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Molecular Mechanisms > Transplantation and therapies > Flashcards

Transplantation and therapies Flashcards

1
Q

how was transplant rejection discovered to be controlled by the immune system?

A

“The fate of skin homografts in man”
- Used skin grafts – solid organ transplant
- If the skin graft was taken from same donor to recipient, from one part of body to another = survival
- If donor and recipient are different, graft is destroyed

First description of rejection, mediated by immune system

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2
Q

what was the first successful kidney transplant?

A

renal transplant between identical Herrick twins:
- Successful: If donor and recipient are the same (identical twins), they survive
- Recipient (Richard) survived for 8 years before dying of an unrelated condition
- Donor (Ronald) died in 2010 at the age of 79
- Living-related transplantation

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3
Q

why is transplantation important?

A

Transplantation is the therapy of choice for end stage organ failure
- Well over 1 million transplants performed to date
- Described as one of the medical miracles of the 20th century

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4
Q

how widespread are transplantations?

A

157000 solid transplants completed in 2022
- Every year has 10% more transplants performed compared to previous year
- Therapeutic option across the world, not just in the US – applicable worldwide
- Kidney is most frequently transplanted, then liver, heart, lung, pancreas, small bowel

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5
Q

what key immunological concepts were discovered via transplantation?

A
  • discovery of MHC: cells from different people reject and kill each other
  • Demonstration that T cells are produced by the thymus (Miller, 1962) – used skin grafts to show these animals lacked T cells
  • Discovery of acquired immune tolerance
  • Discovery of the CD25+CD4+ regulatory T cells (Hall, 1990) in transplantation – found to have suppressive activity
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6
Q

what are the different types of transplant (based on effect of life)?

A

life-saving: renal, liver, heart
- Any transplantable organ: technical limitations, but can be done
- Cellular transplants e.g. bone marrow, islets for diabetes

Life-enhancing:
- e.g. cornea – easy to control rejection as it is in an immune privileged site
- Hemi and full-face transplants
- Arm transplants – 150 of those completed
- Single and double-leg transplants

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7
Q

what are the different types of transplant (based on donor/recipient)?

A

the donor is the recipient = autograft /autologous transplant – e.g. skin, bone marrow

Genetically identical recipient of same species e.g. identical twins = isografts (isogenic or syngeneic transplants) – e.g. renal live organ transplant

Genetically non-identical recipient of same species = allogeneic /allograft transplant

Xenograft/xenogeneic transplant – e.g. heart valve from pig – not very immunogenic

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8
Q

what are the molecular targets of acute rejection?

A

gene products of HLA genes (donor MHC)
- these are the most polymorphic proteins in biology e.g. MHC-DQ has over 34000 alleles
- Polymorphism enables billions of MHC allele combinations which drive rejection

Any protein different between donor and recipient can generate weaker rejection:
- e.g. H-Y is a male antigen which stimulates immune response in females
- Male-female twins will have some rejection due to H-Y

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9
Q

what cells are the most crucial for acute rejection?

A

T cells are required for acute rejection

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10
Q

how were T cells shown to drive acute rejection?

A

Neonatally thymectomised mice do not reject allografts:
- Lacking T cells via thymic removal protects from rejection

Mutants which lack T cells are protected from rejection e.g. nude, RAG-/-

Mice depleted of T cells (using mAbs) do not reject allografts

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11
Q

what other cells are involved during acute rejection?

A

T cells are critical, but also involves innate immunity e.g. neutrophils, NK cells, macrophages
- Dendritic cells are needed for T cell activation
- donor DCs present within the graft have different MHC to the recipient
- donor DCs included in the allograft contain donor MHCs – recipient DCs may internalise these donor MHC molecules – causes rejection as T cells recognise these MHC alloantigens

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12
Q

what are the chain of events leading to acute rejection?

A
  1. ischemia/reperfusion injury
  2. innate immune attack of transplants
  3. dendritic cell trafficking for initiating adaptive immunity
  4. T cell responses to transplants - pathways of recognition
  5. T cell subsets and mechanisms leading to graft rejection
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13
Q

what is ischemia in transplantion?

