(HLA) as they were first defined on the surface of

peripheral blood leukocytes

• HLA Class I in humans is found on virtually all nucleated

cells and platelets

• HLA Class II (constitutive expression) is restricted to

specialized cells of the immune system (macrophages, B

cells, etc.)

• HLA genes are highly polymorphic

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Page 5: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Protein Structure

• Class I is heterodimeric with a

polymorphic alpha chain and a

common beta-2 microglobulin

– Alpha chain is composed of 3

extra-cellular domains (α1, α2,

and α3)

– α1 and α2 form a groove like

structure with a floor of βpleated sheets and ridges of αhelices

– presents peptides derived from

internal cellular proteins to the T

cell receptor of CD8 T cells.

– involved in the immune response

against intra-cellular parasites,

viruses and cancer

5

β2m

α2 α1

α3

plasma membrane

cytosol

Antigen Presenting Cell

plasma membrane

cytosolCD8 Tcell

TCR

CD8β2m

α2 α1

α3

plasma membrane

cytosol

Antigen Presenting Cell

plasma membrane

cytosolCD8 Tcell

TCR

CD8

Page 6: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

A view down the

groove of an HLA-A2

molecule

HLA Class I Molecule

αααα1

domainαααα2

domain

Influenza M1

peptide

αααα3

domain

ββββ2

microglobulin

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Page 7: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Protein Structure (con’t)

• Class II is also heterodimeric with

a polymorphic beta chain and a

much less polymorphic alpha

chain

– both chains are composed of 2

extra-cellular domains (α1, α2,

and β1, β2)

– Together the two first domains

create a peptide binding groove

which presents processed

peptides, from extra cellular

proteins, to CD4+ T cells

– involved in the immune response

against extra cellular infectious

agents and non-self HLA

molecules

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β2

β1

α2

α1

plasma membrane

cytosol

Antigen Presenting Cell

plasma membrane

cytosolCD4 Tcell

TCR

CD4 β2

β1

α2

α1

plasma membrane

cytosol

Antigen Presenting Cell

plasma membrane

cytosolCD4 Tcell

TCR

CD4

Page 8: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

αααα chain

ββββ chain

HLA-A2 peptide

A view down the

groove of an HLA-

DR1 molecule

HLA Class II Molecule

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HLA PolymorphismSerologic methods resolve only a small fraction of all known alleles (est. 3%)

Molecular techniques have emerged as the method of choice for HLA typing,

since strategies have been developed that resolve most/all known alleles and

provide a tool for identification of new alleles.

CLASS I CLASS II

3,107 A alleles 1,726 DRB1 alleles

3,887 B alleles 95 DRB3,B4,B5 alleles

2,623 C alleles 54 DQA1 alleles

780 DQB1 alleles

39 DPA1 alleles

520 DPB1 alleles

(status April 2015)

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Page 10: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Polymorphism

http://hla.alleles.org

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HLA Polymorphism

HLA sequences and a current list of known HLA alleles are

found at the IMGT/HLA database:

http://www.ebi.ac.uk/ipd/imgt/hla/

The IMGT/HLA Database provides a specialist database for sequences of

the human major histocompatibility complex (HLA) and includes the

official sequences for the WHO Nomenclature Committee For Factors

of the HLA System.

The IMGT/HLA Database is part of the international ImMunoGeneTics

project (IMGT).

Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE

The IPD and IMGT/HLA database: allele variant databases

Nucleic Acids Research (2015) 43:D423-431

Page 12: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Nomenclature

• Assignments are made under the auspices of World Health

Organization (WHO) Nomenclature committee

• Aim is to provide unique identification for:

– Loci

– Alleles

– Haplotypes

• Therefore the system must be very conservative but

adaptable to change

– Must not serve personal or national vanity

– Nomenclature should optimize not maximize the amount of

information in the code

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HLA Nomenclature (con’t)

