Lecture 20 - Transplantation

MHC Class I Molecules

• HLA-A, HLA-B, HLA-C

• All nucleated cells

MHC Class II Molecules

• HLA-DP, HLA-DQ, HLA-DR

• APCs, few other cells

Major Histocompatibility Complex (MHC) Molecules

are cell-surface proteins essential for presenting antigens to T cells, which triggers an adaptive immune response

Haplotypes

inherited as a set

Most polymorphic genes….

elicit strong immune response

• MHC genes are the most polymorphic genes in humans meaning they have many variants

  • Different MHC alleles can elicit strong immune responses

Autograft

• Transplantation of your own tissue to yourself

• No immune rejection because MHC molecules match exactly

Syngeneic Graft/Isograft

• Transplant between genetically identical individuals (e.g., identical twins)

• Minimal to no rejection due to identical MHC alleles

Allogeneic Graft

• Transplant between two genetically non-identical people of the same species

• Most common type in clinical practice

• Strong immune response due to differences in MHC alleles

Xenograft

• Transplant between different species (e.g., pig → human)

• Very strong immune response

• Strong response vs. MHC (huge MHC mismatch)

• Highest rejection risk

Histocompatibility Antigens

• MHC

• Minor Histocompatibility Antigens (mHAs)

• ABO blood group antigens

• MHC Class I-Related Chain A (MICA) Antigens

• Killer Immunoglobulin-Like Receptors (KIRs)

Histocompatibility Antigens - MHC

Sibling matches; Haplotypes inherited as set from each parent

HLA-identical

25% chance; both haplotypes shared

Haploidentical

50% chance; one haplotype shared

HLA-nonidentical

25% chance; no haplotypes shared

Minor histocompatibility antigens

• Polymorphic non-MHC self-proteins

  • Normal human proteins that vary between individuals due to genetic differences

  • These differences make their peptides appear “foreign” to another person’s immune system

Why Minor histocompatibility antigens matter in transplants

• Even when MHC is fully matched, the recipient’s T cells can still react to donor peptides derived from mHAs

  • Leads to slower, weaker rejection compared to mismatched MHC

  • mHA peptides are processed normally by donor cells and presented on MHC molecules to recipient T cells

    • Often presented on MHC Class I

Examples of Minor histocompatibility antigens

• Some Y chromosome proteins

• Some autosomal proteins

Why ABO Blood Group Compatibility in Transplantation is critical

• A and B antigens are not only on RBCs — they are also expressed on endothelial cells of solid organs (kidney, heart, liver, etc.).

• If a recipient has pre-existing IgM antibodies against donor ABO antigens → immediate immune attack

ABO Blood Group Compatibility in Transplantation - Mechanism of action

Recipient anti-A or anti-B antibodies:

• Bind to donor A or B antigens

• Activate complement system

• Cause hyperacute rejection

  • Onset: minutes to hours

  • Leads to thrombosis, inflammation, and rapid graft destruction

Therefore:

• Recipient and donor must be ABO identical or compatible for solid-organ transplantation.

Universal RBC Donor

• Type O-

• Lacks A, B, and Rh (D) antigens → least likely to be attacked by recipient antibodies

Universal RBC Recipient

• Type AB+

• Lacks antibodies to A, B, or Rh antigens → can receive RBCs from any type.

ABO Blood Group Chart

MHC Class I-Related Chain A (MICA) Antigens

Function / Immune Role

• Involved in activating certain T cell responses (γδ T cells)

Expressed by various cells

• Endothelial cells, Epithelial cells, DCs, others

• Not expressed on lymphocytes

  • Highly polymorphic, with dozens of alleles

Killer Immunoglobulin-Like Receptors (KIRs)

Activating and inhibiting receptors on NK cells

• Polymorphic

Alloreactive Response

• Immune response against antigens from another individual of the same species.

• In transplantation, this means the recipient’s immune system recognizing the donor’s MHC molecules as foreign

Alloreactive Response - If difference at MHC between donor/recipient

Response vs MHC molecule(s) on transplanted tissues

• If the donor and recipient have different MHC alleles, the recipient’s T cells recognize the donor MHC proteins on the graft.

• This leads to T cell activation → graft rejection.

• Main Targets

  • MHC Class I response on most tissues

  • Both MHC I and II responses can occur

Memory cell vs non-self MHC Molecules

• After the first exposure to donor MHC, the recipient develops memory T cells.

• A second graft from the same donor triggers a secondary response to the graft (faster and stronger response)

• This leads to an accelerated rejection

Why are MHC the main target?

due to the high frequency of T cells vs Non-self MHC molecules

Response to Alloantigens

• Naïve alloreactive T cells become activated when they encounter alloantigens presented by APCs.

• There are two main mechanisms of alloantigen presentation:

  1. Direct allorecognition

  2. Indirect allorecognition

Direct Allorecognition

• The graft contains donor APCs

  • Donor APCs migrate from the graft to recipient lymph nodes and spleen via blood.