A

when donor organ is removed, its vessels are clamped, so it is starved of oxygen and undergoes ischemia:
- this happens for both cadaver and live-organ transplants
- full oxygenation to hypoxia, then anoxia – no oxygen at all in the donor tissue
- there is lack of blood supply
- organ metabolism generates waste products which get stuck in tissue and cause inflammation

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14
Q

do all grafts suffer ischemia/reperfusion injury?

A

All grafts will suffer some form of ischemia /reperfusion injury and surgical trauma
- Some grafts can be transplanted as late as 24 hours after harvest
- Living donor transplants have a better outcome than cadaveric organs.
- Prolonged ischemia can result in delayed graft function and immunological consequences

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15
Q

what is reperfusion injury?

A

when the donor organ is plumbed into the recipient, the recipient blood rapidly flows in, which causes more damage
- first interaction of recipient with donor tissue is via the blood hitting the endothelial cells
- innate immune attack of the transplant

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16
Q

how does ischemia/reperfusion lead to injury and surgical trauma?

A

Hypoxia is detected by transplant cells – leads to ROS production which causes inflammation
- leads to necrosis of graft
- Release of TNFa, IL-1B, IL-6
- Upregulation of donor MHC and chemokines or cytokines
- Endothelial cells within the vessels provide first contact between recipient and donor
– endothelium becomes activated and permeabilised and upregulates adhesion molecules for recipient immune cells to get into the donor organ
- Graft “Flagged” as an Inflammatory Target – no matter being placed on ice to slow metabolism

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17
Q

how are TLRs implicated in transplant rejection?

A

TLRs recognise PAMPs
- These are expressed by endothelial and stromal cells and are modulated upon activation
- TLRs will also recognise DAMPs released by the transplant
- Activation of TLRs 2, 1, 4, RAGE (receptor for HMGB1) and responses to uric acid/ATP
- This leads to inflammation, secretion of cytokines and chemokines
- Caspase 1 activation via uric acid and ATP – inflammasome formation for IL-1B and IL-18 production

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18
Q

how do immune cells react to graft hypoxia and waste products when the graft is plumbed in?

A
  • Leads to leukocytes in blood meeting activated endothelium
  • Graft damage from neutrophils, NK cells, macrophages e.g. ROS, proteolytic enzymes, FAS-FASL, PFN-GZMB
  • but these cells can die off quickly in the absence of T cell response – only cause some graft damage
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19
Q

how do innate immune cells exacerbate immune rejection of the graft?

A
  • they exacerbate inflammation by releasing pro-inflammatory cytokines e.g. TNFa, IL-1B, IL-12
  • Also produce chemokines CXCL9, CXCL10 recognised by CCR3 on activated T cells for recruitment
  • also produce CCL3-5 to recruit immature DCs
  • enable T cell activation against the graft
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20
Q

what are the features of immature DCs?

A
  • Highly pinocytic – pull in environmental antigens
  • Low levels of MHC
  • Low levels of costimulatory molecules
  • Low-level cytokine secretion
  • Poor stimulators of the T cell response
  • Express inflammatory chemokine receptors to localise at sites of inflammation to gather antigen
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21
Q

how do immature DCs become activated and mature?

A

TLR signals
Costimulatory molecules (CD40)
Inflammatory cytokines

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22
Q

what are the features of mature DCs?

A
  • Poorly pinocytic
  • High levels of MHC
  • High levels of costimulatory molecules
  • High-level cytokine secretion
  • Potent stimulators of the T cell response
  • Upregulate lymphoid chemokine receptors and downregulate inflammatory chemokine receptors to enter lymphoid tissue and activate T cells
  • Aiming to stimulate T cells
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23
Q

are donor DCs important for rejection?

A

Kidney transplant into immunosuppressed rat – no rejection, but donor DCs become activated and leave graft into periphery
- donor DCs are now no longer in the graft - DC-free graft
- Retransplanted DC-free graft into normal rat, and it wasn’t acutely rejected – (but did have gradual attrition of graft function due to chronic rejection)
- Absence of donor DCs reduces rejection
- Addition of donor DCs back into the normal rat restores rejection
- donor DCs crucial in acute rejection

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24
Q

overall, what are the first key stages in acute rejection?

A
  • ischemia/reperfusion injury and innate responses act on the graft
  • this causes inflammation and upregulation of cytokines, DAMPs etc
  • these act on resident donor DCs in the graft to mature
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25
Q

what do donor DCs do once they have matured in the graft?

