TRANSPLANTATION IMMUNOLOGY

Mixed Lymphocyte Reaction (MLR) for cell proliferation of alloreactive T cells:

- involves detection by DNA incorporation of radioactive thymidine - Recipient (alloreactive) live T Cells- will proliferate in response to allogeneic MHC antigens - UV irradiated- Donor MNC Stimulate Recipient live T cells - "Stimulator" lymphocytes from potential donor are first killed by uvirradiation Then mixed with live "responder" lymphocytes from the recipient .Mixture of cells incubated in cell culture to permit cell proliferation. - DNA synthesis measured by tritiated thymidine in DNA . Greater the DNA synthesis (proliferation) -indicate the unsatisfactory match.

Tests for Donor - Recipient Matching: for Successful transplantation

1. ABO blood grouping: Allogeneic antigens expressed on red blood cells and endothelial cells- IgM antibodies to ABO/ cause hyper-acute rejection 2. Crossmatching: Cytotoxic antibody assay for preformed antibodies in the recipient serum : reactive against donor's allogenic HLA antigens . Checked by donor lymphocyte lysis induced by the recipient's serum plus complement. Antibodies present in recipient's serum due to previous pregnancies, transfusions or transplantation. If present cause risk for hyper-acute or acute vascular rejection (prior to transplantation of solid organs) 3. Tissue Typing: By serology or PCR B cells (expressing both class I and II) used as target cells in these tests. By PCR method (more accurate method)- MHC genes amplified.Routine HLA typing only for A, B and DR (routinely done for BMT) 4. Mixed Lymphocyte Reaction(MLR): Proliferation test of donor and recipients lymphocytes: Testing for alloreactive T cells of Bone Marrow(BM) donor checked by in vitroDNA synthesis by mixing donor and recipient lymphocytes. This test is performed before bone marrow transplantation(BMT).

what can be done to help optimize chances for survival in transplants

1. Blood group antigens: ABO match: MHC antigens are not present on mature erythrocytes --present only on nucleated cells 2. HLA Class I and II Match: Polymorphic MHC locus (MHC antigens HLA in man- 6 different loci (HLA A, B and DR) 3. Presence of Pre-formed Antibodies: Some recipients having multiple transfusions- develop cytotoxic antibodies- Previous Transplantation history Minor transplantation antigens: these are non ABOblood groups and antigens associated with sex chromosomes - are weaker in reaction than MHC antigens

Bone marrow transplant - Key Take-Away Points

1. Bone marrow transplant requires a series of steps including conditioning, selection of a donor, and prophylactic treatments to prevent graft rejection and GvHD. 2. Chemotherapy drugs, radiation, etc. are used to "condition" the recipients marrow to receive the donor cells. 3. A mismatch of donor and recipient HLA types causes an increased risk of graft rejection and GvHD.

Mainstay of preventing and treating the rejection of organ transplants is immunosuppression that is designed mainly to inhibit T cell activation and effector functions. Challenges:

All immunosuppressive drugs carry the problem of nonspecific immunosuppression- making them susceptible to infections particularly by intracellular microbes- lead to increased cancer incidence by oncogenic viruses.

Acute antibody-mediated rejection

Alloantibodies cause acute rejection by binding to alloantigens, mainly HLA molecules, on vascular endothelial cells, leading to endothelial injury and intravascular thrombosis that results in graft destruction.

ABO blood grouping

Allogeneic antigens expressed on red blood cells and endothelial cells- IgM antibodies to ABO/ cause hyper-acute rejection

Tissue Typing: By serology or PCR

B cells (expressing both class I and II) used as target cells in these tests. By PCR method (more accurate method)- MHC genes amplified.Routine HLA typing only for A, B and DR (routinely done for BMT)

allograft

Between: - different individuals of the same species - allo = different Graft antigens: - different (alloantigens) Results: - rejected

Isograft

Between: - genetically identical individuals (identical twins or inbred mice) - (iso=the same) Graft antigens: - identical Results: - accepted

xenograft

Between: - individuals of different species Graft antigens: - different (xenoantigens) Results: - rejected

autograft

Between: - location in the same individual - (auto=self) Graft antigens: - identical Results: - accepted

Rapamycin

Blocks lymphocyte proliferation by inhibiting mTOR and IL-2 signaling

example for acute rejection

CD8+ T cells and antibodies

what is The principal mechanisms of acute cellular rejection?

CTL-mediated killing of graft parenchymal cells and endothelial cells and inflammation caused by cytokines produced by helper T cells.