• Recipient has a high frequency of T cells that can react to non-self MHC molecules.

• Recipient T cells activated by donor APCs

  • TCR reacts directly to donor MHC molecules

  • Recipient TCR recognizes the donor MHC itself, with or without the bound peptide.

  • Activated T cells back to organ = destruction

• Depletion of donor APCs from the graft delays rejection, because direct recognition is weakened.

Indirect allorecognition

• Driven by recipient’s own APCs.

• Process - Recipient APCs:

  • Take up allogeneic donor proteins

    • Non-self MHC and Minor histocompatibility antigens

  • Peptides are then processed and presented on self-MHC to recipient T cells

Effects on transplanted tissue

• Antibody-mediated damage

  • Complement activation

  • ADCC (NK cell–mediated killing)

• Direct cytotoxicity (T-cell killing)

• Delayed-type hypersensitivity (Type IV reaction) inflammation

Acute Rejection to Allograft

• Occurs within days to months after transplantation; as early as 10–13 days

• T cell (CD8+, CD4+) and Antibody-driven

• Memory cell develops

Acute Transplant Rejection - Pathology

• Tissue and vessel damage

• Many CD8⁺ T cells

  • MHC I expression on most cells = cytotoxicity

• CD4+ T cells and Macrophages

  • Cytokines and delayed-type hypersensitivity

• Antibodies to vessel walls

  • Complement, Inflammation, and transmural necrosis

Hyperacute Transplant Rejection

• Occurs within minutes to hours after the graft’s blood supply is connected

• Pre-existing alloantibodies = rapid rejection

  • Recipient already has antibodies against donor antigens vs.

    • MHC (HLA) antigens, Blood group antigens, and Endothelial cell antigens

Mechanism

• Are complement-mediated

• Antibodies bind/act on donor graft endothelium

Result

• Clotting and complement

• Damage and reduced blood flow

• Enlarged graft; hemorrhaging and deoxygenation

Sources of these pre-exisiting allo-antibodies in Hyperacute rejection

• Previous transplant

• Prior blood transfusions

• Response to paternal antigens in pregnancy

Prevention of Hyperacute rejection

• ABO compatibility testing

• Screening recipients for anti-HLA antibodies

  • Confirmed by Cross-matching of donor and recipient serum to ensure no reactive antibodies

• Because of these steps, hyperacute rejection is now uncommon

Chronic Transplant Rejection

• Late rejection of graft

• Occurs months to years later after transplantation

• Gradual loss of function

• Difficult to detect and treat

Chronic Transplant Rejection Factors

• Lengthy cold ischemia time for the graft

• Ischemia–reperfusion injury during transplantation

• Repeated, subclinical acute rejection events

  • These may go unnoticed but cumulatively damage the graft

• Delayed-type hypersensitivity (DTH) responses to donor MHC molecules

Chronic Transplant Rejection Reaction - Chronic allograft vasculopathy

• CD4+ T cells and alloantibodes

  • Espically in the heart and kidney

  • A progressive arteriosclerosis of graft vessels:

  • Leads to fibrosis, atrophy, and reduced blood flow

• Liver: progressive loss of bile ducts

• Lungs: bronchiole scarring (scarring and obstruction of bronchioles)

Graft-Versus-Host Disease (GVHD) Context: Hematopoietic Cell Transplants (HCTs)

• Used to treat hematopoietic cancers

• Patient’s own bone marrow is depleted (chemotherapy/radiation).

• Repopulated immune response with donor cells

Graft-Versus-Host Disease (GVHD) (Complication HCTs)

• Donor graft contains mature T cells

• These donor T cells recognize recipient tissues as foreign

• Severe inflammation is several tissues

• Because the donor immune system is attacking the host → GVHD

Importance of HLA Matching

• HLA matching is critical, especially for:

• Allogeneic cell transplants

• Sibling donors usually preferred (highest chance of MHC match)

  • Even with good HLA match, donor T cells can react to minor histocompatibility antigens

Benefits of Mature T cells in Hematopoietic cell transplants (HCT)

• Reconstitute immunity quickly (helps fight infections)

• Provide graft-versus-leukemia (GVL) effec

  • Donor T cells can kill cancer cells

GVHD Effects

• “Cytokine Storm”

  • Massive activation of donor T cells

  • High cytokine release → widespread inflammation

• GI track, skin, and liver

• Often within 3 months

  • Later effects = Fibrosis of mucosa

Suppression of GVHD

• Immunosuppressive therapy

• Prior removal of mature T cells from stem cells

  • T cells that develop after transplant (from donor stem cells maturing in the host)

