TRANSPLANTATION IMMUNOLOGY

 TRANSPLANTATION
• OF CRITICAL IMPORTANCE HAS BEEN THE GROWING KNOWLEDGE OF THE IMMUNOLOGIC
MECHANISMS OF GRAFT REJECTION AND GRAFT-VERSUS-HOST DISEASE (GVHD), IN
PARTICULAR THE ROLE OF HUMAN LEUKOCYTE ANTIGENS (HLAS) AND THE DEVELOPMENT OF
PHARMACOLOGICAL AGENTS THAT PROMOTE GRAFT SURVIVAL BY INTERFERING WITH VARIOUS
COMPONENTS OF THE IMMUNE SYSTEM.
• THE HLA SYSTEM IS THE STRONGEST IMMUNOLOGIC BARRIER TO SUCCESSFUL ALLOGENEIC
ORGAN AND HSC TRANSPLANTATION.
• THE HLA SYSTEM CONSISTS OF CELL SURFACE PROTEINS THAT PLAY A CENTRAL ROLE IN
THYMIC EDUCATION OF T LYMPHOCYTES, INITIATION OF ADAPTIVE IMMUNE RESPONSES,
AND REGULATION OF OTHER IMMUNE SYSTEM COMPONENTS. HLA PROTEINS ARE FOUND ON
THE SURFACE OF ALMOST ALL NUCLEATED CELLS AND ARE ANTIGENICALLY VERY DIVERSE IN
THE HUMAN POPULATION

• GENETICS
• HLA GENES ARE INHERITED AS HAPLOTYPES FROM PARENTAL CHROMOSOMES
• A HAPLOTYPE IS A GROUP OF CLOSELY LINKED ALLELES ON A SINGLE CHROMOSOME;
FOR EXAMPLE, HLA-A1, HLA-CW7, HLA-B8, HLA-DR3, AND HLA-DQ2.
• OFFSPRING RECEIVE ONE MATERNAL AND ONE PATERNAL HLA HAPLOTYPE
• BASED ON MENDELIAN INHERITANCE
• THERE IS A 25% CHANCE THAT ANY TWO SIBLINGS WILL INHERIT THE SAME TWO
HAPLOTYPES
• THERE IS A 50% CHANCE OF THEM BEING HLA HAPLOIDENTICAL
• THERE IS A 25% CHANCE OF THEM BEING HLA NONIDENTICAL

• POLYMORPHISM
• A CARDINAL FEATURE OF THE GENES ENCODING HLA PROTEINS IS THE EXTENSIVE
DEGREE OF ALLELIC POLYMORPHISM.
 • POLYMORPHISM REFERS TO THE PRESENCE OF TWO OR MORE DIFFERENT GENETIC
COMPOSITIONS AMONG INDIVIDUALS IN A POPULATION.
• THE HLA SYSTEM IS THE MOST POLYMORPHIC GENETIC SYSTEM IN HUMANS; MANY
HLA GENES EXIST IN THE POPULATION AND NUMEROUS COMBINATIONS OF THESE
GENES ARE POSSIBLE IN INDIVIDUALS.

• MINOR HISTOCOMPATIBILITY COMPLEX

• RESEARCHERS IDENTIFIED A SECOND SET OF TRANSPLANTATION ANTIGENS BASED
ON STUDIES IN MICE AND HUMANS. THESE STUDIES DEMONSTRATED TISSUE REJECTION
IN MHC-IDENTICAL TRANSPLANTS AND DEVELOPMENT OF GVHD IN HLA-IDENTICAL
SIBLING HSC TRANSPLANTS.
• mHAS ARE NON-HLA PROTEINS THAT DEMONSTRATE VARIATION IN THE AMINO
ACID SEQUENCE BETWEEN INDIVIDUALS.
• SLOWER PACE OF REJECTION

