Transplantation

Question 1

Define transplantation

Answer 1

The artificial transfer of cells, tissues or organs from one individual to another to replace lost or failing function.

Question 2

In terms of transplantation rejection, what is the one special case?

Answer 2

• The fetus is a repeatedly tolerated foreign graft
• However, in this case there is a special interface between the fetus and immune system
• It is a physical barrier – keeps the fetus and mother separate, controlling what goes across the boundary
• It is also an immunological barrier – actively trying to avoid immune recognition:
− Complement control proteins
− HLA G, E, F on the surface of the trophoblast – binds inhibitor receptors on NK cells
− FasL
− Absence of classical MHC
− Local non-specific immune suppression, eg) TGFB, IL-10

Question 3

What have been the key advances that have got us from aspiration to therapeutic transplantation?

Answer 3

1. Surgey:
   − 1800s → could perform skin grafts
   − Early 1900s → ability to join blood vessels paved the way for organ transplantation
   − 1930 → First surgical transplant of cadaveric kidney – rejected for ‘unknown’ reasons
   − 1954 → first successful kidney transplant between identical twins

However, obviously there was a lot od demand for transplants in people who weren’t twins – immunosuppression paved the way for successful transplantation.

2. Immune suppression:
   − 1959 → Azathioprine – metabolic inhibitor. Quite broad
   − 1972 → Cyclosporine A – targets T cells specifically

Question 4

How did Sir Peter Medawar define rejection?

Answer 4

an immunological phenomena showing specificity and memory.

Question 5

Describe Sir Peter Medawars experiments showing 1st/2nd set kinetics

Answer 5

• If you take skin from a yellow strain onto another genetically identical mouse → tolerated indefinitely
• Take skin from a yellow mouse and add it onto a blue → 1st set rejection
• Take skin from a yellow mouse and add it onto the same blue → rejected quicker, 2nd set rejection
− The mouse was sensitized
− it wasn’t that the mouse was sensitive to ejection, because if they added a skin from a green strain, it showed 1st set kinetics
− This demonstrates that rejection shows antigen specificity and memory
• It was then shown that graft rejection is mediates by lymphocytes, because T cells transferred from a sensitized donor to a naive recipient cause the naïve recipient to show 2nd set rejection when given the graft.

Question 6

Describe the experiments showing graft rejection is mediated by T cells

Answer 6

• Mice lacking functional T cells are incapable of allograft rejection → demonstrated in Nude mice
• Depletion of T cells using antibodies resulted in reduced graft rejection:

Cobbold et al, Nature 1986:
• Depleting CD8+ T cells had no effect in a mouse skin graft rejection model
• Depleting CD4+ cells alone delayed graft rejection from 15 to 30 days
• Depleting both CD4+ and CD8+ delayed rejection to 60 days

Bolton et al, 1989:
• Rapid rejection of an allogeneic kidney transplant can be induced in a Nude rat by adoptive transfer of CD4+ but not CD8+ T cells

Question 7

What are the two ongoing challenges for organ donation?

Answer 7

1. Organ Supply → demand heavily outweighs supply
   • 2014/2015 – around 7000 wanting a transplant, around 3500 transplants occured from 1200 donors (more transplants than donors because some people donate multiple organs)
   • 507 died whilst waiting for a transplant
   • 807 were taken off the list because they were no longer suitable
   • For the first time in 11 years there were less transplants in 2015 than previous years – one of the reasons being that many people died in a way that made them unsuitable for donation
   • The other reason is that despite having people on the registry, - they still need consent when they take the organs, and they cant seem to get this above 60%
   • For kidney transplant – there were 107 altruistic donors in 2014/15 (living donors)
2. Managing Organ Rejection
   • Long-term immune suppression (although we have improved the risk of short term rejection, the kinetics of long-term rejection have stayed the same)
   • Chronic rejection → may tolerate the organ for a while, but you will eventually need a new one

Question 8

What are some routinely conducted clinical transplants and their survival rates?

Answer 8

Kidney - nephritis and diabetic complications - 17,000 in 2009 - 80%

Liver - cancer, cirrhosis - 6000 - 68%

HSC - leukemia, lymphoma, SCID - 15,000 - 40-60%

Cornea - keratitis - 15,000 - 70%

Skin - burns

Question 9

How do we name the types of graft?

Answer 9

ο Named based on relationship between the donor and recipient
ο Syngeneic/Isogeneic í genetically identical
Autograft - from self
Isograft - between identical twins
ο Allogeneic í non-genetically identical donors
Allograft
ο Xenogeneic í between different species
Xenograft
ο The immune response is named in the same way - eg, allorespnose, xenoresponse to alloantigens, xenoantigens.

Question 10

How was it discovered that the gene loci responsible for the most vigorous allograft rejection are located in the MHC?

Answer 10

• They figured this out using congenic mice:
− Have a mouse of one strain, and by breeding you can end up with a mouse that was originally a different strain, but is now the same strain just with one piece of genomic DNA left that is different
− That is, the two differ in only one locus
− If you take skin from the congenic strain onto the parent strain and it is accepted, you know everything in the bit of genome that is different isn’t seen by the immune system
− But if the graft is rejected, you know something encoded in that bit of retained genome is something recognised by the immune system

Question 11

What are the two key features of the MHC?

Answer 11

• The MHC is polygenic – it contains several different MHCI and MHCII genes
• It is also polymorphic – there are multiple alleles of each gene within the population as a whole

Question 12

Describe the MHC gene cluster

Answer 12

• In humans, the MHC (or HLA) is located on chromosome 6, spans up to 7 million base pairs and contains over 200 genes
• 3 class I molecules → HLA A, B and C
• 3 class II molecules → HLA DP, DQ and DR – in many people, the DR cluster contains a duplication of the beta chain that can pair with the alpha, so this means 3 sets of genes can actually give 4 types of MHC-II
• MHCI presents to CD8
• MHCII presents to CD4

The MHC is in linkage disequilibrium with many other genes:
• They are within the class III region
• Complement components
• TNF
• MICA/B → upregulated under stress, recognized by NKG2D. Also polymorphic
• TAP → associated with antigen processing and presentation
• TAPBP
• LMP → assocated with the proteasome

→ Note that many genes in the MHC-III are associated with innate and adaptive immunity
→ The fact it is in LD with genes for antigen presentation suggests the entire MHC has been selected for during evolution.

Question 13

What are the MHC polymorphisms and their implications?

