histocompatibility & kidney transplantation

Histocompatibility & kidney transplantation

A. Amirzargar PhDTehran University of Medical

SciencesDept. of Immunology

Major histocompatibility complex

Polymorphism in MHC Class I genes

Variation >1% at a single genetic locus in a population of individualsIn the human population, over 4700 MHC class I alleles have been identified - some

are null alleles, synonyms or differ in regions outside the coding region

2069

1519

1016

4721 alleles(potential alleles)

(Jan. 2011)

10 22 46

Class I

A B C

No

ofpo

lym

orph

ism

s

E F G

Polymorphism in MHC Class II genes

Over 9000 human MHC class II alleles have been identified - some are null alleles, synonyms or differ in regions outside the coding region

3

966

28

145

35144

2000 alleles

4 7 12 9

DR DPDQ DM DO

Class II

A B1 A1 B1A1 B1

No

ofpo

lym

orph

ism

s

A B A B

Tissue MHC class I MHC class II

B cells +++ +++Macrophages +++ ++Dendritic Cells +++ +++

Thymus epithelium + +++

Neutrophils +++ -Hepatocytes + -Kidney + -Brain + -Erythrocytes - -

Accessory molecule / cell interaction

Costimulatory blockade

PRETRANSPLANT MONITORING

• 1-ABO typing • 2- Ab screening and Identification • 3-HLA matching • 4- Biochemical and infectious disease

evaluation • 5-Cardiovascular and pulmonary tests

Matching between Recepient And Donor

A- Tissue typing Determined by 6 antigens located on cell surface encoded

for by the HLA gen located on the short arm of chromosom 6.

Class I antigens (HLA-A and HLA-B) are expressed on the surface of most nucleated cells.

Class II antigen (HLA-DR) are expressed on surface of APC and activated lymphocytes.

These 6 antigens are refered to as major transplant antigens. The match between donor and recepient can range from 0

to six.

Matching between Recepient And Donor

B- Cross matching

• A laboratory test that determines weather a potential transplant

recepient has preformed antibodies against the HLA antigens of the

potential donor. (Donor Lymphocytest +Recepient Serum)

• A Final CM is mandatory

C- Compatible ABO blood group.

HLA matching

• It was first noted 40 years ago that HLA matching between donor and recipient was associated with better transplant and patient survival.

• Matching for the class I HLA-A and HLA-B antigens influenced survival, but matching for the class II HLA-DR antigens had the most powerful effect.

HLA matching

Over the years, there has been an overall improvement in transplant survival

In solid organ transplantation, the effects of HLA matching reported are generally based on matching at the HLA-A, HLA-B, and HLA-DR loci

Survival of first cadaver renal allografts by HLA matchgrade.

• The best transplant survival was achieved in transplants that had no mismatches at HLA-A, HLA-B, and HLA-DR (000 mismatch grade).

HLA-matching • The emphasis on HLA matching is declining, because:

– The overall difference in 10 yrs graft survival rates between the best and worst matched kidney txs remains at about 18%

– Only 15% of candidates can expect to receive HLA-matched kidney because of extreme polymorphism of HLA

– Little time to search for a HLA-matched in the case of deceased donors

– The waiting list is growing more and more, especially with adding of older patients in recent yrs

– Increasingly difficult and expensive to maintain the patients in optimal medical condition

– A disadvantage of allocation based on HLA-matching is that patients with rare HLA types are difficult to transplant

HLA-matching • The emphasis on HLA matching is declining, because:

Effective therapeutics for the successful treatment of AMR, utilizing IVIG either alone or in combination with plasmapheresis

– Several IS agents available that provided more anti-B cell activity

– Strategies for the successful removal or inactivation of DSA prior to Transplantation

• If several medically suitable relatives are available, the decision for donation can be made on the basis of histocompatibility testing—an HLA-identical sibling being the ideal choice

HLA-matching • Factors influencing the relevance of HLA-matching are:

– Donor/recipient age, living or deceased donor, IS protocol, sex, cause of death, cold ischemia time, donor/recipient body size

• Long-term outcome is better for a completely mismatched younger donor/recipient kidney compared to an HLA-matched older one

• Results with HLA-mismatched living donors are generally superior to even well-matched cadaver donors, suggesting that the quality of the kidney and short preservation time outweigh the benefit of matching

HLA-matching

• When stronger IS drugs were used, the HLA effect diminished

• More detrimental effect of cold ischemia time than HLA-matching

• Maybe the HLA-matching has more beneficial for long-term survival of deceased donor allograft