• ABO ANTIGENS

• THE ABO SYSTEM IS THE ONLY BLOOD GROUP SYSTEM THAT AFFECTS CLINICAL
TRANSPLANTATION. ABO BLOOD GROUP INCOMPATIBILITY IS A BARRIER TO SOLID-
ORGAN TRANSPLANTATION BECAUSE THESE ANTIBODIES CAN BIND TO THE
CORRESPONDING ANTIGENS THAT ARE EXPRESSED ON THE VASCULAR ENDOTHELIUM
 • HYPERACUTE REJECTION - OCCURS WITHIN MINUTES TO HOURS AFTER THE
VASCULAR SUPPLY TO THE TRANSPLANTED ORGAN IS ESTABLISHED BECAUSE OF
COMPLEMENT ACTIVATION
• TRANSPLANTATION APPROACHES USING PLASMA EXCHANGE AND INTRAVENOUS
IMMUNOGLOBULIN ADMINISTRATION HAVE ALLOWED SUCCESSFUL
TRANSPLANTATION OF KIDNEYS FROM ABO-INCOMPATIBLE DONORS.
• KILLER IMMUNOGLOBULIN LIKE RECEPTORS
• KIRS ARE ONE OF SEVERAL TYPES OF CELL SURFACE MOLECULES THAT REGULATE THE
ACTIVITY OF NK LYMPHOCYTES.
• THE KIRS CONTAIN ACTIVATING AND INHIBITORY RECEPTORS THAT VARY IN NUMBER
AND TYPE ON ANY INDIVIDUAL NK CELL.
• THE BALANCE OF SIGNALS RECEIVED BY THE ACTIVATING AND INHIBITORY RECEPTORS
REGULATES THE ACTIVITY OF THE NK CELL
• THE REGULATORY ROLE OF KIRS HAS BEEN EXPLOITED IN HAPLOIDENTICAL STEM CELL
TRANSPLANTATION.
• STEM CELL DONORS HAVE BEEN SELECTED FOR RECIPIENTS WHO LACK A
CORRESPONDING CLASS I MHC PROTEIN FOR THE DONOR’S INHIBITORY KIR TYPE.
 IMMUNOSUPPRESSIVE AGENTS
• CORTICOSTEROIDS
• ARE POTENT ANTI-INFLAMMATORY AND IMMUNOSUPPRESSIVE AGENTS
USE FOR IMMUNOSUPPRESSION MAINTENANCE
• AT HIGHER DOSES, THEY ARE USED TO TREAT AR EPISODES. STEROIDS ACT BY
BLOCKING PRODUCTION AND SECRETION OF CYTOKINES, INFLAMMATORY
MEDIATORS, CHEMOATTRACTANTS, AND ADHESION MOLECULES
• ANTIMETABOLITES
• ANTIMETABOLITES INTERFERE WITH THE MATURATION OF LYMPHOCYTES
AND KILL PROLIFERATING CELLS.
• AZATHIOPRINE WAS THE FIRST SUCH AGENT EMPLOYED. IT HAS BEEN
REPLACED IN LARGE PART BY MYCOPHENOLATE MOFETIL.
• CALCINEURIN INHIBITORS
• CYCLOSPORINE AND FK-506 (TACROLIMUS) - ARE COMPOUNDS THAT BLOCK
SIGNAL TRANSDUCTION IN T LYMPHOCYTES, RESULTING IN IMPAIRED
SYNTHESIS OF CYTOKINES SUCH AS IL-2, IL-3, IL-4, AND INTERFERON- GAMMA
• RAPAMYCIN (SIROLIMUS) - IS AN AGENT THAT INHIBITS T-CELL
PROLIFERATION BY BINDING TO SPECIFIC INTRACELLULAR PROTEINS,
INCLUDING MAMMALIAN TARGET OF RAPAMYCIN (MTOR)
• MONOCLONAL ANTIBODIES
• TWO ANTI-CD25 MONOCLONAL ANTIBODIES ARE AVAILABLE FOR USE IN
TRANSPLANT PATIENTS.
• BASILIXIMAB AND DACLIZUMAB BOTH BIND THE CD25 (IL-2 RECEPTOR) AND
THUS INTERFERE WITH IL-2– MEDIATED T-CELL ACTIVATION
 • POLYCLONAL ANTIBODIES
• THYMOGLOBULIN IS AN ANTITHYMOCYTE ANTIBODY PREPARED IN RABBITS
AND ATGAM IS A POLYCLONAL ANTISERUM PREPARED FROM THE
IMMUNIZATION OF HORSES.
• BOTH ARE POTENT IMMUNOSUPPRESSIVE AGENTS THAT DEPLETE
LYMPHOCYTES FROM THE CIRCULATION
• MAJOR DRAWBACK: DEVELOPMENT OF SERUM SICKNESS BECAUSE OF
FOREIGN IMMUNOGLOBULINS

 CLINICAL GISTOCOMPATIBILITY TESTING

• HLA TYPING
• HLA TYPING IS THE PHENOTYPIC OR GENOTYPIC IDENTIFICATION OF THE HLA
ANTIGENS OR GENES IN A TRANSPLANT CANDIDATE OR DONOR.
• FOR CLINICAL HLA TESTING, PHENOTYPES OR GENOTYPES OF THE CLASSICAL
TRANSPLANT ANTIGENS OR GENES ARE DETERMINED (HLA-A, HLA-B, HLA-CW,
HLA-DR, HLA-DQ)
• HLA PHENOTYPING
• COMPLEMENT DEPENDENT CYTOTOXICITY (CDC) – PANELS OF ANTISERA OR
MONOCLONAL ANTIBODIES THAT DEFINE INDIVIDUAL OR GROUPS OF
IMMUNOLOGICALLY RELATED HLA ANTIGENS ARE INCUBATED WITH
LYMPHOCYTES FROM THE PERSON TO BE HLA TYPED IN SEPARATE WELLS OF A
MICROTITER PLATE.
• EACH WELL OF THE PLATE CONTAINS A DIFFERENT ANTIBODY
• A COMPLEMENT REAGENT DERIVED FROM RABBIT SERUM IS ADDED TO EACH
WELL. IF THE CELLS POSSESS THE HLA ANTIGEN DEFINED BY THE ANTIBODY IN
THAT WELL, COMPLEMENT IS ACTIVATED AND THE CELLS ARE KILLED.
• A VITAL DYE SUCH AS EOSIN RED OR TRYPAN BLUE IS ADDED TO DISTINGUISH
LIVE CELLS FROM DEAD CELLS WHEN THEY ARE VIEWED MICROSCOPICALLY.
• DEAD CELLS = APPEAR COLORED
• LIVE CELLS = COLORLESS
• HLA GENOTYPING
• MOLECULAR-BASED HLA GENOTYPING METHODS USE POLYMERASE CHAIN
REACTION (PCR)-BASED AMPLIFICATION OF HLA GENES FOLLOWED BY ANALYSIS
OF THE AMPLIFIED DNA TO IDENTIFY THE SPECIFIC HLA ALLELE OR ALLELE
GROUP
• 3 TYPES OF PCR TESTING
• PCR-SSP (SINGLE SPECIFIC PRIMER)
• PCR-SSOP
• SBT