Immunosuppresive drugs to treat allograft rejection mediated by T cells: block the activation of host T cells by what negative regulator?

CTLA4

Crossmatching:

Cytotoxic antibody assay for preformed antibodies in the recipient serum : reactive against donor's allogenic HLA antigens . Checked by donor lymphocyte lysis induced by the recipient's serum plus complement. Antibodies present in recipient's serum due to previous pregnancies, transfusions or transplantation. If present cause risk for hyper-acute or acute vascular rejection (prior to transplantation of solid organs)

Class II MHC is encoded by

DP, DQ, DR

what type of allorecognition results in acute rejection?

Direct alloantigen recognition

who performed the first organ transplant and when?

Dr. Joseph Murray in 1954

transplant tolerance

Foreign cell and tissue grafts are tolerated best when the HLA is most closely matched. Tolerance of the transplanted graft is maintained by the action of regulatory T cells and other regulatory pathways. Rejection of foreign cells or tissues occur in 3 main stages: hyperacute, acute, and chronic.

what is the most genetically heterogeneous sequence in the human genome?

HLA (MHC) complex The "HLA type" of an individual is made up of the composite set of MHC Class I and Class II proteins, encoded by the HLA genes that are present on the maternal and paternal chromosomes.

what is a must for allogenic bone marrow transplants?

HLA matching

Sensitization

In the case of direct allorecognition, donor dendritic cells in the allograft migrate to secondary lymphoid tissues, where they directly present allogeneic MHC molecules to host T cells. In the case of indirect allorecognition, recipient dendritic cells that have entered the allograft transport donor MHC proteins to secondary lymphoid tissues and present peptides derived from these MHC proteins to alloreactive host T cells. This is shown for CD4 + T cells, and indirect recognition of allogeneic MHC by CD8 + T cells is likely less important. After both indirect and direct allorecognition, the T cells become activated and differentiate into effector CD4 + helper T cells and CD8 + CTL cells. In Rejection: , The alloreactive effector T cells migrate into the allograft, become reactivated by alloantigen, and mediate damage. In the graft, direct recognition of allogeneic class I by CD8 + CTL is required for killing of graft parenchymal cells, because these cells express only allogeneic MHC. In contrast, both CD4 + helper T cells that can directly or indirectly recognize allogeneic class II MHC can be activated by donor or host APCs, respectively, and both can promote inflammation that damages the graft.

what type of allorecognition results in chronic rejection?

Indirect alloantigen recognition

Immunosuppression has detrimental effects on transplant patients

Infections • Wide variety of infections • Reactivation of latent viruses -Cytomegalovirus virus (CMV), Herpes virus- patients to get prophylactic antiviral therapy • Opportunistic type intracellular Infections (Pneumocystisis carinii, Candida albicans) Neoplasms • B cell Lymphoma caused by unchecked EBV. • Squamous cell carcinoma-associated with human papillomavirus

CTLA4-Ig

Inhibits T cell activation by blocking B7 costimulator binding to T cell CD28

In clinical transplantation what are a typed for matching to avoid rejection?

MHC alloantigens

Prevention and Treatment of Allograft Rejection

MHC polymorphism - difficult to find exact match Match for 3 of the 6 alleles of an individual referred as one haplotype match. Match for 6 of the 6 alleles of an individual referred as two haplotype match ABO compatible - Prevents hyper-acute rejection in solid transplants. Can induce transfusion reaction in bone marrow and stem cell transplantation Immunosuppressive Drugs - Cyclosporin and Tacrolimus (FK506) - inhibit IL-2 gene expression, few toxic effects - 90% survival for unrelated kidney at 1 year - Biologics/Monoclonal Antibodies to T cell structures - e.g. using anti - CD3 for cytotoxic cells. (Biologic Orthoclone binds-CD3) - used for kidney rejection in patients). Blocking with anti-CD28 antibodies or CTLA4 Ig fusion protein (binds to B7 and interferes with CD28 binding) Anti-inflammatory agents Corticosteroids used for general immunosuppression. New anti-inflammatory agents are soluble cytokine receptors, anti-ICAM-1, anti-TNF alpha

two haplotype match

Match for all six HLA alleles

one haplotype match

Match for three of the six HLA alleles

MHC genes are the most polymorphic in the genome: HLA A, HLA B and HLA DR

Most polymorphic genes are HLA A and HLA B from class I and HLA DR from class II. Six class II MHC alleles are inherited, three from each parent (one set of DP, DQ and DR). Co-dominant Expression: • Alleles inherited from both parents are expressed equally - 6 different class I MHC and 6-8 different class II MHC alleles can be expressed on the same APC • Possibility of heterologous pairing (e.g. DQ-alpha, from one chromosome and DQ-beta from another chromosome). Pre-transplantation Testing for HLA Type: • Match for three of the six alleles - one haplotype match •Match for all six alleles - two haplotype match

who established The U.S. Organ Procurement and Transplantation Network (OPTN) and when?

