HLA TYPING, CROSSMATCHING AND TRANSPLANTATION IN SENSITIZED PATIENTS

DM SeminarDr. Vishal Golay

10/8/2011

MHC Class I molecule with bound peptide

First cadaveric kidney transplantation-1950 (graft failed after 10 months).

First living donor kidney transplantation was performed on December 23rd 1954 in Boston, US by Dr. Joseph Murray between the identical Herrick twins. He received a Nobel prize in 1990 for this work.

TOPIC OVERVIEW

Basic concepts about the MHC.

Methods of HLA typing.

Cross matching and detecting sensitization.

Transplantation in sensitized patients.

MHC and HLA TYPING

THE HUMAN MHC

Located on chromosome 6p21.31 Divided in three regions: Class I, II & III

Classic genesClassic genes

Non-classic genes: E,F,GMICA, MICBPseudogenes

Non-classic genes: DM, DOPseudogenes

HSP

HLA CLASS I AND CLASS II ANTIGENS

• Monomer with non-covalently associated subunit (b2m)

• Presents antigenic peptides to CD8+ T cells

• Expressed by most somatic cells

• Level of expression varies between tissues

• High expression on lymphocytes, inducible on somatic cells

• Heterodimer

• Presents antigenic peptides to CD4+ T cells

• Restricted expression on antigen presenting cells (B cells, monocytes/ macrophages, dendritic cells)

• Inducible on other cells (epithelium, endothelium)

STRUCTURE OF MHC MOLECULE

Peptide binding clefts

MHC Class IBinds to 9-11 amino acids

MHC Class IIBinds to 13-30 amino acids

ALLOANTIGEN PRESENTATION BY MHC Direct Antigen Presentation: Donor APC + TCR of Recipient T

cell Donor MHC

Indirect Antigen Presentation: Recipient APC + TCR of Recipient T

cell Processed Donor MHC

Indirect pathway generally activates CD4 T cells. Acute rejection of allograft is primarily dependent on direct

allorecognition

HLA NOMENCLATURE

In case of renal transplantation, if only HLA-A, B & DR are taken, there are 88 recognized antigens encoded by >2200 distinct alleles.

The genes are prefixed by the letter HLA followed by the loci.

HLA typing can be done by two methods: Serological typing DNA typing

SEROLOGICAL HLA TYPING

Developed by Terasaki and McClelland in 1964

Based on the detection of expressed HLA molecules on the surface of separated T cells (HLA class I) and B cells (HLA class II) using panels of antisera, usually obtained from multiparous women in a complement dependent cytotoxicity test.

Requires sufficient live lymphocytes and panel of sera

HLA antigen identified by serological methods were named in the order in which they were recognized. Eg: A1, A2, A3 and so on

DNA BASED HLA TYPING

The nomenclature has been modified by trying to associate alleles to antigens wherever possible, after DNA based tests became available.

A four digit designation was developed. The antigen makes up the first two digits, and the allele make up the remaining two.

For example: HLA-A*0101 Allele number

Test performed by DNA method Serologic antigen

HLA NOMENCLATURE

In case of the HLA-DR antigen, they are distinguished by their β1 subunit. Therefore, the 1st allele of DR1 will be HLA-DRB1*0101

The most common HLA antigen is A2 (50% of world population) with allelic variations. HLA-B 54 is almost exclusively found in Japan and neighboring areas and HLA-A36 is common in blacks.

BMT requires allele level matching to prevent development of GVHD. However, allele level mismatches have no substantial effect on renal graft survival rates.

DNA BASED HLA TYPING METHODS PCR-Sequence Specific Probes(SSP):

DNA amplification using group specific primers and detecting the amplified product by gel electrophoresis.

Cumbersome and unsuited for typing large number of samples.

PCR-Sequence Specific Oligonucleotide Probes (SSOP): Amplification using group/allele specific primers and then

detecting the hybridization of oligo probes with enzymatic or fluorescent markers.

Useful for typing samples in batches

Sequence Based typing: Uses gene-specific primers and so is used for allelic level

typing for SCT and also to type new alleles

ADVANTAGES OF THE DNA BASED TESTS Greater accuracy and reproducibility of the reagents.

