Monika Ohams, PhD

Department of Clinical Immunology

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MHC complex is present in all vertebrate species

In humans, MHC complex is the equivalent of the HLA system (Human Leukocyte Antigens) and is located on the short arm of chromosome 6

MHC complex genes and their products are highly polimorphic

(these antigens are encoded by several hundred alleles)

HLA antigens are crucial for the induction of the immune response to transplant antigens

HLA antigens are glycoproteins expressed on all nucleated cells

except for: red blood cells, sperm, trophoblast cells, the corneal endothelium

Class I (HLA-A,B) and class II (HLA-DR) antigens are considered in clinical kidney transplantation, while class III antigens are not taken under consideration

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MHC Class I antigens encode peptide-binding proteins

and molecules (such as TAP and Tapasin) involved in

antigen processing.

expression:

α chain, expressed on all nucleated cells

ligands :

CD8 (cytotoxic T cells)

inhibitory receptors of NK cells.

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MHC Class II :

peptide-binding proteins and proteins involved in

antigen loading onto MHC class II molecules (such as MHC II DM, MHC II DQ, MHC II DR, and MHC II DP).

two chains: α & β, expressed on:

- APCs (Antigen Presenting Cells): B cells,

macrophages, monocytes, dendritic cells, Langerhans

cells;

- vascular endothelial cells (kidney epithelial cells!!),

- activated T cells,

- thymic epithelial cells,

- intestinal epithelial cells.

ligands :

CD4 on helper T cells.

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MHC class III antigens include: - complement components: C2,C4 and B factor - cytokines: TNF, LTA, LTB - heat shock proteins, - extracellular matrix proteins, - transcription factors, - serine/threonine nuclear kinases

MICA – MHC class I chain α, expressed on monocytes, keratinocytes, fibroblasts, endothelial cells is recognized by intestine δT cells which activate NK cells

MiHA - Minor histocompatibility antigens are receptors on the cell surface of donated organs which induce an immune response by activating CTL-cytotoxic T lymphocytes which results in GvH;

ex.:

H-Y (male donor – female recipient), arrestin, calgranulin, blood group antigens, cell adhesion molecules, differentiation antigens

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MHC I occurs as an α chain (havy chain) composed of three domains:α1,

α2, and α3 connected with β2-microglobulin

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antigen

alpha-1

alpha-2 beta-1

beta -2

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Type of transplant

Autograft – is self-tissue transferred from one body site

to another in the same individual.self-tissue transfreded

Isograft –is tissue transferred between genetically

identical individuals (twins)

Allograft –is tissue transferred between genetically different

members of the same species.

Xenograft - is tissue transferred between different species

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a) and b) direct stimulation - acute rejection,

c) Indirect stimulation (semi-direct) -chronic rejection

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• Hyper acute rejection - few minutes after

transpalnatation, depends on antibodies anti-

antigens expressed on endothelial cells (mostly

MHC and anti-ABO antigens group), activation of

complement cascade

• Acute rejection - weeks and months, depends on

cellular immunity: DTH-reaction, hypereosynophilia,

(10-30% liver transplants) (10-20% kidney

transplants)

• Chronic rejection -different time after transplantation

(years) dependes on humoral and cellular

respoonse. Non immunological causes: ischemia,

atherosclerosis, blood presure, virus infections, age

of recipient

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Immunology,

fromMale Brostoff & Roth

& Roitt

L.B L.Tc

granzmes /perforins

Allograft cells

complement

inflammatory mediators

Vascular occlusion

cell cytotoxic response

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HLA antigens are inherited according to Mendel’s priciples.