A
  • these upregulate lymphoid receptors and home to recipient lymphoid tissue
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26
Q

what do immature recipient DCs do following transplantation?

A
  • originally, these reside in recipient lymphoid tissue and express inflammatory chemokine receptors
  • immature recipient DCs home to the site of inflammation, so migrate into the transplant
  • these recipient DCs acquire alloantigen in the graft
  • immature DCs become activated and mature, express lymphoid-homing chemokine receptors and migrate to lymph node
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27
Q

what is alloantigen in acute rejection?

A

donor MHC

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28
Q

what DCs are found in the recipient lymphoid tissue following a transplant?

A

mature DCs of donor and recipient origin
- T cells can recognise these DCs in different ways

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29
Q

what are the allorecognition pathways of T cells and DCs?

A
  1. direct pathway
  2. indirect pathway
  3. semidirect pathway
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30
Q

what is the direct allorecognition pathway?

A
  • T cells recognise donor MHC+peptide as an intact molecule, presented by donor DCs
  • this is a cross-reaction
  • T cell precursor frequency activated by donor MHC in this pathway is 1-10%
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31
Q

what is the indirect allorecognition pathway?

A
  • this is normally how any antigen e.g. pathogenic is recognised
  • recipient DC, with recipient MHC, presents peptide from donor MHC to the T cell
  • T cell precursor frequency activated by donor MHC in this pathway is 0.01-0.0001%
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32
Q

what is the semidirect allorecognition pathway?

A
  • in tact donor MHC+peptide is presented by a recipient DC to a T cell
  • T cell precursor frequency activated by donor MHC in this pathway is 1-10%
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33
Q

how can a donor DC with donor MHC+peptide stimulate so many T cells?

A

Hypothesis: Likely to be mostly type 2 and type 3 alloreactive interactions which may be responsible for high frequency of responding T cells to alloantigen

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34
Q

what is a type 1 alloreactive interaction?

A

Self-restricted, peptide-specific = indirect pathway
- contact of TCR and MHC, where the peptide specificity drives the response e.g. in infection

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35
Q

what is a type 2 alloreactive interaction?

A

Allorestricted, peptide-specific:
- MHC interaction with TCR needed with specific peptide
– allorestricted
– TCR transgenic mice are alloreactive: need specific peptide and MHC
- combination of specific peptide with donor MHC that stimulates high frequency of T cell responses

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36
Q
A
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36
Q

what is a type 3 alloreactive interaction/

A

Alloreactive peptide-dependent:
- TCR interacts with MHC, where peptide is needed, but this peptide doesn’t need to be specific
contact with peptide
- TCR interacts with donor MHC, but not specifically with peptide – peptide does need to be there

37
Q

what is a type 4 alloreactive interaction?

A

Alloreactive peptide-independent:
- no contact between the TCR and peptide at all
- completely MHC-dependent, doens’t need peptide interaction
- likely not involved in high T cell stimulation

38
Q

summary of allorecognition:

A

Peptides that we are tolerant to are usually presented by our own, recipient MHC
- When that same peptide is presented by donor MHC, this combination is enough to activate the T cell
- so, the T cell no longer tolerant to that peptide because of the interaction with donor MHC
- these peptides are usually housekeeping peptides, but when presented by donor MHC, they are now immunogenic

This is only a hypothesis - not proven yet

39
Q

how does the semidirect pathway work?

A
  • donor MHC and donor peptide from a donor DC, being presented on a recipient DC
  • this happens via graft itself, or donor DCs, secreting exosomes: blebs of membrane which has sample of membrane that includes donor MHC
  • These blebs stick to recipient APCs
  • These cross-dressed DCs have own recipient MHC, but also have exosome stuck to them with donor MHC to drive alloantigen response
40
Q

how is intact, donor MHC recognised in acute rejection?

A

Recognition of intact donor MHC predominate during acute rejection
- Direct pathway - lots of donor DCs available to stimulate T cells
- Semi-direct pathway – unclear whether this pathway stimulates
rejection or is tolerogenic, and unclear where the exosomes come from – may depend on organ

41
Q

how is donor MHC recognised in chronic rejection?