Viable lymphocytes are not required and typing can be performed on any tissue.

The oligonucleotide reagents are more easily standardized and controlled.

Greater accuracy of the test.

Difficult HLA specificities against whom antisera is not available can also be identified using DNA based tests.

INTERPRETATION OF AN HLA TYPING REPORT

Haplotypes are combination of alleles in different loci that are inherited together. Occasionally (in 2% cases) crossover occurs between the A and B locus resulting in a new haplotype.

Mother Father A B DR A B DR 1 8 17 3 13 11 2 44 4 29 44 7

Sibling 1 Sibling 2 Sibling3 Sibling4A B DR A B DR A B DR A B

DR2 44 4 2 44 4 1 8 17 1 8

173 13 11 29 44 7 3 13 11 29 44

7

Mendelian inheritence25%-two haplotype match25%-one haplotype match25%-zero haplotype match

INTERPRETATION OF AN HLA TYPING REPORT

Example: HLA phenotypeIndividual 1 → A1,A24; B8, B44; DR4, DR15Individual 2 → A1, A3; B7, B8; DR4, DR12

Common Alleles: A1, B8, DR4

If the two individuals are related(siblings, parent-child), these three shared alleles is most probably the common shared haplotype and thus this should be interpreted as: “ONE HAPLOTYPE MATCH”

If they are unrelated then there are three shared alleles. This should then be interpreted as : “THREE ANTIGEN MATCH or THREE NATIGEN MISMATCH”

PRACTICAL ISSUES IN HLA TYPING

Incomplete HLA specificity;

Example: 1. A2, ---; B27, B13; DR17, DR4 2. A2, A3; B8, B14; DR17, ----

In particular situation, This commonly means that the individual is homozygous in

the A and DR loci. If 1 is donor for 2, this should be described as zero A, two B

and one DR mismatch. If 2 is donor for 1, this should be described as one A, two B

and zero DR mismatch.

A2

DR 17

HLA AND TRANSPLANTATION

HLA matched kidneys with longer cold ischemia time fared better than HLA mismatched with lesser ischemia time.

However, it was found that HLA matched kidneys from an ECD fared poorly compared to unmatched standard criteria donor putting the issue of achieving an identical match in doubt.

It was also found that unmatched living kidneys had longer graft survival than a fully matched cadaveric kidney.

The survival of grafts from an unrelated donor was comparable to grafts from a one-haplotype matched sibling/parent to child (64% at 10 years).

Please refer to figure in Danovitch

ANTI HLA ANTIBODIES

Antibody detection tests encompasses two broad categories

1. Tests that detect the level of circulating Anti-HLA antibodies ie. detecting sensitization.

2. Test that cross-match between the donor and the recipient.

CDC-BASED ASSAYS

Lymphocytes (T cells, usually)

Patient serum

+ rabbit complement

Red = deadGreen = alive

LIMITED SENSITIVITY NIH extended CDC:

ENHANCED CDC-BASED ASSAYS

Enhance with anti-human

globulin

(AHG)

Lymphocytes

Patient serum

+ rabbit complement

METHODS(CROSS MATCH)

Complement Dependent Lymphocytotoxicity (CDC): IgG antibodies directed against HLA class I (on both B

and T cells) are the most important. IgM antibody that shows reactivity at 4˚C and removed

by heating to 55˚C or treating with dithiotreithol(DTT) can be ignored.

Flow Cytometry Cross Match: Patients serum+target cells →washed and incubated with

anti-CD3(monoclonal mouse), Anti CD-19 and CD-20 antibodies and AHG conjugated with fluorescent dyes .

The fluorescence is measured by a flow cytometer and recorded in MCS(median channel shift).