A child receives one haplotype from each parent

(complex of genes located on single chromosome inherited together)

Offsprings cells express antigens from both parents

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Mother Father

A B DR A B DR

1 7 4 3 35 1

2 8 3 30 44 11

child I child II child III child IV

A B DR A B DR A B DR A B DR

1 7 4 2 8 3 1 7 4 2 8 3

3 35 1 3 35 1 30 44 11 30 44 11

Phenotypes of HLA haplotype combinations

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HLA typing in transplantology:

- Hematopoietic cell transplantations

- Recipient of liver, kidney, and heart transplant

- Donor of liver, heart, kidney, and pancreas transplant

- HLA-related diseases (analysis of the relationship

between certain HLA alleles and specific disease)

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1. Serological method – utilizes specific reactions between anti-HLA antibodies and HLA antigens on the surface of cells. The assay is carried out by microlimphocytotoxic test

( Terasaki 1964r.)

2. Genetic method – based on PCR techniques to identify the HLA alleles.

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Assay is carried out on a Terasaki plate coated with

monoclonal antibody against specific HLA alleles

Recipient lymphocytes are added onto the plate

and incubated with complement.

Lysed lymphocytes are observed if antigen is present

on the surface of the cells. Reaction is visible after adding a dye (eosin, trypan blue, ethidium bromide).

Percentage of live and dead cells is determined

with inverted contrast-phase microscope with

severity scale response according to NIH

1.Serological method

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Positive reaction Negative reaction

+

barwnik

Tarasaki plate coated with anti-HLA antibodies

+

Recipient cells

+

complement 5 µl

+

Label 5 µl

(acridine orange,

ethidium bromide,

eosin yellowish)

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SSP (Sequence Specific Priming)- DNA replication using the allele-specific primers

SSO (Sequence Specific Oligonucleotide Typing)- hybridization of PCR product with the probes

- Microarrays

- Luminex

RFLP (Restriction Fragment Lenght Polymorphism)

SBT (Sequencing Based Typing) – Sequencing PCR product

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preparation of biological material: disintegration and cell lysis inactivation of cell nucleases with proteinase K separating the nucleic acid from other cellular components preparation of DNA (removal of low molecular weight contaminants)

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PCR -SSP, PCR –SSO methods determine alleles at low resolution

and intermediate resolution at the level of groups of HLA allels,

for example:

A*09

SBT (Sequencing Based Typing)

high resolution method to determine a single allele, Splits for HLA-A9 is

A23 and A24, for HLA-A28 splits are: HLA-A 68 and HLA-A69

for example:

A* 24 : 02 : 01 : 02 L

Genetic methods for HLA typing

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HLA nomenclature (since 2010)

A* 24 : 02 : 01 : 02 L

genetic record allele groups next allele synonyms (synonyms difference

(equivalent of (differences in substitution within intron)

serological the amino acid

allel) sequence) (EPLETS)

Changes in expression:

N- null, an allele which is not expressed

L- low, an allele with a low expression

S- secreted, a gene product which occurs in soluble form only

A-aberrant, an allele of uncertain expression

Q- questionable, different data on an allele expression

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LINKAGE DISEQUILIBRIUM

The difference between the expected and the actual incidence rate

of specific combination of alleles is called LINKAGE

DISEQUILIBRIUM. It sometimes concerns genes = extended haplotype

POSITIVE AND NEGATIVE LINKAGE DISEQUILIBRIUM

Example:

HLA-A-1,-B8,i –DR3 is the most common haplotype in Caucasians

Frequency: 16% for HLA-A*0101, 9% for HLA-B*0801

Frequency of predicted haplotype HLA-A1,B8 is 1,4%, while actual

incidence is 8,8% of population.

Afroamericans in the USA

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MHC molecule is composed of 4-5 regions which

contain different epitopes.

Recipient of a certain MHC recognizes some of these

epitopes as strong (the taboo effect ) and others as poorly immunogenic (permissible mismatch).

CREG (cross - reacting group) –

cross-reactive antibodies which react with epitopes

from HLA-A and -B antigens. Matching for CREG

results in an improved graft outcome and reduces

the requirement for rejection treatment.