• However in the case of sensitized patients, a zero HLA-mismatched organ may be the only option

Factors affecting TX survivalIn a rigorous multivariate analysis of factors

influencing the outcome of primary deceased donor transplants in a cohort of transplants performed in the United Kingdom from 1986 to 1993,

donor and recipient age, waiting time to transplant, diabetes in the recipient, donor cause of death cold ischemia time, HLA mismatching

Allorecognition pathways

• Presentation of foreign antigen to recipient T cells : direct Direct pathway. Recognition of intact foreign MHC on

donor APC primes CD4 and CD8 recipient T cells. CD4 cells then provide T cell help for the effector function of CD8 cells

• direct response dominates the early posttransplant period

• indirect Indirect pathway. Recipient APCs traffic through transplanted organs, phagocytose allogeneic MHC shed from foreign cells through cell necrosis and apoptosis and present the processed peptides in

the context of self-MHC class II to MHC class II-restricted CD4 T cells. • Semi-direct pathway

Allorecognition

Direct and indirect allorecognition

dAPC

rCD4 TrCD8 T

Direct allorecognition Indirect allorecognition

dAPC rAPC

rCD4 T

B cellP cell

Alloantibodies

Indirecrt pathway Indirect alloantigen presentation results in

alloresponses that are dominated by CD4 T cells. As T cell help for B cells to class switch and

differentiate into antibody secreting plasma cells is provided by CD4 T cells that recognize peptides derived from antigens internalized by B cell surface immunoglobulins

the presence of class-switched alloantibodies is indicative of help provided by indirect pathway T cells

Semi-direct pathway.

Cell-to-cell contact between donor and recipient APC transfer intact membrane components including intact allo-MHC . donor APC can release small vesicles, known as ‘exosomes’ containing

intact MHC , which fuse with the membrane of recipient APCs . Recipient APCs, now chimeric for MHC, stimulate direct pathway CD4 and CD8 responses through intact foreign

MHC indirect responses through processing and presentation of peptides of

foreign MHC acquired from necrotic and apoptotic cell material. the same APC stimulates both CD4 and CD8 cells, linked help can occur.

Semi direct allorecognitiondendritic cells are able to acquire intact MHC–peptide complexes from other dendritic cells and endothelial cells and to present them to alloreactive T cells

Acute rejection appears within the first 3 posttransplant months and affects 30% of cadaveric transplants and 27% of transplants from living donors. Approximately 20% of patients with transplants experience recurrent rejection episodes.

Patients present with decreasing urine output, hypertension, rising creatinine, and mild leukocytosis. Fever, graft swelling, pain, and tenderness may be observed with severe rejection episodes.

The final diagnosis depends upon a graft biopsy.

Chronic Rejection

• Usually apparent from 3 months onwards and detected clinically by gradual deteriation in graft fxn.

• Factors associated with chronic rejection are both immunological + non-immunological.

Allorecognition

SENSITIZATIONRoutes of Sensitization

• An individual can become sensitized to HLA alloantigens as a result of blood transfusion, pregnancy, or previous organ transplantation.

• Transplantation of poorly HLA-matched kidneys can result in allosensitization to the mismatched HLA antigens.

• In the United Kingdom, an audit of the national transplant waiting list showed that 20% of patients waiting for a first transplant were sensitized,

• but 77% of patients waiting for a second transplant were sensitized.

Allosensitization Approximately 20% of pregnant women produce

HLA-specific antibodies to paternally derived fetal HLA antigens.

The use of erythropoietin for the treatment of patients with anemia has decreased the use of blood transfusion in renal patients with a consequent decrease in the number of patients becoming sensitized by this route.

idiopathic HLA antibodies may result from cross-reactivity with infectious agents and are usually IgM.

Antibody Detection and SpecificityDefinition

The aim of an antibody screening strategy is to determine whether the patient has developed HLA alloantibodies and, if so, the antibody class and specificity of the antibodies.

detection and definition of HLA antibodies , can be used to define precisely the specificity of HLA antibodies in serum samples and to elucidate a patient’s sensitization profile.

sensitization

• The sensitization profile : the timing of appearance or disappearance of

antibody reactivity, The specificity and antibody class of HLA

antibodies, and the presence or absence of autoreactive

antibodies.

presensitization Kidney transplantation in patients with donor-specific sensitization

has a significant detrimental impact on graft survival, with most transplants undergoing hyperacute or acute humoral

rejection. Recipient antibodies against donor histocompatibility antigens bind

to the vascular endothelium of the transplanted organ, which disrupts the intercellular junctions and causes release of cell surface heparin sulfate and loss of the antithrombotic state, leading to rapid uncontrollable activation of the thrombotic and complement cascades.