SOLID PHASE ASSAYS

These assays use HLA antigens on various platforms. The tests fall into three general categories:

1. Using mixture of the HLA class I or class II antigens from several individuals.

2. Using class I or class II HLA antigens for a single individual.

3. Using a single HLA antigen produced through recombinant DNA technology.

Flow Cytometry(Latex beads)

ELISA

Anti-IgG-PE

Anti-IgG-FITC

HLA alloantibody

Luminex Array(Polystyrene beads)

Anti-IgG

Gebel and Bray. Transplantation Reviews 20: 189-194, 2006

SOLID PHASE ASSAYS

Purified HLA molecules are immobilized onto the surface of the solid surfaces: higher sensitivity than CDC-based assays

Used in HLA ab screening and identification of donor specific antibody

Sensitivity of DSA identification methods

DSA levels

Very high

HighModerateLowDSA negativ

e

Luminex SAB

Flow cytometry

CDC-AHG

CDCELISA

TRANSPLANTATION IN SENSITIZED PATIENTS

The definition of sensitization is variable. One definition defines it as moderately sensitized when PRA is >20% and highly sensitized when the PRA >80%. This is however variable between centres.

The definition of sensitization has undergone a paradigm shift after the adoption of a new concept called CPRA(calculated panel reactive antibodies) by the UNOS in 2009.

INTRODUCTION

Sensitization to HLA antigens occurs mainly through- Pregnancy Blood transfusions Previous organ transplantations

Sensitization significantly increases the waiting time for kidney transplantation.

This significantly increases the number of patients dying each year in want of a transplant (15-20% mortality/yr on HD)

Approx. 35% of patients have PRA>0% and 15% have PRA >80%.

Approx 17% of patients on waiting list have had a previous transplant.

INTRODUCTION

Previously, a positive DSA or a positive cross match was considered a contraindication to transplantation.

In the last decade, advances in diagnostics, pathology and therapeutics have made transplantation in sensitized patients possible.

Most of the current protocols are a modification of high-dose intravenous Ig (IVIG) initiated at Cedars

Sinai Medical Center or

plasmapheresis (PP) with low-dose IVIG initiated at Johns Hopkins Hospital.

Donor typing:

A1,A2 B7,B8

Recipient typing:

A1,A3 B8, B52

+

Anti-A2

Anti-B7

A single sensitizing event can lead to multiple antibody specificities

Anti A2

Anti B7

A28A23

A69

A68

B57

A24B58

B27B60

B61

B13

B42

B54

B55

B48

B41

B47

+

Sensitization to multiple HLA antigens from a single transplant

Due to shared epitopes with donor HLA

PLASMAPHERESIS AND IMMUNOADSORPTION

Both these techniques are aimed at removing alloantibodies.

PP removes all plasma proteins including Ig. IA includes a sepharose-bound staphylococcal protein A

column with a high affinity for binding IgG and developed to remove IgG antibodies.

The advantages of IA over PP include specificity, a greater amount of antibody removal, and the elimination of the need to replace large volumes of plasma.

PLASMAPHERESIS AND IMMUNOADSORPTION

One 3- to 4-hour treatment course with IA results in a 15% to 20% reduction and three to six courses of treatment result in 90% reduction in plasma IgG levels.

However, anti- HLA antibody titers rebound and return to baseline levels within a few weeks after the completion of PP or IA.

Most of the IA columns manufactured in USA and Japan are not approved by the FDA

INHIBITION OF ANTIBODY PRODUCTION & COMPLEMENT INHIBITORS

Based on inhibiting antibody production by B-cells and plasma cells. Rituximab (Anti-CD 20):

off label use in desensitization protocol/treatment of AMR as a single dose of 375mg/m².

Plasma cells and pro-B cells do not have surface CD-20 decreasing the efficacy. B-cell recovery takes 6-12 months.

Bortezomib (Proteasomal Inhibitor): Induces apoptosis of plasma cells. Given in dosage of 1.3 mg/m² and repeated on days 4, 8, and 11 intravenously

over 3 to 5 seconds.

Eculizumab: It is a monoclonal antibody against C5. Binds to C5 protein with high affinity, thereby inhibiting its cleavage to C5a and

C5b and preventing generation of the terminal complement complex C5b-9.

IVIG & SPLENECTOMY

IVIg: Multiple effects on the immune system. Has been a part of the desensitization protocols for ABO incompatible

and cross-match positive patients and also for the Rx of AMR. The dose of IVIG varies among protocols from 100 mg/kg to 2.0 g/kg

and is usually given during a hemodialysis session or as a slow infusion in nondialysis patients.