A

Indirect pathway responses predominate long-term and
may be more important in chronic rejection – recipient DCs present donor MHC and peptides from graft
- Donor DCs eventually die, so recipient DCs indefinitely drive chronic rejection
- later, chronic responses driven by indirect pathway
- May relate more to CD4 rather than CD8 T cell responses as there is evidence that CD8 T cells can be activated by donor endothelium

42
Q

what T cell subsets are implicated in acute rejection?

A

Dominant response is TH1 – cytotoxicity via CD8 T cells via IL-2, and delayed-type hypersensitivity – hyperactivate macrophages to kill graft
- default is TH1

Eosinophils in transplant suggest Th2 response

Neutrophils in grafts indicates th17 response

Removal of IFNy stops Th1 developing, leading to Th2 cells which reject via eosinophils
Block both Th1 and Th2 leads to Th17-driven rejection

43
Q

how are memory T cell responses involved in rejection?

A

Naïve T cell responses via DCs won’t have seen alloantigen before
- primary encounter with antigen A = differentiation and clonal expansion of peptide A-specific T cells with effector function to kill target – most undergo apoptosis and leave behind a small population of memory cells
- Memory cells reactivated with peptide A proliferate quicker and have rapid effector function – larger secondary response

44
Q

what are the features of naive T cells?

A
  • require co-stimulation
  • slow acquisition of function
  • require lymphoid tissues
  • susceptible to Treg suppression
45
Q

what are the features of memory T cells?

A
  • Less dependent on costimulation - easier to activate without DCs
  • Rapid effector function
  • Can enter tissues without activation – circulate between tissues normally
  • Resistant to Treg suppression

better for infection, worse for transplantation

46
Q

why might alloreactive memory T cells arise prior to transplantation?

A

Sensitisation:
- e.g. blood transfusion where donor MHC is the same as a later graft, patient has been sensitised to that donor allele MHC so will have an alloreactive response

47
Q

Do transplant recipients that have not been previously exposed to alloantigen still have alloreactive memory T cells?

A

Took blood from people who have or haven’t had previous exposure to alloantigen:
- Split blood from those that haven’t been exposed into groups of memory and naïve T cell populations
- Challenged blood with alloantigen to see what the precursor frequency was in memory and naive populations
- Both populations responded the same – we have a pre-existing pool of alloreactive memory and naïve cell already acquired, whether exposure to alloantigen has happened before or not

48
Q

what clinical evidence is there for pre-existing alloreactive memory T cells?

A

Looked for alloreactive memory T cells before living kidney transplant (Tx):
- used Elispot to look for memory T cells producing IFNy within 24hrs of activation in vitro
- Patients with low memory cells prior to Tx = less rejection
- Patients with high memory cells prior to Tx = these patients experienced acute rejection

Non-sensitised patients have alloreactive memory T cells which are different depending on immune history
- This correlates with acute rejection – memory T cells are harder to control with conventional than naïve for rejection
- the more memory, the more likely to reject

49
Q

how have immunosuppressants evolved?

A

Use of immunosuppression predates transplantation in the 60s, was used in early 20th century
- irradiation was first used
- Pharmacological agents
- azathioprine - anti-proliferative agent
- Steroids
- Polyclonal lymphocyte-specific sera
- Cyclosporine (calcineurin inhibitor)
- Anti-CD3 – first monoclonal antibody licensed for transplant
- FK506 - calcineurin inhibitor

50
Q

what is the impact of evolving immunosuppressants on transplant rejection?

A

When transplant centres formed with radiation and steroids – 80% grafts were lost in first year
- Azathioprine reduced graft rejection to 60% in first year
- Stepwise reduction in rejection in first year as cyclosporine and anti-CD3 came in
- Monoclonal antibodies, anti-proliferative agents, calcineurin inhibitors reduced rejection even further

51
Q

how were these immunosuppressants discovered?

A

These were discovered in mixed lymphocyte reaction assays between donor and recipient populations and seeing proliferation in response to one another
- Immunosuppressive drugs tend to be aimed at T cells

52
Q

how do immunosuppressants target T cells?