National Organ Transplant Act (NOTA) of 1984.

examples of causes of hyper acute rejections

Preexisting antibodies to ABO or to allogeneic HLA antigens (against ABO or allogeneic antibodies to HLA antigens, Cross matching test)

Immune responses to an Allogeneic Fetus: Various hypotheses related to tolerance

Pregnancy: is a naturally occurring allograft • Cytotoxic lymphocytes and antibodies reactive to fetal tissues are detectable Fetus escapes maternal immune response - ? Regulatory T cells in mother near placenta • Trophoblast (outer layer of placenta) cells fail to express paternal MHC molecules (even if they express - may lackcostimulatory molecules - bearing functional inhibition) • Possibilities: Barrier features of the placenta and local immunosuppression at maternal-fetal interface (secretion of TGF-β, IL-4 and IL-10 and complement regulatory proteins are expressed) • Lack of recognition -uterine decidua an immunologically privileged site (e.g. brain, eye and testis). Here immune responses generally fail to occur. Note: Placenta is a fetal derived tissue -interphase is trophoblast

Evidence showing that rejection of tissue transplants is an immune reaction. What is the evidence?

Prior exposure to donor MHC molecules leads to accelerated graft rejection - graft rejection shows *memory and specificity* - two features of adaptive immunity the ability to reject a graft rapidly can be transferred to a naive individual by lymphocytes from a sensitized individual - graft rejection is medicated by *lymphocytes* depletion or inactivation of t lymphocytes by drugs or antibodies results in reduced graft rejection - graft rejection requires t lymphocytes

Mixed Lymphocyte Reaction(MLR):

Proliferation test of donor and recipients lymphocytes: Testing for alloreactive T cells of Bone Marrow(BM) donor checked by in vitroDNA synthesis by mixing donor and recipient lymphocytes. This test is performed before bone marrow transplantation(BMT).

Graft rejection: mediated by cellular cytotoxicity

T cells must recognize class I MHC molecules on the surface of the graft To kill cytotoxic T cells must also receive : • cytokine stimulus (e.g.-*IL-2*) from activated CD4 helper T cells to proliferate for clonal proliferation CD8 T cells kill by - inserting Perforins /granzymes. These activate caspases and initiate apoptosis finally cell death CD8+ T cell mediate killing by Fas-Fas ligand (fas L) cell interaction and induce cell apoptosis Macrophages and NK cells mediate killing by antibody dependent cell cytotoxicity (ADCC)

Inhibitors of T Cell Signaling Pathways

The calcineurin inhibitors cyclosporine and tacrolimus (FK506) inhibit transcription of certain genes in T cells, most notably genes encoding cytokines such as IL-2 . The immunosuppressive drug rapamycin (sirolimus) inhibits growth factor-mediated T cell proliferation. Antibodies that react with T cell surface structures and deplete or inhibit T cells are used to treat acute rejection episodes. The first anti-T cell antibody used in transplant patients was a mouse monoclonal antibody called OKT3 that is specific for human CD3. Recall that CTLA4-Ig is a recombinant protein composed of the extracellular portion of CTLA4 fused to an IgG Fc domain. A high-affinity form of CTLA4-Ig, called belatacept, which binds to B7 molecules on APCs and prevents them from interacting with T cell CD28 is approved for use in allograft recipients. Clinical studies have shown that belatacept can be as effective as cyclosporine in preventing acute rejection,

what causes transplant rejection?

Transplant Rejection are immune responses caused by genetic differences ( HLA I and HLA II) between donor and the recipient

A dominant lesion of chronic rejection in vascularized grafts is:

arterial occlusion as a result of the proliferation of intimal smooth muscle cells, and the grafts eventually fail mainly because of the resulting ischemic damage.

Class III MHC genes encode for?

complement proteins/cytokine proteins

Sensitization - direct allorecognition

donor dendritic cells in the allograft migrate to secondary lymphoid tissues, where they directly present allogeneic MHC molecules to host T cells.

Acute GVHD

epithelial cell death in the skin , liver (mainly the biliary epithelium), and gastrointestinal tract. It is manifested clinically by rash, jaundice, diarrhea, and gastrointestinal hemorrhage.

