Immuno 4 - Transplantation

Question 1

HLA classes

Expression

Answer 1

HLA Class I (A, B, C) - expressed on all cells
thought to be the most immunogenic

HLA Class II (DR, DQ, DP) - expressed on APC but also upregulated on other cells under stress

Question 2

3 commonest transplanted organs

Answer 2

1 - kidney
2 - liver
3 - heart and lung

Question 3

Phases of T cell mediated Immune Response to Transplanted Graft
+ where do they occur

Answer 3

Phase 1_ recognition of foreign antigens
Lymph node

Phase 2_ activation of antigen-specific lymphocytes
Lymph node

Phase 3_ effector phase of graft rejection
In the graft

Question 4

Which are the most relevant protein variations in clinical transplantation?

Answer 4

ABO blood group

HLA - coded by the MHC on chr 6

Question 5

o Two types of rejection

Answer 5

 Direct
• Donor APC presenting antigen and/or MHC to recipient T cells
• acute rejection mainly involves direct presentation
 Indirect
• Recipient APC presenting antigen to recipient T-cells – i.e. the immune system working normally as it would for an infection
• Chronic rejection mainly involves indirect presentation

Question 6

Most important HLAs to match and why

Answer 6

o Most important to match = DR > B > A

o These are the HLA molecules with the most variation and therefore the most immunogenic

Question 7

Major determinant of the risk of rejection

Answer 7

number of mismatches

Question 8

Number of mismatches in siblings

Answer 8

25% 6MM
50% 3MM
25% 0MM

Question 9

Parent to child - matches

Answer 9

> = 3/6 matched

Question 10

how do we express differences between recipients and the HLA donor ?

Answer 10

o HLA-A: HLA-B: HLA-DR

Question 11

How do we determine an individuals HLA genotype?

Answer 11

PCR based DNA sequence analysis for HLA alleles

Question 12

How do alloreactive T cells get activated?

Answer 12

 Presentation of foreign HLA antigens by APCs (i.e. the donor’s HLA molecule)  both donor and host APC cells are involved

 Co-stimulatory signals – particularly of the IL-2 receptor in T cells through the release of IL-2

Question 13

what happens to the T cell during phase 2 of the transplant rejection?

Answer 13

Phase 2_ activation of antigen-specific lymphocytes

o Proliferation
o Product cytokines (IL2 is important)
o Provide help to CD8+ cells
o Provide help for antibody production by B cells
o Recruit phagocytic cells (monocyte/macrophage lineage cells)

Question 14

Targets for immunosuppressive drugs targeting T cell/cellular activation

Answer 14

Most drugs target T cell activation

calcineurin
MTOR pathways
co-stimulatory pathways

targeting the 3 signals that activate T cells:

APC MHC - TCR
APC CD80/CD86 - T cell CD28
Cytokine IL-2 - T cell CD25

Question 15

Key points of the phase 3 -effector response in rejection

Answer 15

 graft infiltration by alloreactive CD4+ cells
and start attacking the tubules which express the donor HLA molecules
 recruitment of CD8+ T cells + monocytes

o Cytotoxic T cells:
 Granzyme B (toxin)
 Perforin (punch holes)
 Fas-ligand (apoptosis)

o	Macrophages:
	Phagocytosis
	Proteolytic enzymes
	Cytokine release
	O2 and N2 radicals 

o Antibodies bind to graft endothelium

Question 16

Histological features of acute T cell mediated rejection

Answer 16

o Lymphocytic interstitial infiltration
 Immunohistochemistry – some are CD4+ cells, some are CD8+ cells, others are macrophages lineage

o Arteritis

(also see tubulitis in kidney graft rejection - inflammatory cells within the tubule)

Question 17

DDx for rejection in a transplanted kidney

Answer 17

• Calcineurin inhibitor toxicity
o Presentation - raised creatinine
o Nephortoxic
o Mx – reduce immunosuppressive drug