Answer 13

• Because of the polygeny, each person expresses at least 3 different MHCI and II molecules on their cells.
• However, the number of MHC expressed is much greater because of the extreme polymorphisms – there are multiple alleles of each gene within the population
• The number of alleles ranges from 7 in HLA-DRalpha (functionally monomorphic) to over 4000 in HLA-B
• Note – number of alleles doesn’t mean number of proteins, because sometimes new alleles are non-functional.
• One allele is inherited form each parent at each locus, and is co-dominantly expressed.
• Each allele is relatively frequent – so there is only a small chance the gene loci on both homologous chromosomes of an individual will have the same allele – most individuals will be heterozygous.
• The particular combination of MHC alleles found on a chromosome is known as an MHC haplotype
• Because most individuals are heterozygous, most matings will produce offspring that have one of 4 possible combinations → thus siblings only have a 25% chance of being a match
• MHC polymorphisms seem to have been selected for by evolution:
− Point mutations can be classified as replacement (which change the amino acid) or silent (which change the codon, but the amino acid is the same)
− Replacement substitutions occur within the MHC at a higher frequently relative to silent than would be expected, providing evidence that polymorphisms have been actively selected for.

Question 14

What is meant by the MHC haplotype

Answer 14

• The particular combination of MHC alleles found on a chromosome is known as an MHC haplotype
• So, closely linked genes in the HLA are usually inherited as a set – a haplotype
• So families not only inherit the same MHC, but also all the other genes in linkage disequilibrium
• These genes will have polymorphisms too – so this is even more of an advantage with family – you inherit the whole package, not just the MHC
• This is important, because eg) MICA and MICB are polymorphic too – and have been implicated in some forms of rejection.

Question 15

What are minor histocompatibility antigens

Answer 15

• These are the gene loci responsible for less vigorous graft rejection
• MiHA are receptors on the cell surface of donated organs known to give an immunological response in some transplants
• They cause problems of rejection less frequently than those of the MHC
• MiHA are due to normal proteins that are themselves polymorphic in a given population → even when a donor and recipient are compatible in terms of their MHC genes, the amino acid differences in minor proteins can cause the tissue to be slowly rejected

So:
• An allelic different in any gene that leads to the presentation of a peptide that was not previously tolerised against in the host is likely to lead to rejection.
• However, the reactions seem to be restricted to a few epitopes, dubbed immunodominant

Examples:
• Encoded on the Y sex chromosome:
− The most thoroughly studies
− Known as H-Y antigens
− Responses are very frequent (37-50%) in women with previous male pregnancies
− Male anti-female responses aren’t seen, because both males and females express X-derived genes
• Encoded by polymorphisms on autosomes
• Mitochondrial encoded (maternally derived)

Question 16

Define allorecognition and alloresponse

Answer 16

• Allorecognition describes the recognition of transplanted allogeneic tissue by the host
• Alloresponse describes the effector mechanisms recruited in the reaction to the foreign tissue, and the outcome of those effects.

Question 17

Describe direct allorecognition.

Answer 17

• Donor APCs are transplanted along with the graft – these go to draining lymph nodes
• T cells recognise the donor MHC and peptide
• This is likely to happen very early in the response – however the donor MHCs are not being continually replaced, as the graft is away from the donor
• So as the response continues, the number of donor MHCs decrease
• However, by then we already have activated T cells

But! From what we know about MHC restricted, T cells should only recognise peptide presented on self MHC?

• Remember, donor MHC and peptide weren’t available during central tolerance in the thymus. The donor MHC, if presented, may have been positively or negative selected against – but it wasn’t presented, so there was no negative selection against it. Just because donor MHCs arent selected against doesn’t mean it cant bind – it just wasn’t shown it, you only get shown self MHC.

• MHC-dominant binding (‘high determinant density model’):
− Donor MHC resembles self-MHC → so together resembles self MHC, foreign peptide
− This model predicts that that every MHC molecule on a cell surface can serve as a ligand for an allospecific T cell, then the antigen density on the cell surface would be extremely high in contrast with the density of a specific peptide + specific MHC.
− The high ligand density available for stimulating alloreactive T cells implies that receptors of much lower affinities would be able to respond to foreign MHC, leading to a high frequency of allreactivity.
• Peptide-dominant binding (‘multiple binary complex model’):
− Proposes that recognition of the peptide bound is of primary importance
− Multiple different bound peptides, in combination with one allogenic MHC, may produce determinants recognized by different cross-reactive T cells
− This model predicts that if each bound peptide is an essential component of the determinant recognized by alloreactive T cells, each peptide-allo-MHC complex will be recognized by a different alloreactive T cell, and a single MHC incompatibility can stimulate a wide diversity of T cells

→ Vigorous response, because a high frequency of T cells can recognise the graft, and could be high affinity because no negative selection has occurred.
→ Also fast, because don’t need to wait for antigen processing

Question 18

Describe indirect allorecognition.

Answer 18

• Similar to normal reaction to foreign antigens
• Involves antigen processing of donor peptides by recipient MHCs
• These can be any process allelilc antigen (minor antigen) or processed allo-MHC
• Tends to be from necrotic and apoptotic cell material

Question 19

Describe semi-direct allorecognition

Answer 19

• Donor APC interacts with recipient APC (cell-cell contact)
• Recipient APC pulls some MHC off the donor APC (cross-dressing) and presents the donor APC
• It can also pick up donor MHC released from exosomes
• Now you have both direct and indirect together

Question 20

Describe the relative contribution of each method of allorecognition.

Answer 20

• Passenger APCs in donor tissue at the time of transplantation dictates the direct method is vigorous during early-post engraftment period. It diminishes with death of donor ACPs over time
• The indirect method requires antigen processing – so is less rapid, but continues for the life of the graft as the donor antigens will always be present.
• The relative contribution of semi-direct is unkown
• Of clinical relevance is the fact that rejection is most common in the early period, while tolerance develops later – fits in with the fact TRegs have indirect alloreactivity.

Question 21

What is the role of innate immunity in rejection (what releases DAMPs, evidence for role of TLRs, role of DCs, macrophges and NK)

Answer 21

• Because transplantion of some tissues is inherently non-sterile, and ischaemia and surgical trauma release PAMPs and DAMps, these systems are relevant to transplantation biology
• TLRs are a well studies family of PRRs – found on immune cells but also endothelial cells and organ parenchyma cells – these recognise the PAMPs and DAMPs

What releases the DAMPs?
• Organ procurement → physical stress and damage
• Ischaemic reperfusion injury →
− Ischaemia: restricted blood flow to isolated organs can cause shortage of oxygen and nutrients such as glucose. Causes cell death and build up of metabolic intermediates
− Reperfusion: Restoration of blood flow results in inflammation and cell damage – ROS, complement

• Specific examples include hyaluronan, heparin, fibronectin and byglycan
• Several studies have examined a role for TLR signals in allorecognition:
− Using minor Ag (H-Y Ag) disparate skin transplants, it was shown that MyD88 KO female recipients (adaptor needed for IL-1 and TLR signaling) did not reject the graft.
− One study reports a model by which absence of MyD88 impairs DC production of IL-6, rendering alloreactive T cells more susceptible to suppression by TRegs
− In the clinic, it has been found that lung and kidney transplant recipients heterozygouts for TLR4 funcitonal polymorphisms had a reduced incidence of acute allograft rejection → however increased incidence of infection.