The resultant intravascular coagulation and interstitial hemorrhage can lead to graft destruction within minutes or hours after revascularization.

• Living donor transplants fxn immediately after transplant

• 30% of cadaveric transplants have delayed graft fxn because of more prolonged ischaemic cold preservation. These pts need continued dialysis support until the kidney starts to fxn.

HLA-matching• Despite improvement in IS during the past 50 yrs

ago, the more number of HLA-mismatches, the more incidence of immune complications

• HLA-matching for the class l influenced survival, but for class ll had the most powerful effect

• Matching for HLA-DQ has been variously reported as having either a beneficial effect or no effect on tx outcome

• Matching for HLA-DP has an effect on the tx survival of regrafts, but not of first txs

Allosensitization • Routes of sensitization are blood transfusion, pregnancy, and

previous organ transplantation• Then the immune system is stimulated in a helper T-cell

dependent process → class switching from IgM to IgG. This has 2 implications:

– Upon re-exposure, memory B-lymphocytes proliferate rapidly and differentiate into plasma cells that produce Abs → AMR(Ab mediated rejection) (even if the Ab titer has decreased to a level not detectable with current assays prior to tx )

Allosensitization (con.)• Approximately 20% of pregnant women produce HLA-specific Abs to

paternally derived fetal HLA antigens

• A study in UK showed that 20% of patients waiting for a first tx and 77% of patients waiting for a second tx were sensitized

• The use of erythropoietin for the treatment of anemia has decreased the use of blood transfusion in renal patients with a consequent decrease of sensitized patients

• The use of leukocyte-depleted blood would prevent allosensitization

• There are some idiopathic HLA antibodies which may result from cross-reactivity with infectious agents and are usually IgM

Allosensitization (con.)• There is a casual link/risk/predictive factor between preexisting anti-HLA

Abs and – Hyper acute rejection (which is obvious from 4 decades ago)– More rapid onset of CAN in the case of subclinical rejection– C4d-positive acute Ab-mediated rejection– Mixed cellular and humeral acute rejection and can be treated by IS– Chronic allograft function– Post-tx lymphoproliferative disease (skin cancer related to Anti-HLA-B)

• Anti-class ll Abs found to appear at later times than class l and are associated with late graft function

• Mechanism of injury by Anti-HLA Abs:– IgG Anti-HLA class l: complement lysis– Anti-HLA class ll: leukocyte activation– Anti-MICA: complement lysis of ECs

Ab Detection and SpecificityDefinition

To perform effective antibody screening, samples should be obtained regularly from patients on the waiting list, on at least a 3-monthly basis.

Information about the nature and timing of potential sensitizing events is important in a patient’s sensitization profile.

If sensitization occurs, additional samples are required (e.g., 14 and 28 days after a transfusion with blood products).

Technologies to elucidate patient sensitization profile

• This profile enables the definition of unacceptable HLA-mismatches

• Complement-dependent cytotoxicity (CDC)– The first technique for detection of complement-

fixing IgG and IgM in PRA and in crossmatch test– Dithiothreitol (DTT) for differentiation between

IgG and IgM– Presence/absence as well as the specificity

definition is possible with CDC

Complement-Dependent Cytotoxicity

Complement-dependent cytotoxicity (CDC) was the first technique routinely used for HLA antibody detection and for the cross match test .

In this assay, lymphocyte target cells(lymphocytes isolated from peripheral blood, lymph node, or spleen)

are used to detect complement-fixing IgG1&3 and IgM antibodies present in patient’s serum samples after the addition of rabbit complement.

Lymphocytotoxic cross match test Panel or donor lymphocytes are incubated

with recipient serum in the wells of a microtiter (Terasaki) tray.

followed by the addition of rabbit complement.

After a second incubation period, vital stains (e.g., eosin-y or acridine orange and ethidium bromide) are added, and the wells are viewed using inverted or fluorescent (ultraviolet) microscopy to determine cell viability.