• Viral infections (reactivation of latent viruses e.g. polyoma viruses)
o BK nephrotpathy
o Mx – reduce immunosuppressive drugs

•	Vascular disease
o	Arterial intermural thickening
o	Small lumen
o	Hypertension 
o	Mx -BP control, vascular stent if larger arteries are affected

• Post transplant lymphoproliferative disease
o Some of this is virally driven
o Mx -  immunosuppressive drug
o Depending on the malignancy of the infiltrate they might need chemo

• Recurrent
glomerulonephritis
o Depends on nature GN

Question 18

Phases of antibody mediated Immune Response to Transplanted Graft + where do they occur

Answer 18

o Phase 1: B cell exposure to foreign antigen/HLA epitope
Lymph nodes

o Phase 2: proliferation and maturation of B cells with antibody production + start producing anti-HLA ab
Lymph nodes

o Phase 3: effector phase – antibodies in the circulation fix on the endothelial cell surface (bind to graft endothelium) + cause damage + recruit inflammatory cells to the endothelium (capillaries of glomerulus and around tubules)
Graft

Question 19

Main difference between T cell mediated rejection + antibody mediated rejection

Answer 19

T cells – crawl out of the circulation + go to interstitium + tubules of the kidney

B cell mediated rejection = intra-vascular disease

 T cells  interstitial damage
 Antibodies  endothelial damage

Question 20

Anti-ABO ab
Anti-HLA ab

naturally occurring or not

Answer 20

Anti-ABO ab - naturally occurring

Anti-HLA ab - not naturally occurring

Question 21

Action of antibodies in infection/ transplant rejection

Answer 21

o Neutralise toxins

o Opsonise (aid phagocytosis)

o Antibody-dependant cellular cytotoxicity

o Recruitment of inflammatory cells through complement independent mechanisms

o Complement activation: (complement fixes on the antibodies and then gets activated)
 MAC lysis
 Phagocytosis of microbes opsonized wit h complement fragments (e.g. C3b)
 Inflammation

Question 22

• Antibody mediated rejection histopathology

Answer 22

o Inflammatory cell infiltrate

o Complement fragments on the endothelial cell surface  activation of the complement

o capillaritis  Inflammatory cells in the microcirculation – a cardinal feature of antibody mediated rejection

Question 23

Describe phase 3 of the antibody mediated rejection

Answer 23

o Antibodies bind to antigens (HLA) on the endothelium of the blood vessels in the transplanted organ

o Antibodies fix/activate complement which assembles to:
 Form MAC  endothelial cell lysis
 Recruit inflammatory cells to the microcirculation

o Antibodies can
 Crosslink the MHC molecules  activating them
 Directly recruit mononuclear cells, NK cells and neutrophils  capillaritis

Question 24

how can mismatch positive transplantation take place?

Answer 24

requires a lot of preparation

Plasma exchange to remove ab
IVIG
*IVIG reduce antibody production through feedback and displaces troublesome ABs so they cannot exert their harmful effects

Question 25

Which 3 assays can be used before transplantation to minimise rejection?

Answer 25

o Cytotoxicity assays [CDC – Complement depended cytotoxicity]:
 Inspects if recipient’s serum will kill the lymphocytes of the donor, in the presence of complement
• No antibodies  lymphocytes remain viable = negative crossmatch
• Antibodies present  lymphocytes are killed = positive crossmatch
 Positive crossmatch suggests that there is cell lysis (i.e. +ve is a bad thing) = contraindication for transplantation

o Flow cytometry [FACS]
 Inspects if recipient’s serum binds to the donor’s lymphocytes
 Uses fluorescently labelled anti-human immunoglobulin
 Detection of whether there is human Ig from the recipients serum bound on the donor lymphocytes
 Negative flow crossmatch  donor specific antibody not detected
 Positive flow crossmatch  donor specific antibody detected

o Solid phase assays (uses a series of synthetic beads containing all the possible HLA epitopes) [Luminex]:
 Does the recipient’s serum bind to recombinant single HLA molecules attached to a solid support such as beads
 Recipient’s serum is mixed with beads and fluorescently labelled immunoglobulin is used to determine which HLA epitopes the antibodies bind to
 Patients that have antibodies to lots of different types of antibodies are regarded as highly sensitised