Roles of the cells of the innate immune system:
• DCs:
− Mature DCs convey Ag and stimulate T cells
− However, it is recognized that in the absence of danger signals, immature DCs expressing no co-stimulatory molecules results in anergy or apoptosis of the T cells
− Given this, there is a potential use of immature DCs to promote T cell tolerance as a therapeutic → a study has shown injection of immature DCs prolonged survival of a fully mismatched graft.
• Macrophages:
− Unlike DCs, don’t play a direct role in induction of allrecognition because they ineffectively prime naïve T cells
− Nontheless, within 24h of transplantation, macrophages of both donor and recipient origin infiltrate the allograft and proliferate
− In the absence of rejection, number decreases, but in acute rejection they accumulate to comprose 40-60% of the infiltrate
• NK:
− Their activation is based on a balance between activating and inhibitor signals
− Due to ‘missing self’ on allografts, NK cells are cytotoxic to target cells mismatched for MHC-I
− They are not sufficient for solid organ rejection on their own, but they act as facilitators by amplifying early graft inflammation and supporting the activity of allroeactive cytotoxic T cells.

Question 22

Describe hyperacute rejection

Answer 22

• Occurs within minutes to hours (usually within the first 48 hours)
• Is a result of pre-formed donor antibodies (usually high affinity IgG against HLA antigens or low affinity IgM against ABO blood groups) from a previous sensitizing event
• These days, because of pre-screening, it is a rare occurrence
• Sensitising events include: pregnancy, blood transfusion, previous transplant
• Antibodies bind to the graft blood vessel endothelium and recruit effector cells, triggering clotting, complement and kinin cascades leading to vascular thrombosis
• Complement activation → inflammation → vascular damage → platelet aggregation → activation of the clotting cascade → blockage of the vessel

Question 23

Describe acute rejection

Answer 23

• The equivalent of a response to a pathogen
• Usually occurs between 5 days and 3 months after the transplant
• With acute rejection – there are different stages of immune response against the graft

Sensitisation phase:

1. Allorecognition and activation of CD4+ helper cells
2. Generation of effector cells

Effector phase:
3. Attack on the graft:
− DTH → giving soluble mediators and macrophage-mediated immunity
− Generation of anti-graft antibody → resulting in complement and NK-mediated ADCC
− Direct killing of graft cells → recognition of allo-MHCI on the graft and direct killing of the target cell by apoptosis (perforin/granzyme; Fas/FasL)

Question 24

Describe chronic rejection

Answer 24

• Slow progressive loss of graft function (years)
• Eventually leads to blockage of vessels and ischaemia
• The mechanism isn’t fully understood, is a complex process involving components of vascular and immune system
• A chronic inflammatory reaction in the vessel wall leads to intimal smooth muscle proliferation and vessel occlusion
− Mainly caused by:
➢ Cytokines released from activated T cells (indirect recognition)
➢ Allo-specific antibodies reactive to the vessel endothelium (often associated with C4d deposition) → many cases of antibody-mediated rejection misclassified as drug toxicity because they are C4d negative, but it is probably playing quite a large role.
➢ Drug toxicity
• Both cell-mediated and allo-antibody reponses implicated
• Current kidney graft half life is 10 years
• Responds poorly to immunosuppressive theraoy → these tend to stop the T cell response, but if it isn’t mediated much by T cells then they wont have an effect.

Factors increasing the risk of chronic rejection:
• Previous episode of acute rejection
• Inadequate immunesuppression
• Initial delayed graft function
• Donor-related factors (old age, hypertension)
• Reperfusion injury
• Long cold ischaemia time
• Recipient related factors (diabetes, hypertension, hyperlipidemia)
• Post-transplant infection (cytomegalovirus)

Question 25

What are the applications of a HSC transplant?

Answer 25

• To treat disease involving haematopoetic cells, eg) primary immune deficiency, blood cancers, aplastic anaemia
• Previously involved transfer of whole bone marrow by aspirating from iliac crest – now can transfer HSCs from peripheral or cord blood.

Question 26

Describe the sources of stem cells for HSC transplant.

Answer 26

Cord blood:
• First done in the 1980’s
• Used in children as it was thought cell dose will be limited, however now dual cord blood is routinely used for adults, providing sufficient cell dose
• Achieves high engraftment rates and outcomes equivalent to unrelated donor transplants
• Readily available
• Lower risk of GvHD reported
• High cost

Peripheral blood:
• Mobilise blood with GM-CSF and collect using apheresis (outpatient procedure – severy hours on each of several consecutive days until adequate quantities reached)
• Cyclophasphamide, a non-stem cell toxic agent is frequently used for mobilisaton and can lead to a 50-fold increased in GM-CFUs
• Yields high numbers that can be cryopreserved, and generates faster reconstitution after transplantation
• Stem cell quantitation now performed with flow cytometry and monoclonal antibodies to identify the stem cell antigen CD24 – allows for reproducible means of quantifying stem cells
• Problem – contains a greater number of T cells, having implications in GvHD

Question 27

Describe the features of autologous HSC transplant.

Answer 27

• Potential risk of tumour contamination in the graft

* As treatment-related mortality rates are

Question 28

Describe the features of allogenic HSC transplant

Answer 28

• HSC transplants using bone marrow from normal donors first done in late 1960’s
• Allogeneic transplantation using HLA-matched sibling donors is now the treatment of choice in young patients with severe aplastic anemia, with long-term survival rates of >80%
• Beyond simply restoring haemopoeisis in primary hypocllular disorders, HSC transplants allow the administration of highdose myeloablative chemotherapy to eradicate malgingnay
• It is highly important to HLA match (donors often siblings) because you are implanting a full foreign immune system. If you don’t, GvHD WILL happen – the donor T cells will attack the repicient

Indications for allogeneic HSC transplant:
• Severe aplastic anaemia → new diagnosed patients under 40 with a sibling donor
• ALL → Philadelphia+
• CML → imatinib resistant, advanced phase

Question 29

Describe conditioning for HSC transplant

Answer 29

• Chemotherapy used to ablate underling haematological disease
• Immunodeficiency ensures (can be 12 months to fully reconstitute)
• Possibility of ‘mixed chimerism’ – only partial ablation to ‘make space’”
− Total ablation of host haemtopoetic system was presumed to provide the greatest chance of reducing relapse risk
− Advances in tecniques for chimerism detection have demonstrated that complete donor chimerism is not as common as thought, and may not be necessary for long-term disease free survival
− Mix chimerism – a state where donor and recipient haemtopoetic cells persist together may be desirable by decreasing the risk of GvHD

Question 30

Describe the features of GvHD after HSC transplant.