• PCR SSP
• THE MOST COMMON APPROACHES FOR ANALYSIS INVOLVE PCR
AMPLIFICATION OF HLA GENES WITH PANELS OF PRIMER PAIRS, EACH
OF WHICH AMPLIFIES SPECIFIC ALLELES OR RELATED ALLELE GROUPS
(PCR-SSP).

• ONLY THOSE PRIMER PAIRS THAT BIND PERFECTLY TO THE TARGET

GENE RESULT IN DETECTION OF AN AMPLIFICATION PRODUCT
• AMPLIFICATION IS DETECTED BY AGAROSE GEL ELECTROPHORESIS.
 • PCR SSOP (SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBE)
• A SECOND COMMON APPROACH FOR HLA GENOTYPING IS TO PERFORM PCR-
SSOP. THIS INVOLVES A SINGLE PCR REACTION THAT WILL AMPLIFY ALL HLA
GENE VARIANTS AT A SPECIFIC LOCUS (REFERRED TO AS A GENERIC
AMPLIFICATION).
• THE AMPLIFIED GENE IS THEN SUBJECTED TO HYBRIDIZATION WITH A PANEL
OF DNA PROBES, EACH SPECIFIC FOR A UNIQUE HLA ALLELE OR ALLELE GROUP.
• ONLY THOSE PROBES THAT SPECIFICALLY HYBRIDIZE TO THE AMPLIFIED DNA
WILL BE DETECTED

• PCR SBT (SEQUENCE BASED TYPING)

• A THIRD COMMON METHOD FOR HLA GENOTYPING IS SBT, WHICH INVOLVES
SEQUENCING OF PCR- AMPLIFIED HLA GENES. SBT IS TYPICALLY CARRIED OUT
USING SANGER DIDEOXY CHAIN TERMINATOR SEQUENCING.
• A GENERIC AMPLIFICATION OF THE HLA GENE OF INTEREST IS CONDUCTED,
FOLLOWED BY A SEQUENCING REACTION USING DIDEOXY NUCLEOTIDES.
• THE DIDEOXY TERMINATORS ARE FLUORESCENTLY LABELED.
INCORPORATION INTO THE SYNTHESIZED DNA MOLECULE IS DETECTED USING
AUTOMATED DNA SEQUENCERS WITH FLUORESCENT DETECTORS.
• THE SEQUENCE OF THE TARGET GENE IS COMPARED WITH AN HLA
SEQUENCE
DATABASE TO DETERMINE THE SPECIFIC HLA ALLELE FOR THE PATIENT.
• SBT FOR HLA TYPING IS CONSIDERED THE GOLD STANDARD AND IS ABLE TO
DETECT NEW ALLELIC VARIANTS

• HLA AB SCREENING, IDENTIFICATION AND CROSSMATCHING

• ANTIBODIES TO HLA ANTIGENS CAN BE DETECTED IN CANDIDATES AND
RECIPIENTS OF SOLID-ORGAN TRANSPLANTS BY PERFORMANCE OF A
CROSSMATCH TEST.
• THESE ANTIBODIES CAN DEVELOP IN RESPONSE TO MULTIPLE BLOOD
TRANSFUSIONS OR TO PRIOR HLA- MISMATCHED TRANSPLANTS.
• FLOW CYTOMETRY
• ANOTHER APPROACH FOR ANTIBODY DETECTION AND IDENTIFICATION IS
FLOW CYTOMETRY.
• ANTIBODY IN PATIENT SERUM CAN BE INCUBATED WITH BEADS THAT ARE
COATED WITH PURIFIED HLA ANTIGENS, EITHER FROM A POOL OF DONORS, AN
INDIVIDUAL DONOR, OR A SINGLE PURIFIED OR RECOMBINANT HLA PROTEIN.
• BEADS COATED WITH POOLED HLA PROTEINS ARE A SENSITIVE QUALITATIVE
SCREEN FOR THE PRESENCE OF
HLA ANTIBODY BECAUSE THEY WILL DETECT ANTIBODIES TO THE MAJORITY OF
COMMON HLA ANTIGENS.