PRACTICAL TRANSPLANT IMMUNOLOGY Dr Nisar Anwar FRCP(Edin),Accredited Renal Physician (UK) Professor & Head : Department Of Nephrology KTH, Peshawar

TERMINOLOGY OF TRANSPLANTATION Auto graft (e.g. skin grafts for burns, etc.) Syngraft (genetically identical twins; some stem cell therapies: nuclear transfer, iPS approaches) – No immunologic rejection for these two types
 Allograft (genetically distinct member of same species; almost all medical transplants) Xenograft (different species; attractive due to shortage of organs for transplants) – Very severe rejection problems.

IMMUNOLOGY OF TRANSPLANTATION Major barrier to organ/tissue transplantation is immunological Immune system recognizes transplant as foreign and destroys it Memory and specificity have properties of adaptive immune response Memory is transferred by T cells (antibodies also can contribute).

HISTOCOMPATIBILITY ANTIGENS ABO blood group antigens
Pre-existing antibodies to A or B antigens lead to “hyperacute rejection” if not matched properly Major histocompatibility antigens Encoded by the MHC, originally discovered for their role in skin graft rejection in mice Minor histocompatibility antigens Allelic variants of normal cellular proteins.

Transplantation antigens Major Histocompatibility Complex (MHC): Class I antigens: constitutively expressed on surface of most cells Class II antigens: expressed on cells of lymphoid system Expression of MHC molecules can be up regulated by ischemia, etc. nomenclature HLA (human) class I: A, B, C; class II: DR, DQ H-2 (mouse) class I: K, D, L; class II: IA, IE 5

MHC NOMENCLATURE Identified & characterized over 40 years ago Antigens were identified using antisera obtained from multiparous women, new antisera can split HLA antigens in to narrower specificities DNA based techniques are more accurate in describing alleles Frequency of antigens, alleles and combinations vary considerably Most common HLA antigen A2 is found in 50% of the population.

MHC STRUCTURE

MHC CHARACTERISTICS Polymorphism Genes are co-dominantly expressed Each MHC locus can express any one of hundreds of different molecules Set of different MHC molecules or alleles expressed on one chromosome is called a Haplotype A Genotype is the sum of two haplotypes Genes are co-dominantly expressed An individual expresses alleles from both chromosomes at each locus

INHERITANCE Each parental chromosome 6 provides a haplotype or linked set of MHC genes to the offspring. Haplotypes are usually inherited intact from each parent The child carries one representative antigen from each of class I & class II loci of each parent A child is a one haplotype match to each parent HLA haplotypes are inherited in a mendelian fashion 25% siblings will share two haplotype match, 50% will share one haplotype and 25% will have zero match

HLA-sensitization Exposure to non-self HLA antigens can cause production of HLA-directed antibodies Common causes of HLA-sensitization include blood transfusions, pregnancies, previous transplants In infants, tissue patches implanted during cardiac surgery cause sensitization 11

Does MHC (HLA) ‘Matching’ Prevent Rejection? Reduces rejection but there are still ‘minor histocompatibility antigens’ (MiHA) MiHA are probably polymorphisms affecting peptides in the grooves But we cannot MHC-match most grafts: too much polymorphism, too little time, too few donors Therefore need immunosuppression 12

Matching and Cross-Matching Matching: finding a donor who shares the HLA antigens of the recipient, to minimize antigen disparities requires donor and recipient antigens to be identified Cross-matching: testing the SERUM of the recipient for antibodies against the donor antigens 14

Identifying MHC Polymorphisms (‘tissue typing’) Formerly determined by antibodies against MHC molecules HLA typing MLR Now by DNA testing: allele-specific PCR, sequencing 15

Tissue Typing(or HLA-typing) Used to identify HLA molecules on cells Ab against HLA1 + complement Recipient Donor Cells die, appear blue

MIXED LYMPHOCYTE REACTION: Recipient Donor + (Irradiate) Cell Proliferation Strong Proliferation--->High incompatibility Weak proliferation--->Low incompatibility No proliferation---> 100% compatibility Helps to identify any antigenic differences between donor and recipient

CD4 & CD8 Subsets Of T Cells CD 4 or Helper subset of T cells directly bind MHC class II molecules CD 8 or Cytolytic subset of T cells directly bind MHC class I molecules CD 4 cells mediate initial recognition of an allograft CD 4 deficient mice are unable to reject grafts while CD 8 deficient mice do reject grafts.

TCR SPECIFICITY AND SELF-MHC MHC molecules are highly polymorphic During development in the thymus, T cells are selected 
for moderate/weak binding to self-MHC + a self-peptide (“positive selection”) During development in the thymus, T cells are killed (or become regulatory T cells) if they have strong binding to self-MHC + a self-peptide (“negative selection”) Do the T cells of one person “see” MHC molecules from another person?
Actually about 1% of T cells bind such MHC with self peptide strongly--like a microbial antigen/MHC complex-- and become activated (“alloreactive T cells”)

DIRECT PRESENTATION Direct presentation in transplantation (MHC + peptide: MHC is from graft; peptides are from cellular proteins, graft and/or host) Graft dendritic cell is induced to mature, express co-stimulatory molecules (B7) and migrate to lymph node by stress of surgery, etc. DC in lymph node activates allo-MHC recognizing CD4+ T cells and CD8+ T cells Activated T cells expand and then home to sites of inflammation where they find allo-MHC on graft cells and secrete cytokines/kill graft cells This is the critical pathway for CD8 T cell response. 