„Taboo combinations” - forbidden combinations (difference in eplets) - increase the risk of graft loss.

The mechanism by which „taboo”HLA mismatches diminish allograft survival is still unknow.

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Poland law regulations:

Living donor may be a relative in a straight line,

a sibling, an adopted child, a spouse, or, following

the court consent, an unrelated person,

in case of specific personal reasons.

USA: Donor Recipient Pair Match Program

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1. Donor report

2.Transfer of material from the donor to the laboratory

(lymph nodes, spleen fragment, anticogulated blood)

3.Examination of histocompatibility antigens of the donor

(using polymerase chain reaction-PCR)

4. Isolation of donor lymphocytes

5. Selection of potential recipients (virtual cross-match)

6. Crossmatch

7. Result issue

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Donor lymphocytes 1µl

+

Recipient Serum 1µl

+

complement 5 µl

Positive reaction Negative reaction

+

Label 5 µl

(TMB)

CROSS-MATCH

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Live cells

Dead cells

Points % of dead cells Interpretation

1 1 - 10 negative

2 11 - 20 questionable

negative

4 21- 50 poorly positive

6 51 - 80 positive

8 81 -100 highly positive

0 difficult to evaluate

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Serological cross match is performed before organ transplantation to detect anti-HLA antibodies

Cross match from living kidney donors is performed by flow cytometry method (FACS) to detect complement-dependent or complement-independent antibodies (IgG or IgM class)

SEROLOGICAL CROSS-MATCH IS MANDATORY BEFORE

RENAL TRANSPLANTATION,

AND ITS RESULT HAS TO BE NEGATIVE

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Blood group of donor

Blood group of recipient

identical

compatible

O O A, B, AB

A A AB

B B AB

AB AB -

Cadaveric donors - identical blood group,

Living donors - compatible blood group

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If there are several potential donors with the same or

compatible blood group, a transplanted organ should be from

a donor with the lowest number of incompatible antigens

with the recipient

HLA phenotype of the recipient with blood group A

HLA -A 1,2; B 7,8 ; DR 3,4;

Potential living donors:

blood group

HLA -A 1, 2; B 7, 44; DR 1, 7 ; A

HLA -A 1, 3; B 7, 27; DR 3, 7; O

HLA -A 1, 11; B 7, 15; DR 3, 10; B

HLA -A 1, 2; B 7,8; DR 3, 4; AB

Second donor should not have the same incompatibility as the first donor. Ex.:

The phenotype of the recipient: HLA-A 1.2, B 7.8, DR 3.4;

The phenotype of the first organ donor HLA-A1, 25, B 7, 44, DR 3, 11; Incompatibilities: HLA A 25 B 44 DR 11

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Sera of recipients waiting for transplantation are tested

every 12 weeks to detect the presence of anti-HLA antibodies.

The assay is carried out using microlimphocytotoxic test .

Recipient serum is incubated with lymphocytes of 30 healthy

blood donors in the presence of complement. Lysed cells are

an evidence of anti-HLA antibodies.

„Cold antibodies”- IgM low affinity antibodies, give reaction in room

temperature,

„Warm antibodies” – IgG high affinity antibodies, give reaction in 370C,

responsible for hyperacute rejection

Increase of PRA is caused by blood transfusions, previous transplant,

pregnancy; patients highly sensitive to MHC antigens,

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highly immunized recipients (PRA>80%)

compatibility of 6 HLA –ABDR antigens (or zero mismatches)

paediatric recipients of kidney from a donor under age of 16

Recipients over 60 years from donors over 65 years

recipients of kidney and another organ transplanted

simultaneously

Priority groups of recipients for transplantation, regardless of

the number of preferential points:

The organ recipient is selected based on greater number of

preferential points

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SEROLOGICAL CROSS-MATCH IS MANDATORY

BEFORE RENAL TRANSPLANTATION,

AND ITS RESULT HAS TO BE NEGATIVE