Answer 30

• Donor cells attack cells and tissues of the host
• HSC transplantation involves transfer of a competent donor immune system
o Mature donor T cells contaminating the HSC prep are responsible
o Particularly relevant if mismatches at MHC I or II (therefore carful matching)
o Also occurs in context of minor mismatches – so can develop even if the donor pair are matched for HLA antigens.
o Immune suppression needed

• The old definition of acute GVHD was GVHD within 100 days of transplant; however, the new definition relies more on symptoms.
• It generally affects three primary organ systems: the skin, liver and gut. These organs normally act as integral barriers to infectious stimuli that can enhance local inflammatory reactions, hence are not surprising target organs for GVHD.
• Epithelial damage leads to rashes and exfoliation of the skin, bile duct degeneration in the liver with increased conjugated bilirubin and intestinal crypt cell necrosis typically leading to profuse, watery diarrhoea.
• The incidence of GVHD is dependent on a host of factors, the most important of which is donor–recipient HLA disparity.
− In HLA-matched sibling transplants, the incidence of acute GVHD is 30–60%. The high incidence of acute GVHD with HLA-MUDs, and its occurrence even in HLA-identical twin donors (syngeneic transplants), points to the importance of discrepancy between minor antigens outside the MHC.
− Other factors that increase the incidence of GVHD include sex mismatching and donor parity, greater number of donor T cells infused and older recipient age.
− Advanced severity of GVHD predicts for poor outcome.
− Prophylactic immunosuppression significantly decreases the risk and severity of acute GVHD and is an integral component of post-transplant care. Without prophylaxis, virtually all patients will develop some degree of acute GVHD

• Although most frequent following allogeneic BMT (91) GvHD does also occur in association with solid organ transplants including liver (92), intestine (93), lung (94) pancreas (95) and kidney (96).
• The relative risk of developing GvHD (higher in BMT than in solid organ transplants) is thought to be related to the number of donor lymphocytes which are transferred along with the allografted tissue
− Way more donor lymphocytes transplanted in BMT obviously!

→ Deplete T cells prior to transplantation? Would stop GvHD, but…

Graft vs. Leukemia:
• In some cases, the donor T cells attack the recipient leukemia cells

So you want a balance beween GvL and GvH

Question 31

Describe the potential of xenotransplants

Answer 31

• Thought could be a way to deal with demand for organs – people thought pig organs were the answer
• Problems:
− Hyperacute rejection → gal-a1,3galactose is absent from humans, and so preexisting human abs initiate rapid hypercute rejection
− Failure to regulate human complement – no complement control ability – so complement was attacking
− Concerns regarding transfer of pig virsues

Strategies to circumvent hyperacute rejection:
• Knockout the sugar from the pig
• Make the pig transgenic for human complement control proteins DAF and CD59
• Transfect the cells with acetyl transferase to change the sugar

Reardon, “new life for pig-to-human transplants’, Nov 2015, Nature:
• Thanks to improved immunosuppressant durgs and advances in genome editing technologies such as CRISPR/Cas9
• Can edit pig genes which could cause rejection or inection
• The first a-gal knockout in pigs took 3 full years, now they can do it in 150 days
• CRISPR has even been used to knockout two genes simultaneously
• Researches have transplanted CRISPR modified pig organs into monkeys, one of which has survived for more than 3 months
• Some researches expect to see human trials with solid organs such as kidneys from genetically modified pigs
• United Therapeutics, a biotech company has spent $100million to speed up the process of making transgenic pigs for lung transplants → first major investment in more than a decade. They want to see clinical trials by 2020.

Question 32

Describe how bioengineering is advancing the field of transplantation

Answer 32

MSCs for Trachea Repair
• In 2008, a stem cell based tissue engineerd windpipe was transplanted in woman with end-stage post tuberculosis left main bronchus collapse
• In this therapy, they harvested a cadaver trachea and decelluarised it – left the matrix
• They then recellularised it with the patients own MSCs
• No need for a donor, and no risk of rejection
• Treatment invented by Paolo Macchiarini
• This was also performed on a 10 year old – and due to the urgency of his operation, they didn’t wait and culture his stem cells in the lab, they allowed them to culture on the trachea in his body
• They are now able to use synthetic scaffolds, rather than relying on decellularised cadaveric material.

Question 33

How is the demand for organs changing the source of organs away from fit and healthy donors?

Answer 33

Cancer after receiving lung from a smoker
• Death of a transplant patient reveals that 4 in 10 lungs com form donors who smoke
• Cystic fibrosis sufferer was given double lung transplant but died from cancer, after being told her donor was a smoker.
• Her family said if she had been told, she would have refused the lungs
• New figures show 35% of all organs used in transplants now come from higher risk donors
• Surgeons have even usd the organs of those over 80, drug addics and cancer sufferers.

“Risk of transfer of infection:
• “Man has to choose between possible death or HIV after being offered life-saving kidney from donor who may have been infected”
• Waited 4 years for a donor kidney and pancreas
• Found out donor had died from a drug overdose
• HIV test result would take 2 weeks, but transplant had to be immediate
• He took the gamble, and was later told the donor did not have HIV.

“Make best use of available donors”
• “HIV positive donors can transplant to HIV positive patients”
• Johns Hopkins University School of Medicine first to perform the first kidney transplants
• Estimated that 500-600 organs from HIV positive donors go to waste every year
• Allowing these donations could save 1000 people
• Because these transplants have never been done before, most medical facilities do not have systems in place to handle them.

Question 34

Other than the health of the donor, what types of donor are considered for transplant (donor, brain function, timing of transplant and caveats)

Answer 34

Cadaveric No – no heart beat or breathing. Organ damage occurs within minutes. Determination of legal time of death, rapidity of organ removal. Not much time to sort out paperwork. Organ must be transplanted immediately

Brain-dead No – breathing with respirator Within hours Trauma for family disconnecting the respirator. But have time to sort out paperwork and get consent.

Living related Yes Pre-arranged Must be altruistic, potential surgical risk to donor

Living non-related Yes Pre-arranges Must be altruistic, potential surgical risk to donor

Living heart donor Yes - healthy heart, assisted breathing Domino transplant Donor trades perfectly functional heart for a donated one/

Question 35

Describe how optimal donor management helps avoid organ rejection at the point of the donor.

Answer 35

The health of the organ starts at the point someone is identified as a potential donor –> as soon as identified, they are monitored.
Can monitor salt balances, treat inflammation in response to brain death.

Early identification of donors:

• requires cooperation with colleagues in the ICU and emergency department
• early applications of advanced resus. techniques, can produce good results for patients in previously unsurvivable circumstances.
• appropriate tecniques for diagnosis of brain death required.

Specific donor treatment:

• Methylprednisolone given for anti-inflammatoyr effects, inflam processes triggered by brain death worsens outcome.
• However, multi-organ procurements shown to be associated with rises in both pro and anti inflammatory cytokines even when MP given.
• Early arginine vasopressin advocated in many guidelines

Question 36

Describe how organ preservation techniques helps avoid organ rejection at the point of the donor.