Question 26

T cell mediated transplant rejection prevention

Pre- transplant induction agents

Answer 26

Inhibitors of cell surface receptors

Alemtuzumab

• Anti- CD52
• Causes lysis of T cells + T cell depletion
• Dampens down immune response

Basiliximab

• Anti-CD25
• Targets IL-2 R
• Prevents proliferation

OKT3
- Anti-CD3 ab

ATG - anti-thymocyte globulin

Question 27

T cell mediated transplant rejection prevention

Post- transplant drugs

Answer 27

• Steroids
  Prevent general T cell mediated rejection
• Calcineurin inhibitors (tacrolimus, cyclosporin)
  Inhibit cell signalling
• Anti-proliferative agents (mycophenolate mofetil, azahioprine)
  Act downstream of the MTOR activation + block T cell proliferation in response to antigen

Question 28

Episodes of acute rejection mx

Answer 28

Steroids

OKT3
- Anti-CD3 ab

ATG - anti-thymocyte globulin

Question 29

B cell mediated rejection prevention (7)

Answer 29

 Rituximab (anti-CD20)  B cell depletion

 BAFF inhibitors  target cytokines that promote B cell activation and growth

 Proteasome inhibitors (i.e. bortezomib)  block production of antibodies by plasma cells

 Complement inhibitors (i.e. eculizumab, anti-C5)  block complement binding to endothelial cells

 Corticosteroids

 IVIG

 Plasma exchange

Question 30

GvHD

prophylaxis
sx
mx

Answer 30

prophylaxis

• Methotrexate
• Cyclosporin

Sx

• Skin (rash)
• GI (N, V, D, abdo pain, bloody stool)
• Liver (jaundice)

Mx
- CS

Question 31

Post-transplantation complications

Answer 31

• Infection
o Increased risk of conventional infections – bacterial, viral, fungal
o Opportunistic infections – CMV, BK virus, Pneumocystis carinii

• Malignancy
o Viral associated (x100) – Kaposi’s sarcoma (HHV8), Lymphoproliferative disease (EBV)
o Skin cancer (x20)
o Other cancers e.g. lung, colon (x2-3)

• Atherosclerosis
o HTN, hyperlipidaemia
o X20 increase risk in death from MI compared to age-matched general population

Question 32

rejection prevention by tissue typing

Answer 32

o ABO/HLA tissue matching

o Screening for anti-HLA antibodies

Question 33

Post kidney transplant patient presenting with raised creatinine mx

Answer 33

o Rejection  increase
immunosuppresive drugs

o Nephrotoxicity  decrease immunosuppressive drugs

Question 34

• The most important antigenic determinant of rejection in current clinical practice for kidney transplantation is

Answer 34

o Human leucocyte antigens/MHC

Question 35

• A potential donor is described as being 1:1:0 MM what does this mean

Answer 35

o 1MM A locus, 1MM B locus, 0 DR locus

(• The way we express differences between recipients and the HLA donor 
o HLA-A: HLA-B: HLA-DR)

Question 36

• A patient has an episode of acute T cell-mediated rejection 2 months post transplantation. What additional drugs would most commonly be administered?

Answer 36

o Steroids

o (tacrolimus, cyclosporin, azathioprine are used for baseline immunosuppression)

Question 37

What type of hypersensitivity reaction is acute cellular rejection of the transplant by T cells?

Answer 37

Type IV

Question 38

Hyperacute vs acute vs chronic rejection + cause

Answer 38

Hyperacute - mins hrs
Pre formed ab activates complement

Acute- -<6m
antibody or t cell mediated

Chronic >6m
immune and non immune mechanisms