Chronic GVHD

fibrosis and atrophy of one or more of the same organs, without evidence of acute cell death. Chronic GVHD may also involve the lungs and produce obliteration of small airways, called bronchiolitis obliterans, similar to what is seen in chronic rejection of lung allografts. When it is severe, chronic GVHD leads to complete dysfunction of the affected organ.

Rejection of foreign cells or tissues occur in 3 main stages:

hyperacute acute chronic

anti-IL 2 receptors

inhibits T cell proliferation by blocking IL-2 binding may also opsonize and help eliminate activated IL-2R expressing t cells

The mainstay of preventing and treating the rejection of organ transplants is what?

is immunosuppresion (before & after transplant) designed mainly to inhibit T cell activation and its effector functions

Cyclosporin and Tacrolimus (FK506)

nhibit IL-2 gene expression, few toxic effects - 90% survival for unrelated kidney at 1 year

Direct alloantigen recognition

occurs when T cells bind directly to an intact allogeneic MHC molecule on a graft (donor) antigen-presenting dendritic cell (passenger APC) - Acute Rejection. - donor APC to recipient T cells

what is a natural occurring allograft?

pregnancy

what T cell Immunosuppression in clinical use for transplantation patients do we need to know?

rapamycin corticosteroids anti-IL 2 receptors CTLA4-IG

GVHD is caused by

reaction of grafted mature T cells in the HSC inoculum with alloantigens of the host . It occurs when the host is immunocompromised and therefore unable to reject the allogeneic cells in the graft. In most cases, the reaction is directed against minor histocompatibility antigens of the host because bone marrow transplantation is not usually performed when the donor and recipient have differences in MHC molecules.

Sensitization - indirect allorecognition

recipient dendritic cells that have entered the allograft transport donor MHC proteins to secondary lymphoid tissues and present peptides derived from these MHC proteins to alloreactive host T cells. This is shown for CD4 + T cells, and indirect recognition of allogeneic MHC by CD8 + T cells is likely less important. After both indirect and direct allorecognition, the T cells become activated and differentiate into effector CD4 + helper T cells and CD8 + CTL cells.

Corticosteroids

reduce inflammation by effects on multiple cell types

Allorecognition

the ability of an individual organism to distinguish its own tissues from those of another Recipient DC transporting alloantigen to nearest Lymph node 1. transport of alloantigens to lymph node 2. activation of t cells, generation of effector t cells by direct and indirect antigen presentation 3. sensitization of alloantigen specific t cells 4. inflammatory cytokine secretion 5. killing of graft cells 6. rejection

what is the major barrier to survival of transplanted tissues or organs?

the immune response against grafted tissues

who is the donor and who is the recipient during transplantation?

the individual who provides the graft is called the donorand the individual who receives the graft is called either the recipient or the host.

MHC gene function

the physiologic function of MHC molecules is to display peptide antigens for recognition by T lymphocytes. • MHC molecules serve as histocompatibility antigens : are membrane proteins on antigen presenting cells (APC) that display peptides for immune recognition by antigen specific T lymphocytes • Protein antigens derived from the extracellular space or the cytosol are processed into peptides and loaded onto MHC molecules • Physiological Function: Peptide presentation to T lymphocytes • Highly polymorphic class I and Class II MHC genes - More than 250 alleles identified in the population by serology using antisera or monoclonal antibody. • Single serologically defined HLA further tested by molecular sequencing .show multiple variants differing slightly.

transfusion

the transfer of circulating blood cells or plasma from one individual to another

Class I MHC is encoded by:

three loci A, B and C

what is the physiologic function of MHC molecule?

to display peptide antigens for recognition by T lymphocytes.

Indirect alloantigen recognition

when allogeneic MHC molecules from graft cells are taken up and processed by recipient's APCs. Peptide fragments of the allogeneic MHC molecules are bound and presented by the recipient (self) MHC molecules - Chronic Rejection - recipient APC to recipient T cells

MHC Detection : HLA typing and matching before transplantation

• By serologic test (lymphocytotoxicity test) using anti sera or monoclonal antibody-complement-mediated assay • For some HLA loci more than 250 alleles identified in the population by serology (First HLA typing methodology) • Single serologically defined HLA further tested by molecular sequencing show multiple variants differing slightly • Now, molecular methods are used- to identify specific nucleotide sequence polymorphism distinctive of a specific HLA antigen