Answer 36

• Speed of delivery → all organs in the UK sent out to people by the UK registry. Location will be an issue – determines the speed you can get it there
• Perfusion → keeping a liquid going through the organ so you don’t get buildup of molecules that can lead to reperfusion injury

There has been some research in this area in terms of lung transplants.
• 175 lung transplants performed in the UK in 2011-2012
• Average wait for a lung = 412 days; but 25% mortality on the waiting list
• Only 15-20% of donated lungs were deemed suitable for transplantation in 2012
• “Ex vivo lung perfusion” – artificial perfusion and ventilation of donated lungs
• Facilitates assessment, resuscitation and potential repair of suboptimal donor lungs
• Dampening of stress signals and washing out of donor-derived leukocytes

Stone (2016) Altered immunogenicity of Donor Lungs via Removal of Passenger Leukocytes Using Ex Vivo Lung Perfusion → Dr James Fildes research group in the MCCIR, Manhcester.
• Passenger leukocyte transfer from the donor lung to the recipient is intrinsically involved in acute rejection. Direct presentation of alloantigen expressed on donor leukocytes is recognized by recipient T cells, promoting acute cellular rejection.
• We utilized ex vivo lung perfusion (EVLP) to study passenger leukocyte migration from donor lungs into the recipient and to evaluate the effects of donor leukocyte depletion prior to transplantation.
• EVLP-reduced donor leukocyte transfer into the recipient and migration to recipient lymph nodes was markedly reduced.
• Recipient T cell infiltration of the donor lung was significantly diminished via EVLP. Donor leukocyte removal during EVLP reduces direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection.

Question 37

Describe blood typing as a method of screening to avoid graft rejection

Answer 37

Blood Typing
• The first transplantations were blood transfusions
• Recipient anti-blood group antibodies react with blood group alloantigens on endothelial cells of the graft → hyperacute rejection
• Blood group antigens are carbohydrates – A and B differ in terminal sugar, O doesn’t have a terminal sugar
• This is less of an issue for transfer of HSCs as there is no vasculature

ABO typing:
• Mix recipient erythrocytes with known anti-A or anti-B serum
• Antibody mediated agglutination indicates the recipient blood type

Reverse ABO typing:
• Recipient serum with serum with erythrocytes of known A or B blood type
• Agglutination indicates the presence of recipient anti-A, B or both
• Reveals anti-blood group antibodies generated by sensitization

Question 38

Describe HLA typing as a method of screening to avoid graft rejection

Answer 38

• Solid organ → HLA-A, B and DR considered – DR being the most significant
• HSCT → HLA-A, B, C, DR&DQ (umbilical cord A, B & DR)
• Typing is performed twice prior to transplantation
• As you get an allele from each parent, each match is worth 2. So if you match all HLA types for solid organ transplant, you get a score of 6. Max score for HSCT is 10.
• You need to minimise number of HLA mismatches to:
− Avoid allorecognition and rejection
− Avoid sensitization
− This is where they may not be a full match, so you produce antibodies against the organ, but you have enough immunosuppression so the organ isn’t rejected
− This means if you have another transplant, you are sensitized and it may reject

Reducing HLA mismatching:
• From renal transplant data, the more mismatches, the reduced chance of graft survival and the increased amount of people receving treatment for rejection
• From HSCT data, it seems matching is much more critical. There is a big difference between a 10/10 mismatch and a 2-4 mismatch.
• With solid organs, it is still better to be match but you can do more with the immunosuppression, and GvHD is less of an issue.

Choice of donor:
• Remember! The MHC is inherited as a haplotype, and you not only inherit the MHC, but some minor antigens too
• Therefore, in order to get the best match you would go:
1. HLA identical sibling
2. Other HLA identical related donor
3. Unrelated donor

How is HLA typing performed?
• Sequence specific primer HLA typing → PCR amplification using HLA allele specific primer set, if you see a band on the blot you have your HLA type
• Sequence specific oligonucleotide probes → PCR amplification of HLA loci using loci specific primer sets, include fluorescently tagged probes that will identify specific alleles
• Sequencing (high resolution matching) → PCR amplification of HLA loci using loci specific primer sets, then sequence the amplified products.

Question 39

Describe HLA-specific antibody screening as a method of screening to avoid graft rejection

Answer 39

• As well as testing to make sure they don’t have antibodies before getting the graft, the recipient is repeatedly tested to check they aren’t becoming sensitized
• Sensitisation is defined by the detectable present of antibodies to allo-antigens (eg, anti-HLA)
• Sensitising events:
− Pregnancy
− Transfusion of blood
− Previous transplant
• Not everyone that has a sensitization event will become sensitized

HLA-Specific antibody screening using Luminex:
• Get a bead, coat it with a HLA allele
• The antibodies bind to the bead – which is a mix of two dyes
• There are different intensities of the two dyes within each microbead, with a max of 100 microbeads per reaction
• You can make a range of these beads with a slightly different colour, each with a different HLA allele on
• You can mix these beads with a small amount of serum, and anti antibodies recognizing any of those allleles will bind
• You then add fluorescently labelled anti-humanIg and use two colour FACS to analyse

Question 40

Describe the CDC cross match reaction as a method of screening to avoid graft rejection

Answer 40

• Is a functional assay – the other assays tell you if you have presence of antibodies or particular assays,, this is actually a functional assay proving the rejection would happen

The complement dependent cytotoxicity cross-match reaction:
1. Patients serum mixed with donor lymphocytes in wells that contain a stain, and left to incubate for 1 hour
2. During the hour, if there are antibodies in the patient serum directed towards the donor, they will bind
3. After 1 hour, complement is added to the reaction and binds patient antibodies if they have bound to donor cells
4. If bound, complement will activate MAC complexes, making the donor cell membranes leaky
− Positive reaction → cell membrane disrupted, allowing the stain to enter and intercalate with the DNA
− Negative reaection → stain gets trapped in the unbroken cell membranes

→ With this, don’t need to know the identiies of the antibodies doing the damage, it is enough to know they are present
→ This one takes a little longer, and you need access to both donor and recipient.

Question 41

What is the difference between induction and maintenance immunosuppression?

Answer 41

• Induction therapy → Drugs given at high dose before, or at the time of, the transplant to prevent sensitization and inhibit initiation of alloresponses
• Maintenance therapy → Lower dose of drugs to ensure continued graft survival. If there is sign of rejection, can change the dose and make it higher until the episode is resolved.

Question 42

Describe corticosteroids as a conventional immunosuppressive drug.

Answer 42

eg) Prednisone (analogue of cortisol)
• Act via translocation of the GC receptor transcription factor to the nucleus, affecting numerous genes
• Have side effects, so often used in combination with other drugs to lower dose

Decrease IL1, TNFa, GM-CSF, IL-3, IL-4, IL-5, CXCL8 Decreased inflammation caused by cytokines

Decreased NOS Decreased NO

Decreased
phospholipase A2 and COX
Decreased prostaglandins

Decreased adhesion molecules Reduced leukocyte migration

Increased endonucleases Induction of apoptosis in lymphocytes and eosinophils

Question 43

Describe anti-proliferative agents as a conventional immunosuppressive drug.