• Haplotype

– The combination of alleles from two or more loci located on the same

chromosome

– Does NOT imply any linkage disequilibrium

• Genotype

– The combination of two haplotypes; one from each parent, inherited

by an individual

• Linkage Disequilibrium

– Presence of two alleles that are inherited together more frequently

than would be expected based on the gene frequencies

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HLA Nomenclature (con’t)• Each allele is initially identified by a letter(s) indicating “locus” A, B, C, DR,

DQ, and DP

• Then identified by individual specificity e.g. A1, B27, DR8, etc. (numbered

in order of discovery)

– Specificities were initially defined by using antisera (antibodies)

• Historically these were sera obtained from multiparous women

• Initially HLA Labs maintained large banks of specific sera

– Patient cells were mixed with various sera, incubated and then Complement

and a vital dye were added.

• If a cell had the corresponding antigen, then the antibody would bind,

complement fixed and cell death ensued.

• Most sera were “cross reactive” such that a technologist had to interpret

results, e.g.,: One well had antibody reactive with A23, A24 (Pos); but

another well with anti-A2, A23 might be negative; thus suggesting a

patient typing of A24

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Page 15: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Nomenclature (con’t)• HLA specificities can also be determined by genetic analysis

by identifying the presence/absence of the gene encoding the

HLA protein.

• Techniques include direct DNA sequencing, PCR using

Sequence Specific Primers (SSP) and PCR using sequence

specific oligonucleotide probes (SSO)

– Technique specifics will be discussed later

• Again the initial identification is the locus letter(s): A, B, C, etc.

– To differentiate from serological identification we use an asterisk (*)

and a place marker, such as: A*01, B*27, etc.

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HLA Nomenclature (con’t)

• Class II molecular specificities are identified at the level of the

gene encoding a particular chain, that is alpha or beta. But

remember most of the polymorphism for Class II is in the beta

chain thus common identification reflects that: DRB1*01,

DQB1*02, etc.

• The molecular two digit specificity is referred to as “Low

Resolution” typing e.g., A*01, B*27, etc.

• “High Resolution” typing are sub-specificities of the main

group e.g., A*01:01, A*01:02, B*27:01, B*27:100

– Each representing a change in at least one AA

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Page 17: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Nomenclature (con’t)

Expanded Allele Nomenclature:

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LOCUS

Molecular Typing

Antigen Level or Allele Family

Allele Level

“Subtype”

Synonymous “Non-

Coding” Substitutions

Suffix to

denote

changes in Expression

eg N=Null

HLA-DRB4*01 03 01 02N: ::

Intron

Substitutions

Colon

Delimiters

“MHC”

http://hla.alleles.org/nomenclature/naming.html

Page 18: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Nomenclature (con’t)

• Quiz Question - What does the “--” (Blank) in the following typing indicate?

A*01, --; B*08, --; DRB1*03, DRB1*15

• Most molecular and certainly all serological typing can NOT distinguish between a single versus a double presence of any particular allele

• Given that both A*01 and B*08 are common in the N. Amer. population it is highly likely that the “real” typing is as follows:

A*01, A*01; B*08, B*08; DRB1*03, DRB1*15

• Another but much, much less likely possibility is that the individual is carrying an allele that can not be detected by the specific molecular technique employed

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HLA Nomenclature (con’t)

PROBLEM:

• Many registry donors have been tested by serological

methods

– The vast majority of these do not have good documentation of which

antigens were tested for and which were not

• The majority of HPC transplant candidates have been tested

by molecular (DNA-based) methodologies

• The nomenclature of antigens (serology) and alleles (DNA) is

in some cases NOT concordant

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Page 20: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Nomenclature (con’t)

• Quiz Question – Is this Donor/Recipient pair a good match from an HLA perspective?