DIRECT PRESENTATION IN GRAFT REJECTION Blue: host cell; pink: graft cell see also Abbas et al. Fig. 10-8.

Semi-direct Antigen Presentation the Membrane Patch Pathway Host CD8+ cytotoxic cell Host CD8+ T cell Allogeneic Donor Cell Class I Shed membrane with donor MHC Intact donor MHC Host APC 24

INDIRECT PRESENTATION Indirect presentation in transplantation (MHC + peptide: MHC is from host, peptide may be from graft MHC) Dendritic cell pick up antigens (e.g. allo-MHCs), and load peptides from these allo-MHC onto their own MHC II, migrate to lymph nodes and activate T cells CD4+ T cells are activated and then migrate to sites of inflammation where they are activated by host macrophages or DCs presenting graft antigens, leads to inflammatory reaction and tissue damage. The indirect pathway is probably responsible for chronic rejection (rejection after 1 year)

INDIRECT PRESENTATION IN GRAFT REJECTION

Allorecognition: Indirect Pathways CD8+ cytotoxic cell CD4+ T cell IL-2 Class I Shed Allogeneic MHC (donor class I-derived peptide presented by host class II molecules) Allogeneic Donor Cell Donor MHC molecules are taken up and processed by host antigen presenting cell Host antigen presenting cell 27

3 Signals for T Cell Responses APC Ag signal 2 costimulation B7 CD28 cytokines IL- 15 etc signal 1 IL-2 signal 3 T cell activation G1 S M G2 replication key genes e.g.IL-2, CD40L Expression of effector activity 28

Three-Signal Model Halloran, N Eng J Med, 2004;351:3715

ROLE OF CO-STIMULATORY PATHWAY Do Alloreactive T cells need co stimulation? Ordinarily T cell immune responses require co-stimulation (B7/CD28) Blocking co stimulation with CTLA4-Ig, “belatacept” protects against acute graft rejection in kidney patients (phase III clinical trials published in 2010; FDA approval in 2011) Most likely explanation:
-ischemia, stress to organ, release of DAMPs can induce expression of co-stimulators
-may explain why live donor organs have superior outcomes for kidney transplants compared to cadaver donor organs.

CNIs Halloran, N Eng J Med, 2004;351:3715

CALCINEURIN PATHWAY

MECHANISM OF IMMUNOSUPPRESSION BY CYCLOSPORINE NF-AT: nuclear factor of activated T cells NF-AT is required for transcription of the IL-2 gene and some other cytokine genes NF-AT cannot be activated in presence of cyclosporine.

Corticosteroids Corticosteroids Halloran, N Eng J Med, 2004;351:3715

Mycophenolates Halloran, N Eng J Med, 2004;351:3715

m-TOR inhibitors Halloran, N Eng J Med, 2004;351:3715

ROLE OF B LYMPHOCYTES Key role in hyper acute rejection. B cells are less important than T cells in initiating an allo response but antibody formation can play a role in the effecter phase of graft destruction and chronic rejection.

LEUKOCYTE RECRUITMENT

Transplantation Antigens ABO system ABH antigens are complex carbohydrate (polysaccharide) structures on surface of many cell types including graft cells & RBC; genes encode production of specific glycosyltransferases catalyze addition of terminal trisaccharide nomenclature H antigen: base chain; defines blood type O A trisaccharide on H chain: blood type A or A1 B trisaccharide on H chain: blood type B A and B trisaccharides on H chains: blood type AB 42

The ABO Blood Group Barrier in Organ Transplantation ‘ABO’ antigens: carbohydrate structures expressed on many tissues and organs, including endothelium of organ transplants Recipient pre-formed ‘natural’ anti-A or anti-B antibodies to non-self A/B antigens Transplantation of ABO-incompatible organs: ‘Hyperacute’ rejection 43

© 2007 New Science Press Ltd new-science-press.com ‘isohemagglutinins’ Figure 14-11 The ABO blood cell antigen system Top: schematic representation of the carbohydrate structures corresponding to the A, B and H antigens. The H antigen is modified by the addition of a single sugar residue by the allelic glycosyltransferase enzymes that create the A and B antigens. The H antigen is present on the surface of A, B, AB and O individuals, as indicated in the lower panel, because not all H antigens are modified by the A or B glycosyltransferase. The H antigen is only recognized by antibodies made in rare individuals who lack enzymes that make this oligosaccharide structure, which is present on glycolipids and glycoproteins of red blood cells and some other cell types including endothelial cells. Also shown in the lower panel is the antibody to the A and B antigens present in individuals of different blood types. ABO compatibility between donor and recipient is crucial to avoid rapid graft rejection © 2007 New Science Press Ltd new-science-press.com 44

ABO Incompatible Renal Transplantation Plasmapharesis IVIG CMV-IVIG Splenectomy Pre Transplant Immunosuppression

TYPES OF TRANSPLANT GRAFT REJECTION Antibody-mediated rejection (AMR) Hyperacute rejection Acute or delayed AMR Cellular rejection ‘Chronic’ rejection 47