Answer 43

• Developed originally to treat cancer
• Targets rapidly dividing cells
• In addition to other side effect, have GI tox

eg) Azathioprine
• First drug developed to allow allo-transplants to occur
• A purine analogue → interferes with purine synthesis
• First introduced in 1962 in combination with steroids
• Standard agent (50% graft survival) for use in transplants until CsA came around
• Works because it targets all highly proliferative cells – so it targets the T cells
• However, has GI tox as it targets the proliferating cells in the gut
• Being replaced in some centres by mycophenolate

eg) Cyclophosphamide
• Alkylating agent – crosslinks DNA so cant separate
• Very toxic

Question 44

Describe immunophilin binders as a conventional immunosuppressive drug.

Answer 44

• An immunophilin is a protein that binds with high specificity to immunosuppressive agents
• These inhibitors start to target in on the T cells

Calcineurin inhibitors:
eg) Cyclosporin A and Tacrolimus
• Bind to the cylophilin A and FKBP immunophilins respectively
• Target calcineurin, blocking production of NFAT and hence preventing transcription of IL-2
• Tacrolimus has the same effect as cyclosporine but at a lower dose
• These have become the primary immunosuppressive agents for liver transplantation – rejection free rates of 40 and 55% respectively
• Reduce TReg activity

Non-calcineurin inhibitors:
eg) Rapamycin
• Also binds the FKBP immunophilin, but in this case the target is mTOR
• This blocks the cell cycle
• Faciliates the survival and development of TRegs

→ If you tip the balance to TReg rather than TEff, youll get tolerance
→ So for a tolerising protocol, you would add Rapamycin

Question 45

How can therapeutic mAbs me manipulated to reduce immunogenicity?

Answer 45

Fc function manipulation:
• Select ideal recombinant isotype → often, IgG4 doenst bind much
• Mutation of the glycosylation site Asn297 of human IgG1 → Acglycosl form
• Mutation of two Leu residues to Ala residues → Non-FcR binder
• Avoids side effects of unwanted effector functions

Question 46

Describe the T lymphocyte depleting induction agents as biological immunosuppresive drugs

Answer 46

rATG (rabbit or horse-derived anti-thymoglobulin)
o Polyclonal anti-lymphocyte antibody
o Widely used in solid organ and HSC transplant protocols
o It also modulates the immune response through effects on other immune cells – B cells, DCs, NK, endothelial cells, disrupts trafficking
o Selective preservation of Foxp3+ Treg (reduction of CD4/CD8 Teff)

Drawbacks;
o Each batch is slightly different (new immunisation)
o Contains many foreign proteins - cytokine release

Alemtuzumab (anti-CD52)
o Humanised monoclonal
o Depletion of lymphocytes
o The ones that come back are okay around the new organ.

Question 47

Describe the non-lymphocyte depleting induction agents as biological immunosuppresive drugs

Answer 47

Daclizumab (Anti-IL2R a chain/CD25)
o CD25 expressed by activated but not resting T cells
o Blocks IL2 mediated clonal expansion of T cells
o Both approved for induction therapy
o Efficacy in reducing acute rejection similar to OKT3 and rATG, with fewer side effects

Belatacept (CTLA-4-Ig receptor fusion protein)
o Engineered to bind both co-stimulatory molecules CD80 & CD86
o Co-stimulation blockade
o Blocks sensitisation and tips the balance of activation to T cell anergy
o Clinical trials in renal transplantation indicated lower toxicity than CNI but higher incidence of acute rejection in yr 1
o Used in a CNI-sparing immunosuppressive regime

Note: Tregs need CTLA-4 to become activated. So cant get tolerance if you use this.

Question 48

Describe targeting the B-cell mediated rejection using biological immunosuppresive drugs

Answer 48

• → You may have an organ for someone, but you’ve done all the screening and you know they have antibodies against it. So people are trying to come up with ways to overcome this (as it causes hyperacute rejection).
• B cells are also increasingly being found to have a role in chronic rejection too.
• B cells are involved in the sensitisation – they produce allo-Abs, meaning that if you have a subsequent graft, you will get hyperacute rejection.

Plasmapheresis:
• Plasmapheresis (PP)-based removal of circulating antibodies and immune complexes has been utilized to treat systemic and kidney diseases since the 1970s.
• Upon cessation of PP, however, patients had rebound increase in their antibody levels which was ameliorated by maintenance cyclophosphamide, azathioprine, and corticosteroid therapy.
• These experiences provided the first evidence of rebound in antibody levels following PP, which remains a significant barrier today in desensitization.
• PP removes alloantibodies from the circulation; however, PP also removes all plasma proteins including clotting factors.

Immunoadsorption:
• Immunoadsorption (IA) provides an alternative approach for removing alloantibodies allowing selective removal of IgG antibodies while sparing other plasma proteins including clotting factors.
• However, most IA columns are not FDA approved and are unavailable in the United States.

Rituximab (anti-CD20)
o Depletion of all B cells except early pro and plasma cells
o Rituximab can deplete B cells via ADC) by natural killer (NK) cells, complement-dependent cytotoxicity (CDC), and apoptosis → polymorphisms in the FcyRIII gene affect the effectiveness

Joosten et al: Performed a randomized double blind study:
Affect of Rituximab on acute rejection:
• Immunologically high-risk patients (PRA N6% or re-transplant) not receiving rituximab had a significantly higher incidence of rejection (13/34, 38.2%) compared to other treatment groups.
• Although most rejections were T cell mediated, rituximab treated patients tended to have less antibody mediated rejections, compared to placebo-treated patients.
• After 24 months, the cumulative incidence of infections and malignancies was comparable. We concluded that a single dose of rituximab as induction therapy did not reduce the overall incidence of acute rejection, but might be beneficial in immunologically high-risk patients to reduce the incidence of BPAR in immunologically high-risk patients to a level comparable to that in immunologically low risk patients.

IVIG
o Compete with allo-ab for binding FcRn → decrease half life
o Role in up-regulating inhibitory FcRγIIB

Newer potential B cell specific targets
o Anti-BAFF → BAFF/BAFFR interaction is essential for B cell activation and plasma cell development
o Bortezomib → proteasome inhibitor, targets 26S proteasome (normally actively degrading excess protein in active plasma cells - build up → plasma cell death)
o Eculizumab → humanised anti-C5; blocks cleavage to C5a (anaphylatoxin) and C5b, preventing generation of MAC. Prevents complement mediated effector functions.

Question 49

How do TRegs regulate?