Donor: A19, A10; B12, B62; DR5, DR2

Recipient: A*29, A*26; B*44, B*15; DRB1*11, DRB1*15

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• Answer: Maybe but possibly yes– A19 (serological) was split: A*29, *30, *31, *32, *33, *74

– A10 (serological) was split: A*25, *26, *34, *66

– B12 (serological) was split: B*44, *45

– All the others are also splits although the molecular B*15 includes

the very different serological specificities B62, 63, 70, 71, 72, 75, 76

and 77

Page 21: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

DRB Haplotype Variations

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Almost always inherited as a predictable haplotype

DRB1

DRB1

DRB1

DRB1

DRB1 DRB6

DRB6

DRB6

DRB6

DRB2

DRB7

DRB3 (52)

DRB8 DRB4 (53)

DRB5 (51)

DRB9

DRB9

DRB9

DRB9

DRB9

DQB1DQA1DPB1DPA1 DRA

DR1, DR10

DR15,16, 51

DR11, 12, 13, 14, 3

(17,18), 52

DR4, 7*, 9, 53

DR8

Pseudo-genes

Expressed Genes

*about 1/3 of DR7

haplotypes lack an expressed

DRB4 (DR53) gene

Page 22: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

How do we do HLA typing?

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Historically, HLA typing has been done at the

protein level, microlymphocytoxicity test being the

standard method.

HLA typing at the DNA level has become the

method of choice for clinical laboratories. DNA

methods are more robust and reproducible and

provide more information.

Page 23: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

5’UTR E1 E2 E3 E4 E5 E6 E7 E8 3’UTR

PCR

Hybridization with set of

oligonucleotide probes

corresponding to the

polymorphic sites

Sequencing

SSO SBTSSPSequence

Specific

PCR amplification

Genomic DNA: HLA-A, B or C locus

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PCR Amplification

The majority of molecular assays for HLA typing

require the amplification of the target sequence of

DNA via Polymerase Chain Reaction, or PCR. A

sample of the patient’s DNA, called the template, is

allowed to react with specific primers. The primers

bind to the template DNA just upstream of the

sequence of DNA that is intended for amplification.

Page 25: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

PCR AmplificationLet’s say the DNA sequence below is located on the

Major Histocompatibility Complex on the short arm

of chromosome 6, at exon 2 of the –DRB1 gene. The

region we want to amplify is the sequence

‘GTTTAACGGCAT’. The primer would bind

immediately upstream of the target sequence.

Template DNA

3’ 5’

Target sequence

C A T G G C G T T T A A C G G C A T A C A G G G A C

Page 26: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

PCR Amplification

3’ 5’

Template DNA

A DNA polymerase enzyme (most frequently Taq),

recognizes the primer bound to the template, and

begins to add nucleotides to the 3’ end. When it

reaches the end of the template, it adds a final

adenine to complete the reaction.

G T A C C G

Primer5’ 3’

C A A A T T G C C G T A T G T C C C T G A

Target sequence

Taq

C A T G G C G T T T A A C G G C A T A C A G G G A C

Page 27: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

There are three major steps to PCR amplification. Once the

required buffers, templates, nucleotides, polymerase, and primers

have been added to a reaction tube, the temperature is raised to

about 94 degrees Celsius to denature the double stranded DNA.

This step is called ‘melting’.

Template DNA

Note: The template DNA shown above has been inverted. The 5’

– 3’ strand is on the bottom, and the 3’ – 5’ strand is on top.

PCR Amplification

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Template DNA

Next, the temperature is reduced to 50-60 degrees to allow the bind-ing of the primers to the template DNA. Two primers are needed for amplification. One binds upstream to the target sequence on the sense strand of the template, and one binds upstream to the target sequence on the antisense strand. This step is called ‘annealing’.

After enough time to allow for the binding of the primers (roughly 30 seconds), the temperature is raised to 72 degrees to allow the DNA polymerase to extend the primer in the step called ‘extension’.

PCR Amplification

After extension, the temperature is ramped up once again to about 94 degrees, and melting occurs again.

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The temperature is again reduced to 50-60 degrees to allow new

primers to anneal to both the original template DNA, and the newly

created DNA strands from the previous PCR cycle.

This is followed by another round of extension.