Chronic rejection

• Can occur months to years after the transplant by recognition of alloantigen by CD8+ and CD4+ T cells and by macrophages. Alloantibodies are also involved. • Chronic delayed type hypersensitivity (DTH ) reaction in vessel wall (arteriosclerosis of vascular endothelium) leading to luminal occlusion. • Recurrent episodes of acute rejection lead to chronic inflammation- principal cause of graft failure - Fibrosis/scarring of vessel wall occlude the vessel

The U.S. Organ Procurement and Transplantation Network (OPTN) helps success and efficiency of the U.S. organ transplant system. OPTN responsibilities include:

• Facilitating the organ matching and placement process through the use of the computer system and a fully staffed Organ Center operating 24 hours a day • Collecting and managing scientific data about organ donation and transplantation. • Providing data to the government, the public, students, researchers

Bone Marrow Transplantation Rejections: Graft-Versus-Host Disease (GVHD): due to graft mature T cells in BM

• GVHD is the principal limitation to the successful Bone Marrow transplantation. • Caused by reaction of grafted mature T cells in the marrow inoculum: to alloantigens of the immunocompromised host. • Reaction directed against minor histocompatibility antigens of the host. Careful HLA matching required. • Acute GVHD - epithelial cell death induced by killer cells (in skin, intestinal tract and liver) - takes 7-10 days - condition is fatal. • Chronic GVHD - fibrosis and atrophy of one or more same target organs - complete dysfunction of affected organ - takes 100 days - 5 years. May be fatal.

Human MHC Gene Complex

• Human MHC molecules are called human leukocyte antigens (HLA) • Located on chromosome 6 • All nucleated cells express Class I MHC • Only APCs (DCs, macrophages, B cells, endothelial cells express class II) • The HLA locus is inherited as a block on Chromosome 6 and encodes the Class I and Class II MHC molecules. We have Different 6 class I HLA and 6-8 different class II HLA can be expressed on the same APC

Scientific Registry of Transplant Recipients:

• Improvement in the field of solid organ: organ allocation and transplantation. • Developing (1999) and maintaining a secure Web-based computer system -maintains the nation's organ transplant waiting list and recipient/donor organ characteristics. • Providing professional and public education : about donation and transplantation.

Xenogeneic Transplantation

• Lack of availability of donor organsfor transplantation have lead to attempts of transplantation of organs from other mammals -(pigs, baboons) into human recipients • Challenges: Presence of natural antibodies reactive with carbohydrate determinants expressed by cells of evolutionarily distant species(cause hyper - acute rejection) • Cell mediated immune responses - to xenoantigens • Risk of transfer of unknown infections from these animals

Acute rejection

• Occurs in 11-14 days to months, marked reduction in circulation and MNC infiltration, eventual necrosis Called primary (first-set) rejection • Mediated by CD8+ T cells reactive to alloantigens on the graft endothelial cells and parenchymal cells. CD4 +T cells induce inflammation (DTH reaction); - (*A type IV hypersensitivity*) • Donor class II HLA on donor antigen presenting cells that remain in the graft are recognized by CD4+ T cells causing the T cells to become activated. • Alloreactive antibodies may also contribute to vascular injury by ADCC - (*Type II Hypersensitivity reaction*) • Direct allorecognition by recipients T cell

Hyperacute rejection

• Occurs minutes to days after transplant. • Caused by binding of *pre-formed antibodies* to cells in the graft (i.e. endothelial cells), leading to complement fixation, inflammation, clotting, and blood vessel occlusion in the graft. - A type II hypersensitivity reaction. • Antibodies form due to previous transplants, blood transfusions, or pregnancies. • Can cause a systemic inflammatory response syndrome.

Hematopoietic Stem cells (HSCs) from Bone Marrow

• Transplantation of pluripotent hematopoietic stem cells : (Aspirated bone marrow (BM) to restore bone marrow cells damaged by chemotherapy or treat leukemia's • Also performed for Immunodeficient Recipients (SCID, Wiskoll -Aldrich syndrome, Advance Leukemia etc.) • Preparatory Immunosuppression by depletion of recipients bone marrow ( by radiation / chemotherapy drugs) • Mature T lymphocytes in bone marrow graft respond to host alloantigensto produce Graft -Verses -Host Disease (GvHD) • Bone Marrow rejection (not completely understood) *Due to Mature T cells in the graft*

what is transplantation?

• widely used for replacing nonfunctioning organs and tissues with healthy organs or tissues. • It involves process of taking cells ,tissues or organs called a graft from one individual and placing them into a (usually) genetically different individual.