    • Become tolerant to recipient antigens

    • Lower risk of GVHD

Immunosuppressive Agents Purpose and Risks

Purpose

• Inhibit rejection responses after transplantation

• Essential for graft survival

Risks

• Increased susceptibility to infection and cancer

• Toxic side effects depending on the drug

Major categories of Immunosuppressive Agents

• Corticosteroids

• Calcneurin inhibitors

• Antimetabolites

• Monoclonal antibodies

• Other Non-antibody drugs

Immunosuppressive Agents - Corticosteroids

• Anti-inflammatory and immunosuppressive

• Blocks cytokines, inflammatory molecules, and cell adhesion molecules

Immunosuppressive Agents - Calcneurin inhibitors

Blocks signaling for T cell proliferation and differentiation

Immunosuppressive Agents - Antimetabolites

Blocks lymphocyte maturation and destroys proliferating cells

Immunosuppressive Agents - Monoclonal Antibodies

• Depletion of Mature T Cells (Anti-CD52)

  • Given at time of transplant

    • Removes mature T cells from circulation

• Used also in bone marrow transplant to deplete donor mature T cells

• Inhibits TCR Signaling (Anti-CD3)

• Reduces activation by APCs

  • Anti-CD4, Anti-CD28, Anti-CD40L

• Reduce T cell activation (Anti-CD25)

  • Blocks IL-2 signaling

Immunosuppressive Agents - Non-antibody drugs to target

• Cell cycle

• Translocation of nuclear factors

• Differentiation cascades (in T and B cells)

Histocompatibility Testing - Mixed Lymphocytes

Purpose

• Detects alloreactive donor T cells

• Assesses the likelihood of T-cell–mediated rejection or GVHD

• Especially important in hematopoietic cell transplantation

Cells Used

• Donor lymphocytes (responding cells)

• Recipient cells (stimulator cells)

  • Recipient T cells or APCs

  • Irradiated to prevent proliferation

Why Irradiate Recipient Cells?

• Prevents recipient cells from dividing

• Ensures that any observed response comes from donor T cells only

Mechanism

1. Donor T cells are mixed with irradiated recipient cells

2. Donor T cells recognize recipient alloantigens (non-self MHC)

3. This recognition triggers donor T-cell activation

Recognition of recipient allo-antigens by donor cells

• Proliferation - CD4⁺ T cells respond mainly to MHC class II

• Measured by increased DNA synthesis or cell division

Cytotoxicity -

• CD8⁺ T cells recognize MHC class I

• Kill recipient target cells

Histocompatibility Testing: HLA Typing

• Determines HLA antigens or genes of a donor or recipient

• Used to assess histocompatibility before transplantation

Complement-dependent cytotoxicity (CDC) test

Purpose

• Determine HLA phenotype

Cells Used

• Lymphocytes from the individual being typed

Cell type by HLA class

• MHC Class I typing:

  • T cells and B cells (both express MHC I)

• MHC Class II typing:

  • B cells only (express MHC II)

Reagents/Procedure

• Antisera with known HLA specificities

• Add reagent Complement

• Add dye; taken in/enters dead cells only

• Measure the proportion of dead cells using standard scale

Histocompatibility Testing: HLA Typing — Molecular Methods

Purpose

• Determine HLA genotype

• More precise than serologic (CDC) typing

• PCR amplification of HLA genes

  • Specific HLA alleles and allele groups identified

PCR with Sequence-Specific Primers (PCR-SSP)

Principle

• Uses panels of primers, each specific for a particular HLA allele or allele group

• Perfect base-pair matching is required for amplification

• Only reactions with matching primers → DNA amplification

• Can ID HLA genotype based on primers that led to amplification

PCR with Sequence-Specific Oligonucleotide Probes (PCR-SSOP)

Principle

• Single PCR reaction amplifies all relevant HLA genes

• Amplified DNA is then tested with a panel of labeled DNA probes

Process

• Each probe is specific for a particular HLA allele

• Hybridization of probe to PCR product indicates presence of that allele

  • HLA genotype is then determined

Sequence-Based Typing (SBT)

Principle

• Direct sequencing of HLA genes

Key Features

• Considered the gold standard for HLA typing

• Can identify new alleles

Histocompatibility Testing: HLA Antibody Screening

Purpose

• Detect antibodies against HLA antigens in patient serum

• Critical for transplant planning and monitoring

• Informs about potential donors

When & Why Testing Is Done Patients on Transplant Waiting Lists

• Patients on waiting list tested periodically

Transplant Recipients

• Tested periodically to:

  • Assess rejection

  • Monitor effectiveness of anti-rejection therapy

Complement-dependent cytotoxicity test

• Uses panels of lymphocytes with known HLA antigens

• Add:

  • Patient serum

  • Complement

  • Vital dye (enters dead cells)

Interpretation

• Antibody binding → complement activation → cell death

• Cell death indicates patient serum contains Abs to that HLA antigen

ELISA (Indirect)

• HLA antigens coated onto microtiter wells

• Add patient serum

• Add enzyme-labeled secondary antibody

Detection

• Color change indicates presence of anti-HLA antibodies

Flow Cytometry

• Beads coated with HLA antigens

• Incubated with patient serum

• Add fluorescently labeled secondary antibody

Multiplex Bead Array

Allows detection of antibodies against many HLA antigens in one tube