Addition of Lymphoflot

Centifuge for 20 minutes at 1000 x g without braking.The lymphocytes will settle as a white ring at the boundary between the

plasma and the Lymphoflot

White ringof Lymphocytes

Isolation of Lymphocytes

Using a pipette, carefully transfer the ring to a fresh centrifuge tube,fill the tube with Hanks Solution and mix.

Washing of Lyphocytes

Centrifuge for 10 minutes at 230 x g, pour off the supernatant, resuspend the sediment,fill the tube with Hanks Solution and mix. Centrifuge again for 10 minutes at 110 x g.Pour off the supernatant.For HLA-ABC typing, resuspend the lymphocyte sediment in Lymphostabil.The lymphocyte suspension should contain approximately 2000 - 3000 lymphocytes/μl.

Performing MLCT

Thaw the microplates MP at room temperature- Carefully add 1 μl of the adjusted lymphocyte suspension to the HLAreagent into each well just under the oil.- incubate at room temperature (20...24 °C) for:HLA-ABC typing 30 minutesHLA-DR/DQ 60 minutes

CDC

• A high percentage of cell death above background levels is interpreted as a “positive cross match” with the potential to damage a transplanted kidney.

• A positive CDC is C/I

Positive CDC due to auto-ab

During the 1970s, it emerged that not all lymphocytotoxic antibodies that cause a positive crossmatch are specific for donor histocompatibility antigens,

some antibodies display autoreactivity, causing in vitro lysis of the patient’s own cells in the CDC assay.

Auto-Ab a positive crossmatch caused by non-HLA

(autoreactive) lymphocytotoxic antibodies could be safely ignored , with transplant survival rates being equivalent to, transplants with a negative crossmatch.

these autoantibodies as polyreactive IgM,

capable of low-affinity binding to multiple antigens owing to weak electrostatic interactions.

Determination of IgM ab

• IgM antibodies can be differentiated from IgG antibodies by the use of dithiothreitol(DTT).

• DTT reduces the disulfide bonds in the IgM pentamer and consequently renders negative a reaction due to IgM.

• A positive cross match with IgM is safe• A positive cross match due to IgG is C/I

Panel Reactive Ab (PRA)

Serum samples are tested against panels of cells that can be “random” or alternatively can be “selected” to represent the spectrum of HLA types in the population.

The technique can be used for specificity definition, but the results are often expressed as the percentage of the panel to which the sample has reacted (% panel reactive antibody [%PRA]).

PRA its use is now strongly discouraged because the figure entirely depends on the

composition of the panel used for testing. If a patient has a monospecific antibody to a

specificity that is common in a population, and a random panel is used, the %PRA is high,

but if the panel has been carefully selected to cover rare and common specificities, the %PRA value for the same antibody may be low.

Values for %PRA cannot be compared between panels or laboratories.

Technologies to elucidate patient sensitization profile (con.)

• %PRA (which is done by CDC) usage is now strongly discouraged because:– The figure entirely depends on the composition of the panel used for

testing– Values for %PRA can not be compared between panels or laboratories– Only complement fixing Abs are detected– Both HLA and non-HLA Abs are detected– The sensitivity of test depends on the viability of the target cells

• To increase the sensitivity of CDC:– Increase the incubation times– Augmentation with Antihuman Globulin (AHG)

limitations of the CDC technique

Only complement-fixing antibodies are detected, the sensitivity of the technique depends on the

viability of the target cells the particular batch of rabbit complement Both HLA and non-HLA antibodies are detected. Although the use of DTT can differentiate IgM

from IgG antibodies, this does not indicate the specificity of the antibody.

Reactivity resulting from an IgM HLA-specific antibody would be indistinguishable from reactivity of an IgM autoreactive antibody.

B cell cross match Most studies did not differentiate between

nondamaging (autoreactive) and potentially harmful (HLA specific) B lymphocyte–reactive antibodies.

The clinical interpretation of a B cell crossmatch result is impossible without definition of the specificity of the antibody;

most positive donor B cell crossmatches are not caused by HLA-DR–specific antibodies.

most positive B cell crossmatches are caused by non–HLA-specific, usually B cell–autoreactive antibodies

The B cell cross match Ab is not harmful to a transplant.

approaches used to increasethe sensitivity of the CDC test

increasing the incubation times, the wash technique, - unbound serum is washed from the cell

suspension before the addition of rabbit complement, removing the anti complement

factors in the serum. - preferentially detect IgG antibodies because

the lower affinity IgM antibodies become detached during the washing process.

augmentation with antihuman globulin

Crossmatch techniques and their clinical relevance (con.)