PCR Amplification

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The third cycle of melting, annealing, and extension produces the “short product”, a

length of double stranded DNA that is the exact length of the target sequence

bordered by a primer on each side. With each subsequent cycle, the short product is

doubled.

Short Products

After 25 to 35 cycles are completed a single DNA template may generate over a billion

target DNA short products. It is these products that are utilized in molecular assays for

HLA typing.

PCR Amplification

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HLA Typing using SSO(SSO = Sequence specific oligonucleotides)

• Using PCR and generic primers amplify large amounts of

virtually all alleles (versions) of, for e.g., HLA-A

• Then using heat to again separate the dsDNA into single

strands (ss)

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• Allow these to interact with ss specific oligo-

nucleotide probes bound to a solid matrix

(microarray Beads)

• Based on the pattern of which probes were bound

deduce the HLA type of the specimen

Page 32: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

PCR-SSO / Microarray Beads

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1. PCR amplification 2. Denaturation

4. Labeling / Detection3. Hybridization

Page 33: HLA –Basics (or how to recognize “self”) · HLA –Basics (or how to recognize “self”) Directors’ Affairs Committee plus others (with our gratitude) Overview • Some

HLA Typing using SSO

Interpretation

is based on

both negative

and positive

beads when

compared

against a panel

of control

beads

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SSP: Sequence Specific Primers

• As suggested by the name, SSP, does NOT use

generic primers but rather “Sequence Specific

Primers”

• Therefore the only DNA that is amplified is that

which matches the primers

– However this technique is quite labor-intensive and

unlike SSO can not be used in a “batch” mode i.e.,

only one sample at a time

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DNA SSP Gel Electrophoresis

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None +

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Based on well position of positive reactions

on the SSP gel, the type can be assigned

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Sequence Based Typing

• Both alleles (one from each copy of chromo-some 6) are sequenced simultaneously

• Ambiguities arise when particular allele combinations are present due to sequences in common across multiple alleles. These cis/trans ambiguities can be resolved.

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Sequence Based Typing

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Polymorphism: Two different nucleotides at the same position

on the individual’s two different chromosomes

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Polymorphisms

• R = A + G

• M = A + C

• W = A + T

• S = G + C

• K = G + T

• Y = C + T

Nucleotide Base Codes

A = Adenine

G = Guanine

C = Cytosine

T = Thymine

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The MHC in Transplant

• Allogeneic cellular response

• Alloantibody response

• Long-term graft survival correlated to degree of HLA antigen mismatch for both solid organ and BMT

• Antigens consist of epitopes (T cell or B cell) that can trigger a cellular or humoral response

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HLA-A+B+DR Mismatches

Deceased Donor, First Kidney Tx, 2000-2011

CTS Collaborative Transplant Study

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Opelz G. Transplantation 2013; 95:4-7

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Even a single nucleotide/amino acid

change can be immunogenic

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HLA Matching & Immunogenicity

• T cell immunogenicity: A nascent science and almost non-

existent with regard to HLA antigens

– Likely to be the most important aspect of HLA immunogenicity in

BMTx

• Current knowledge is driven by vaccine development

– While T cell repertoire is important, most work is looking at HLA Class I

and II supertypes

– HLA is extremely polymorphic (concentrated in the peptide binding

region) with each variant believed to be capable of binding a unique

set of peptide ligands

– BUT most HLA molecules are clustered into groups (Supertypes - 12)

with overlapping peptide binding specificities

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HLA Matching & Immunogenicity

• With all of the above it becomes almost impossible for a

transplant program to determine the best mismatch (from a

group of MM donors)

• Currently the best we can do is:

– List the number and kind of aa MM between various alleles

(within a single antigen group – See http://histocheck.org)

or

– Use known/suspected serological epitopes as an

approximation of T cell epitopes

• An important advancement in this area came with Dr. Rene Duquesnoy’s

MatchMaker program (www.hlamatchmaker.net)

• This program tallies the number of likely epitope (eplets - B cell)

differences between different antigens and alleles

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Patient is A*02:07

No exact match but …

A*02:07 vs A*02:01

One aa difference

Total Dissimilarity: 1.83

A*02:07 vs A*02:03

Four aa difference

Total Dissimilarity: 5.81

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http://www.mh-hannover.de/institute/transfusion/histocheck/

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Knowing Structure of MHC

• Allows re-examination of the nature of the

allo-immune response

• Not an antigenic response

• But an epitope response

• If you cannot match antigens… can you match

epitopes?