Answer 49

• Production of anti-inflammatory, pro-regulatory cytokines: TGF-B, IL-10, IL-35
• Expression of inhibitor receptors (CTLA-4)
• Modulation of DCs
• Outcompete effector T cells for resources (CD25 mops up IL-2, so not available for proliferation → cytokine sink)
• Direct killing of T cells – using granzyme and perforin
• More recently, context dependent inhibition has emerged as strategy of regulation:
− Treg cells appropriate partial or “aborted” forms of the transcriptional programs of respective target T helper (Th) cell types by expressing their master transcription factors and co-opting their function
− For example, in the case of a Th1 response, Treg cells upregulate the expression of T-bet which in turn induces the expression of some Th1 related genes such as CX3CR1 but not others, enabling Treg cells to migrate to sites of Th1 cell mediated inflammation while restraining their differentiation into Th1 cells.

Question 50

Describe the experiments that allowed us to understand tolerance

Answer 50

• Recipient A receiving a graft from B → graft rejected
• Repicient A treated with anti-CD4/CD8 antibody → graft accepted
• Take Recipient A 60 days after the transplant and give it a 2nd graft → graft is still accepted
− Long-term acceptance, A has been tolerised to B
• However, if you take recipient A 60 days after the first transplant and give it a graft from C → rejected
− So, it isn’t that they’ve just got used to the inflammation from the first graft, as another graft later is accepted
− They also haven’t lost the ability to reject, because a graft from a new strain is rejected
− You therefore have long-term, antigen specific tolerance
• The induced tolerance is dominant and infectious:
− Dominant tolerance → If you take competent CD4+ cell that should induce rejection, and inject them into a tolerised mouse along with a graft – the graft is still accepted
− Infectious tolerance → If you take tolerised T cells that wont reject, and put it in a new naïve recipient along with a graft, they accept the graft
• The induced tolerance also shows linked suppression:
− Linked tolerance means tolerance to one set of antigens can be extended to others co-existing in the same tissue, without need for therapeutic intervention
− If you take a graft from an F1 mouse (ie, B and C cross) into recipient A that is tolerised to B, the mouse will be able to accept grafts from both A and B

Question 51

Define operational tolerance

Answer 51

• This occurs when you have non-compliance, or physician-driven withdrawal of immune suppression, but the patient is fine
• This form of tolerance is defined as ‘a well-functioning graft lacking histological signs of rejection in the absence of any immunosuppressive drugs (at least for a year) in an immunocompetent host

Question 52

What are the potential biomarkers of operational tolerance that have been found for kidney and liver transplants?

Answer 52

From Mastoridis et al, 2015:
• a small, selected group of patients in whom immunosuppressive treatment has been discontinued for one reason or another without adverse effects on graft function have become the focus of intense study.
• This group of patients, described as displaying ‘clinical operational tolerance’ (COT), are few in number, but their very existence has instigated efforts to reproducibly induce a comparable state of immunological unresponsiveness to donor alloantigens (‘induced tolerance’) in a larger number of transplant recipients, and thereby to expand the pool of candidates eligible for weaning off immunotherapy.
• The induction of immunological tolerance is held by many to be the Holy Grail of transplant research

Biomarkers in kidney transplantation:
• Approximately 100 cases of COT have been reported thus far in kidney transplant recipients
• Broadly, the cross-platform signature identified from consortia studies is dominated by B-cell features.
• There is agreement across different centres that both the percentage and absolute number of B cells are increased in COT
• One hallmark feature of all these studies is the absence of detectable donor-specific antibody.
• B cells overexpress the receptor transducing inhibitory signal FCgRIIb
• Additionally, the increased ratio of TGFb/IFNg and the elevated numbers of IL10-producing B cells also point towards a more tolerogenic functionality in the B cells of COTs.
• Across sites, with different cohort characteristics and with different microarray platforms, 35 genes were found to be specifically enriched in COTs. Of these, 24 (69%) were B cell related
• Regulatory T (Treg) cells dominated the early investigations of operational tolerance. The widely held belief that tolerance represented a tipping of the balance of effector to regulatory cells was partially supported by a study showing increased Treg numbers in a tolerant group of kidney transplant recipients compared with recipients undergoing chronic rejection.
• Nevertheless, it is apparent that measurement of Treg levels alone is an inadequate biomarker of tolerance. More promising are Treg-associated markers: in particular, a high ratio of expression of FoxP3 to a-1,2-mannosidase expression has been described in tolerant patients. This ratio has also previously been demonstrated as being associated with good graft function in animal models.

Biomarkers in liver transplantation:
• Reports of spontaneous operational tolerance are common in liver transplant recipients. Until very recently, an average estimate was that around 20% of liver allograft recipients were operationally tolerant if weaned off immunosuppression.
• However, in a trial of immunosuppression withdrawal recently completed by Benitez et al. 79.2% of patients who had had their graft for over 10.6 years were successfully weaned off immunosuppression.
• Comparison of the biomarker profiles of tolerant kidney and liver transplant recipients does not reveal significant overlap. Unlike the B-cell signature associated with operational tolerance following kidney transplantation, spontaneously tolerant liver transplant recipients have been characterized by transcriptional patterns enriched for the expression of genes related to specific T-cell subsets, as well as NK cells
• Regarding T cells, microarray and realtime PCR (RT-PCR) assays performed by MartinezLlordella et al. defined a tolerance signature which included the upregulation of gdTCRþ T cells, whereas phenotypic analysis of peripheral blood showed an increase in Tregs
• The ability to offer a mechanistic interpretation from these studies, however, has been hampered by their retrospective design and their reliance on the analysis of peripheral blood without concurrent allograft tissue analysis → so they can see what markers are in the blood, but cant see what is actually happening to the graft.
• The mechanisms of allograft tolerance may in fact be of a similar nature to those of self-tolerance in an inflammatory environment. Hence, the location in which to detect a biomarker signature of tolerance is likely to be the allograft tissue itself.

Question 53

Describe how graft tolerance could be induced in the clinic by altering the balance of Effector T cells

Answer 53

• Deletion – during induction therapy (eg, rATG or Alemtuzumab) to remove lymphocytes → protects against allorecognition and rejection
• Deletion alone is insufficient for sustained acceptance
• The body doesn’t like being lymphocyte depleted – so it trys to reexpand the population → homeostatic repopulation
• However, when this happens, you don’t always get an equal balance
− Alemtuzumab → rebound of Tmemory and Teffector cells
− Rapamycin → rebound of TRegs

So:
• Essentially, after the induction therapy and lymphodepletion, you get homeostatic repopulation, but this is tipped in devour of Teffector cells, so you get rejection → this is an IL-7 mediated effect
• Waldman and colleages suggest that if you have physician intervention at this stage, you could selectively favour TReg proliferation and survival over Teffector, and tip the balance towards tolerance
− This is known as physician guided reconstitution
− Has been attempted in mice using an anti-IL7 receptor blocking antibody and added Rapamycin

Question 54

Describe how graft tolerance could be induced in the clinic by co-stimulation blockade

Answer 54

CTLA-4-Ig
• Belatacept (binds both CD80 and CD86)
• Co-stimulation inhibition
• Reduced toxicity compared with calcineurin inhibitors, and a better 5 year renal function, but higher episodes of acute rejection.
• However, it also blocks both CD28 and CTLA-4 (ie, inhibits both co-stimulatory and co-inhibitory signalling).
• Therefore, CTLA-4 signals important for the activation and function of TRegs are blocked.
• Binding of CD80 to PDL1, which also acts to inhibit T cell activation, is also blocked.