• CDC crossmatch (con.)

CDC significantly reduces the incidence of hyper acute rejection

Poor outcome is seen in sensitized and regrafts despite –ve crossmatch

• Predictive value of CDC crossmatch is 80%, the reason is:– ↓ Ab levels below the threshold of CDC definition– Non complement fixing Abs (IgG2, IgG4)– The problem can be resolved by:

» Extended post complement incubation time» Addition of Antihuman globulin (AHG)

Enzyme-Linked Immunosorbent Assays(ELISA)

• The targets in an enzyme-linked immunosorbent assay (ELISA) are soluble HLA antigens coated onto plastic, and this is termed a solid-phase assay

• These commercially available kits have immediate advantages over CDC in that the test does not rely on viable cells,

• only HLA antibodies are detected.


• ELISA, A solid phase assay (Ag coated plates)

ELISA for Ab sreening The overall sensitivity of ELISA is greater than CDC. Two different types of ELISA are routinely used 1- assays to detect the presence or absence of HLA

antibodies that can be used as a prescreen of a patient’s serum sample

2- assays that are designed for antibody specification. The assays have been shown to be reliable for the

detection of IgG antibodies, but less so for the detection of IgM, probably because of the washing steps required and the lower affinity of IgM antibodies.

Ab screening strategy• Because many patients are unsensitized, first

screening the samples is done by a sensitive method to detect presence/absence of HLA Abs

• Then the specificity of positive samples is determined

• Samples should be obtained regularly on at lest 3-monthly basis

• If sensitization occurs, additional samples are required (eg. 14 and 28 days after a transfusion with blood products

ELISA metod Antibody screening using solid-phase binding assays (enzyme-linked

immunosorbent assay [ELISA] 1- Purified HLA proteins (either pooled HLA specificities or single

antigen specificities) coated onto a solid phase (microtiter tray [ELISA] or microparticles [flow cytometry/Luminex]) are incubated with patient serum.

2- HLA-specific antibodies bind to the antigen-coated solid phase, and nonspecific antibodies are washed off.

3,-IgG HLA specific antibodies bound to the antigen-coated solid phase are detected using a conjugated (e.g., alkaline phosphatase [ELISA] or fluorescein isothiocyanate [FITC] [flow cytometry/Luminex]) anti–human IgG secondary antibody

and detected by colorimetric analyses (ELISA, e.g., using p-nitrophenyl phosphate)


• Advantages of ELISA over CDC:– Does not rely on viable cells– Only Anti-HLA abs are detected– More sensitivity of ELISA compared to CDC

• To detect the presence/absence as well as for Ab specification

• Reliable for IgG detection

Flow Cytometry

The original use of flow cytometry in antibody screening was as a test to determine the presence or absence of antibody.

Flow cytometry is more sensitive than CDC and primarily detects IgG antibodies; this offers the advantage that IgM autoreactive antibodies are not detected.

Although less frequent, however, IgG autoreactive antibodies will be detected by this method.

B screening by flowcytometry

• Patient sera + donor lymphocyte + rabbit antihuman FITC conjugate

• Positive control serum• Negative control serum


• Flow cytometry, A solid phase assay (Ag coated microparticles)

Ab screening by flowcytometry


• Flow cytometry advantages:– To detect Ab presence/absence as well as

specification– More sensitive than CDC– Specific microplates coated with a single Ag are

used to elucidate highly complex Ab profiles


• Luminex, A solid phase assay– Uses multiplexed microparticles and allowes the

simultaneous detection and specification of multiple Abs in a serum sample

Strategies for transplanting sensitized or likely sensitized patients

• Ab removal (con.)– High-dose of IVIG for living and deceased donors when there is a

preformed low-titer < 1/16– The main difficulty with high-dose IVIG is that not all patients

respond and not all anti-HLA antibodies are suppressed equally

– Plasmapheresis combined with low-dose IVIG for living donors in the case of higher titer > 1/16

• allows for more consistent and universally reliable removal of anti-donor Ab• As a rule of thumb, each PP/IVIG treatment reduces the DSA titer by one dilution

– Ab removal is slower In the case of:• higher number of mismatches and previous txs• Anti DRB3/4/5> Anti DRB1, DQB> Anti class l• +ve CDC> +ve flow, -ve CDC

– Ab removal usually occurs before tx, so monitoring is necessary to indicate additional removal after tx

histocompatibility & kidney transplantation

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