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HLA Matchmaker Concept

The HLA type of the antibody producer

determines what structural

components of an immunizing HLA

antigen can be “seen” as

non-self

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Structural Basis of a HLA-B51 Mismatch

Polymorphic

Residues on B51

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“Seen” by

A2, A68;

B27, B44

“Seen” by

A2, A68;

B35, B44

“Seen” by

A2, A24;

B7, B8

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HLA Matchmaker Concept

Matching at the Epitope Level Provides

an Additional Assessment of HLA

Compatibility

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PRA- Panel Reactive Antibody

- Percent Reactive Antibody

• PRA can be a qualitative and/or quantitative assessment of allo-immunization in transplant patients. PRA only reflects the breadth of the response. Optimally, PRA testing should identify the specificity of an antibody and provide the “transplantability” of a patient.

• More importantly, PRA testing should correlate with (i.e:, Predict) the final crossmatch.

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Bead Based PRA

• Antibodies to HLA (actual not putative as in cell-based assay) are

detected by a panel of beads each with its own fluorometric

signature

• Commercially available beads with bound HLA molecules from

more than 50 different cell lines covering the range of typical N.

American donors

• Antibodies are detected using fluorescent labeled second

antibody

• Can now perform the equivalent of a 50 cell PRA for any

individual within one week

• Software can also assist in identifying antibody specificities

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PRA – Bead Based

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Sample Number: 08-151553 Session Number: 2008-02-29-I-ID