→ We may need to consider selective blockade of CD28. Monovalent Fabs and PEGylated Fab forms are being developed.

Question 55

Describe how graft tolerance could be induced in the clinic by adoptive cell transfer of TRegs.
What are the decisions for this?

Answer 55

• It seems plausible that TRegs expanded in vitro could be used to induce tolerance in humans.
• To test this hypothesis outside of the clinic, a number of humanized preclinical mouse models (21) have tested the efficacy of ex vivo expanded human Tregs for the prevention of transplant rejection.
− In these models, immunodeficient miceare ‘‘reconstituted’’ with human immune cells and receive an allogeneic human transplant, such as islet cells, blood vessel, or skin. Coinjection of ex vivo expanded Tregs in to these models has clearly shown that Tregs prevent the rejection of transplants and development of vascular injury.
• First case of in-man study of ex vivo expanded Treg to treat GvHD demonstrated [Trzonkowski et al (2009)]
• Used in umbilical cord blood transplantation - Patient receiving Tregs showed lower incidence of acute grade GvHD, with no toxicity associated
with transfusion of Tregs nor increased infective episodes.

• No reports of use in solid organ transplantation, but recruitment of patients to kidney (ONE study – multicentre trial across Eurpoe) and liver (ThRIL study – singe centre, Kings Collage) phase I trials plus others.

Hurdles/decisions:
• Which Treg to use? (multiple subpopulations; so do you use bulk or specified sub populations)
• Foxp3+ TRegs
• TR1 cells
• Th3
• iTr35
• How to isolate them? (there are small numbers and poor markers in vivo)
• Magnetic bead
• Flow cytometry
• Surface markers that differentiate TReg subpopulations:
− CD127
− CD45RA → marker for naïve T cells
− Helios → thymic Tregs
− Nrp-1 → thymic Tregs
• Polyclonal or alloag specific?
• Protocols for expansion of antigen specific Treg?
• [Putnam et al, Am J Transplant 13:3010-3020 (2013) – alloag reactive Treg, expanded under GMP (good manufacturing practice)]
• TRegs isolated using a FACSAria based on the phenotype of CD4+ CD127lo CD25+
• Considerations of instability of isolated/expanded Treg? (how do you make them stay as Tregs, and not have reversion to TH17?!)
• Tregs and Th17 cells are diametrically op- posed functional lineages (suppressive vs. proinflammatory, respectively), with the latter implicated in transplant rejection
• Studies of pTRegs and Th17 cell differentiation has enabled data supporting the assertion that Tregs retain the ability to express Il-17 and Th17 transcriptional profiles in inflammatory environments. Such plasticity in Tregs could be potentially hazardous in the context of Treg-based therapy for human transplantation.
• Murine studies on Treg-Th17 plasticity have yielded contrasting data sets (79). Notably, Komatsu et al. observed that CD25+ cells resisted conversion after extensive proliferation under polarizing conditions and exhibited stable Foxp3 expression.
• In humans, the data are more robust, with clear demonstration that cells both functionally and phenotypically indistinguishable from bona fide Tregs can be induced to express IL-17. Initial observations suggested that ‘‘Th17-plasticity’’ was restricted to CD25hiCD127loCD27+ CD45RACD45RO+ memory Tregs. Notably, these Tregs retained their ability to suppress Teffs when induced to produce IL-17(48, 85), suggesting that IL-17 production is compatible with suppressive ability.
• Expense of individualised cell therapies?
• Regulatory issues?

Question 56

Describe how graft tolerance could be induced in the clinic by adoptive cell transfer of tolerogenic APCs.
What are the decisions for this?

Answer 56

• Implied role in infectious tolerance
• Delivery of antigen to immature DC (anti-Dec205) induces tolerance
• Immature DC and DC treated in vitro with agents such as TGFβ, IL10, vitamin D3, rapamycin have all been described as tolerogenic (low MHC and co-stimulatory molecule expression & resist activation by danger signals)
• study has shown injection of immature DCs prolonged survival of a fully mismatched cardiac allograft

Hurdles;

• No uniform criteria for in vitro tolerogenic APC
• Stability of tolerogenic state when introduced in vivo?
• Migration in vivo?
• Expense of individualised cell therapies?
• Regulatory issues?

Other cell types being developed as potential immunoregulatory cell-based therapies:
• regulatory macrophages
• myeloid derived suppressor cells

Question 57

Describe how graft tolerance could be induced by mixed donor chimerism.

Answer 57

• Mixed chimerism refers to a hybrid immune system.
• Perform a conditioning partial myeloablative regiment to ablate some of the recipient immune cells
• Transplant donor bone marrow or HSCs alongside graft
• Donor HSCs engraft and coexist with recipient stem cells → gives permanent and stable donor specific tolerance.
• Because this results in repopulation of the thymus with donor-derived dendritic cells, donor-reactive T cells will be deleted by negative selection. Because such donor-derived stem cells have the ability to replicate perpetually, they theoretically continue to provide donor-type DCs indefinitely.
• A number of successful clinical cases in have been reported whereby patients with hematological indications for bone marrow ablation who also require renal transplantation have received a BMT and a kidney transplant from the same donor, resulting in long-term donor-specific tolerance.
• Nevertheless, the morbidity and mortality of myeloablative therapy and risk of graft-versushost disease (GvHD) in most transplant recipients makes this mode of therapy unacceptable to those without a hematological indication for bone marrow ablation. On the other hand, mixed chimerism, where donor cells represent a varying proportion (but not 100%) of the total hematopoietic pool is a more promising area of research (Kawai et al., 2011). Mixed chimerism can be established using non-myeloablative conditioning regimens, therefore maintaining immunocompetence and reducing the risk of GvHD.

There is evidence for the operation of both central deletional and peripheral regulatory mechanisms in mixed chimerism.
• Peripheral regulation also takes place whereby newly developing donor-reactive and recipient-reactive T cells that escape negative selection in the thymus are suppressed in the periphery by regulatory T cells
• A series of promising clinical trials utilizing mixed chimerism for the induction of tolerance have been performed.
• An initial trial enrolled six patients with renal failure consequent to multiple myeloma (Fudaba et al., 2006). Patients received nonmyeloablative BMTs and renal transplants from an HLA-identical sibling followed by a donor leukocyte infusion as treatment for both the multiple myeloma and renal failure. Four patients transiently developed mixed chimerism, which was later lost, while the other two patients eventually developed full donor chimerism. Interestingly, all patients successfully accepted their renal transplants long-term (up to >9 years) without any immunosuppression.