Splenectomy: It has been used in desensitization protocols of ABO-incompatible

kidney transplant recipients. Splenectomy removes a major source of lymphocytes, including

antibody-secreting B cells, B cell precursor cells, and plasma cells. It has also been used in the treatment of refractory AMR

DESENSITIZATION PROTOCOLS

PP WITH LOW DOSE IVIG This protocol was first used in 1998 at Johns Hopkins Hospital in

cross-match incompatible living-donor kidney transplant candidates

Patients received PP and CMVIg at 100 mg/kg after each PP along with tacrolimus and MMF treatment for desensitization starting 2 to 3 weeks before transplantation.

The start of therapy depended on the DSA titers so that patients with low titers (<1:8) required two to three sessions of PP, whereas patients with higher titers (1:128) had six to 10 sessions.

Patients received transplantation if the cross-match became negative with the use of daclizumab induction therapy and continued for two to five sessions of PP after transplantation depending on the titers of DSA.

Pediatr Transplant 8: 535–542, 2004

PP WITH LOW DOSE IVIG Johns Hopkins Hospital used this protocol but all 4 patients

had AMR which responded to Rx and had 100% 1 yr graft survival.

This was slightly modified by using OKT3 induction. Lower AMR (36%) and 100 % I yr graft survival

Transplantation 70: 1531–1536, 2000

Mayo Clinic used ATG induction with rituximab and splenectomy with not so favorable outcomes.

Brigham and Women’s Hospital Transplant Center & Univ of Illinois also used this protocol but the AMR rate was high.

The latest report is from Univ of Maryland. Their patients, with this protocol and ATG/OKT3 induction had lower acute AMR (12%) compared with the studies discussed above, but patient survival and graft survival at 4 years were only 78% and 66%, respectively.

Am J Transplant 9: 536–542, 2009

HIGH-DOSE IVIG

First pioneered in Cedars Sinai Medical Centre, IVIg @2g/kg was given till the crossmatch was negative and then transplanted. The primary advantage was its applicability in deseased-donor kidney transplant.

This group also compared ATG + IVIg vs those without these, in CDC- but low level DSA+ pts and found significantly lower AMR when agents were used

COMPARISON OF PP/LOW DOSE IVIG VS HIGH DOSE IVIGPP/low-dose IVIG and rituximab demonstrated more success in abrogating positive cross-match and lower acute rejection rates, but no regimen was completely effective in preventing AMR.

CDC T cell CXM+

CDC T cell CXM-CDC B cell and/or flow cytometry T and/or B cell CXM+

The acute rejection rate decreased to 7% from 44% in the following 14 patients receiving PP.

HIGH AMR RATE (PROBLEM WITH DESENSITIZATION PROTOCOLS)According to a recent review of studies published between 2000-

2010, The patient and graft survival were 95% and 86%,

respectively, at a 2-year median follow-up.

Despite acceptable short term patient and graft survivals, the AMR was 36% and acute AMR was 28%, which is significantly higher than in nonsensitized patients (<10%).

The acute AMR rate was high regardless of which PP/low-dose IVIG or high-dose IVIG was applied or which types of induction agents were used (daclizumab, ATG, or alemtuzumab).

The addition of rituximab or splenectomy did not appear to decrease the acute AMR rate. Clin J Am Soc Nephrol 6: 922–936, 2011

SUMMARY AND MESSAGE

Acute, subclinical, and chronic AMR rates are unacceptably high with current desensitization protocols despite acceptable short time graft survival.

There is also a concern about lower long term graft survival compared with nonsensitized patients.

The strongest predictor for development of AMR is the pretransplant strength of DSAs and those patients should be evaluated by Luminex single-antigen beads MFI and flow cytometry MCS values.

Patients with strong DSA’s should not be considered for transplantation

SUMMARY AND MESSAGE

There are presently no clear scientific data to recommend a certain protocol, but PP should be used in patients with strong DSAs to decrease the titers, and higher dose IVIG might have more immunomodulatory effect.

All cross-match incompatible living-donor candidates should be considered first for paired exchange programs.

Patients with low level DSA’s can be Tx with IVIg and induction with ATG/Campath

The role that novel agents such as bortezomib and eculizumab will play is not clear and requires more clinical experience

SO IS DESENSITIZATION REALLY HELPFUL?

THANK YOU