%PRA: 34 Lot ID: 111507-LMI

Draw Date: 2/26/2008 Expiration Date: 11/7/2008

Patient HLA Type: A26,30; B13,38 Negative Controls: 128, 95, 229.5, 142

Reviewer: ______________ Positive Control: 13558.5

Bead DonorID AdjVal1 AdjVal2 AdjVal3 AdjValC Assigned Raw Val Class I Antigens

148 432 45.82 60.22 23.17 33.02 Positive 6182 A2 A36 B7 B72 Bw6 Cw2 Cw7

142 372 43.26 57.06 21.79 30.69 Positive 5900 A2 A23 B35 B61 Bw6 Cw15 Cw4

154 633 42.52 55.66 21.06 29.74 Positive 5824 A2 A23 B63 B65 Bw4 Bw6 Cw16 Cw8

139 404 41.87 54.6 20.32 29.02 Positive 5778 A2 A24 B51 B52 Bw4 Cw1 Cw12

144 215 40.51 52.75 19.51 27.52 Positive 5586 A2 A3 B50 B57 Bw4 Bw6 Cw18 Cw6

161 587 37.54 49 18.37 25.92 Positive 5159 A68 A74 B42 B57 Bw4 Bw6 Cw17 Cw7

113 2BW 36.56 47.87 17.95 25.58 Positive 4998 A2 A3 B13 B56 Bw4 Bw6 Cw1 Cw6

166 850 35.22 45.88 16.7 24.03 Positive 4895 A2 A24 B27 B44 Bw4 Cw1 Cw5

136 44DFW 34.41 45 16.53 23.75 Positive 4776 A2 A25 B44 B47 Bw4 Cw16 Cw6

152 258 32.43 42.41 15.6 22.26 Positive 4519 A11 A68 B18 B38 Bw4 Bw6 Cw4 Cw6

157 765 31.98 41.64 15.36 21.7 Positive 4452 A24 A68 B39 B55 Bw6 Cw3 Cw7

155 79LB 31.08 40.44 14.84 21.2 Positive 4300 A2 A33 B49 B71 Bw4 Bw6 Cw3 Cw7

147 247 19.94 25.16 8.62 12.41 Positive 2896 A1 A69 B35 B8 Bw6 Cw12 Cw7

133 17LB 13.32 16.25 4.41 6.51 Positive 2087 A1 A6602 B52 B58 Bw4 Cw12 Cw7

162 480 3.66 3.61 -0.34 -0.46 Positive 819 A29 A3 B58 B7 Bw4 Bw6 Cw7

138 60LB 1.82 0.97 -1.74 -2.41 Positive 589 A33 B49 B58 Bw4 Cw7

129 397 1.31 0.48 -1.4 -2.22 Positive 506 A3 A34 B57 B71 Bw4 Bw6 Cw3 Cw7

128 103 -0.17 -2.44 -3.29 -4.59 Negative 376 A24 A29 B37 B7 Bw4 Bw6 Cw6 Cw7

149 416 -0.27 -1.74 -2.37 -3.92 Negative 332 A29 A33 B78 B81 Bw6 Cw16 Cw18

124 531 0.24 -1.2 -2.11 -2.76 Negative 320 A34 A80 B18 B53 Bw4 Bw6 Cw2 Cw6

114 109 -0.7 -2.77 -3.47 -4.57 Negative 303 A31 A33 B35 B46 Bw6 Cw1 Cw4

165 206 -0.82 -2.29 -2.9 -3.95 Negative 285 A3 A74 B45 B8 Bw6 Cw16 Cw7

PRA can be calculated automatically

Antibody identification is interpreted from the results

34%

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Class I:

100% PRA

Specificity???

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Single Antigen Bead Assay

Specificity:

A2,68,69

B8,13,15,18,35,37,39,40,41,42,44,45,47,51,52,53,54,58,

78,81,82

Include?:

B27,48,49,50,55, 57

How to Report

Allele Specific Antibodies?

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Calculated PRA (cPRA)

• With Single Antigen Bead Testing we can identify the

antibodies a patient has, but we can no longer have a

measured PRA

• However if PRA is “Percent Reactive Antibody” with a panel

of cells (or beads) representative of the donor population to

give a measure of “transplantability” then:

• Knowing both the antibodies in a patient and the HLA typing

of actual donors locally, regionally, or even nationally then

we can Calculate the PRA by asking how many of these

previous donors would this patient have had antibodies to?

i.e. Calculated PRA

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cPRA calculator

http://optn.transplant.hrsa.gov/resources/allocationcalculators.asp

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Antigenic Structure of HLA

• Serologically defined epitopes that were originally thought to

occur on only one gene product such as HLA-A2 were

referred to as “Private Epitopes”

• Other anti-HLA antibodies that reacted with more than one

gene product, e.g., anti-HLA A2, A9, and A28, were thought

to detect a shared or cross reactive epitope, termed “Public

Epitope”

• Antibodies to public epitopes have been used to categorize

HLA molecules into major Cross Reactive Groups (CREG’s)

– Each CREG can contain more than one Public Epitope

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Same Donor - Different Patients

2C CREG = A2; A9 (23, 24); A28 (68, 69); B17 (57, 58)

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Immunizer Patient Antibody

A2, 2; B27, 27 A24, 28; B27, 57 A2

A1, 24; B27, 57 A2

A2, 28

A1, 3; B27, 51 A2

A2, 28

A2, 28, 24

A2, B57

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“New” Issues in Antibody Nomenclature

• Reminder: Class II HLA antigens are comprised of

two different polypeptides – the alpha and beta

chains – each coded for by different polymorphic

genes

• Anti-HLA antibodies were thought to have been

directed to the more polymorphic beta chain were

named accordingly

• Thus anti-DQ2 antibodies are reactive with antigens

containing DQB1*02 proteins

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“New” Issues in Antibody Nomenclature

(continued)

• BUT some antibodies are highly specific for the alpha chain

• So for example: Patient is:

DQB1*02:01 with DQA1*02:01

while the donor is:

DQB1*02:01 with DQA1*05:01

• And the patient has “anti-DQ2” antibodies but which are

reactive exclusively with the alpha “05:01”

• There is NO STANDARD Nomenclature with which to identify

such antibodies!

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HLA Testing for other Clinical Purposes

• Disease Risk Assessment

• Pharmacogenomics

• Immunotherapy

• Infectious Disease Vaccines

• Tumor Vaccines

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HLA Association with Disease Risk

Certain diseases have a strong association with certain HLA types. Examples include:

• HLA-B27: Ankylosing Spondylitis and Acute Anterior Uveitis

• HLA-A29: Birdshot Retinopathy• HLA-B51: Behçet's Disease• HLA-Cw6: Psoriasis• HLA-DQ2,8: Celiac Disease• HLA-DR15,DQ6: Narcolepsy• HLA-DR3,4-DQ2,8: Diabetes• HLA-DR4: Rheumatoid Arthritis

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HLA Association with Narcolepsy

• Our knowledge evolving over the years:

• HLA-DR2-DQ1

• HLA-DR15-DQ6

• HLA-DRB1*15:01-DQB1*06:02

• HLA-DQA1*01:02-DQB1*06:02

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HLA Association with Celiac Disease

Our knowledge evolving over the years:

• HLA-A1

• HLA-A1/B8/DR3

• HLA-DR3/DQ2

• HLA-DQ2 and DQ8

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HLA Association with Celiac Disease

• Association with: HLA-DQ2 and HLA-DQ8

usually with the “DQ2 cis” heterodimer:

DQA1*05, DQB1*02 coded on a DRB1*03:01 (DR17) haplotype

but can be “trans”:

DQA1*05 coded on a DRB1*11, DRB1*12 or DRB1*13 haplotype

and the DQB1*02 coded on a DRB1*07 haplotype

• The risk with DQ8 haplotype is usually:

DQA1*03 with DQB1*03:02 coded on a DR4 haplotype

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HLA and Pharmacogenetics

• Severe allergic or hypersensitivity reaction to drugs– Stevens-Johnson Syndrome (SJS)– Toxic Epidermal Necrolysis (TEN)

• Association between allergy or hypersensitivity to a medication and HLA type

• HLA typing allows risk stratification of the patients

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Drugs Associated with

Hypersensitivity Reactions

• Antiepileptic agents: Carbamazepine, Phenytoin, Phenobarbital, Lamotrigine

• Allopurinol

• Nevirapine

• Anti-inflammatories in oxicam family

• Sulfonamides

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HLA and Drug Adverse Reactions

• HLA-B*57:01 hypersensitivity to Abacavir

• HLA-B*15:02 carbamazepine induced SJS or TEN

• HLA-B*58:01 allopurinol induced SJS or TEN

• HLA-DRB1*01 hypersensitivity to nevirapine

• HLA-DRB1*07 ximelagatran induced hepatotoxicity

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HLA and Vaccine Development

• Vaccines producing cellular immunity require

peptide HLA binding

• Cancer cells can express “tumor specific antigens”

• Infectious disease agents have immunogenic

peptides

• Vaccine trials use peptides binding to common HLA

alleles (e.g., A*02:01)

• After proof of principal, trials include peptides

binding to other HLA alleles

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Review and Conclusions

• MHC = Major Histocompatibility Complex which in humans is called the “human leukocyte antigens”

• Class I: A, B, and C; Class II: DR, DQ, and DP– Highly polymorphic and co-dominantly expressed

• Nomenclature: letter designating the locus and a number designating specificity– Low resolution: one field; High resolution: two or more

fields separated by colons– Differences in serological vs molecular designations

• Typing can be done serologically or molecularly• Antibody Identification is critical in the proper interpretation

of PRA• HLA typing to help diagnosis of certain diseases• HLA typing to help prevent adverse drug reactions

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