A
  • Interrupt/suppress TCR signals e.g. calcineurin inhibitors – stops dephosphorylation of NFAT so it can’t enter nucleus and act as TF – stops cell cycle initiation and IL-2 production
  • MPA, MMF and LFL prevent T cell clonal expansion – interrupt nucleotide synthesis to inhibit proliferation
    Rapamycin (sirolimus) binds FKBP and inhibits mTOR activation (downstream of IL-2) – prevents T cell growth factor signalling
53
Q

what are current depleting and induction therapies?

A

After transplant, donor DCs stimulate immune response via direct pathway, where up to 10% T cells recognise the graft - need to inhibit this, so the induction therapy is harsh:
- Semi-targeted anti-CD25, anti-thymocyte globulin – depletes T cells, anti-CD3 depletes T cells, anti-CD52 depletes haematopoeitic cells to clear immune system before transplant

Avoids initial large response to transplant to keep transplant in place

54
Q

following initial transplantation, what therapy are patients moved on to? how is this therapy limited?

A

Double or triple therapy:
- Calcineurin inhibitor e.g. cyclosporine or tacrolimus, corticosteroid, anti-proliferative agent (MPA/MMF)
- Want to remove calcineurin inhibitors as they damage the kidney - nephrotoxic

55
Q

how effective are immunosuppressants?

A

95% 1 year graft survival for renal transplant, but not 100% effective
- efficiency also differs according to tissue, liver is more resistant to rejection compared to the heart as it is larger and stays in place
- Improved 1 year graft survival with every decade due to immunosuppressants
- there is still high attrition/loss of graft over 15 years: only 50% of heart transplants survive for 15 years
- immunosuppressives are useful for the first year graft survival, but have no impact on long-term, chronic rejection

56
Q

why are immunosuppressants limited as a therapy?

A

they are non-specific, so they suppress all T cells regardless of their specificity
- they don’t distinguish between protective responses against pathogens and destructive responses against the graft
- Only a small number of T cells are alloreactive, but immunosuppressants target the total T cell pool
- This reduces infection and tumour immunity - causes increased risk of infection and malignancy

57
Q

what are the main causes of death for heart transplants in the 1st year vs >10 years?

A

In first year: 12% deaths due to acute rejection, 30% due to infection - immunosuppressants leaving patients susceptible to fatal infection

In >10 years:
- Cardioallograft vasculopathy (chronic rejection) causes death in 15%
- 20% dying due to malignancy
- 10% dying due to infection

Immunosuppressants increase risk of infection and cancer: major cause of mortality in patients

58
Q

what non-immune complications are caused by immunosuppressants?

A

Nephrotoxicity – calcineurin inhibitors
Hypertension
Hyperlipidemia
Diabetes disposition
Patients take immunosuppressants for life – long-term
- £10,000 per year per patient
- non-compliance: when patient stops taking the meds, leading to rejection
- The meds are unpleasant – many tablets

59
Q

what is transplant tolerance?

A

Allograft acceptance without the need for continuous immunosuppression

60
Q

how should transplant tolerance be generated?

A

Develop reagents/strategies to keep graft in place without long-term meds:
- Only target alloreactive T cells, so that cancer and infection T cells are unaffected
- Short-term therapy to prevent long-term rejection
- establish immune regulatory networks with Tregs to maintain the graft

61
Q

what happens when transplant tolerance is induced in trial renal transplant patients?

A

5/8 patients had no immunosuppression after 12 months and retained graft - became tolerant

3/8 patients had good grafts but had withdrawal, so stayed on low dose immunosuppressants

62
Q

is transplant tolerance achievable in humans? what are the drawbacks?

A

It is achievable, but only applicable to certain patients
- Chemotherapy, cyclosporine, total irradiation - intense short-term treatment
- The patients had a living donor renal transplant, as well as a BM transplant
- More cyclosporine
- all these treatments in conjunction with MMF and tacrolimus – anti-proliferatives
- patients evenutally weaned off MMF and tacrolimus to be independent of immunosuppression

Highly invasive and not applicable to everyone e.g. can’t have living heart transplant

63
Q

how was transplant tolerance discovered?

A

In cows:
- Cows often bear twins which are dizygotic, so have different dads
- anastomoses form between the blood vessels of respective placentas of the twins, so the twins have shared blood supply
- If cow has a male twin and female twin, male hormones enter female, so she becomes sterile – this won’t be known until female is at reproductive age
- used blood group antigens to identify cows that had anastamoses

64
Q

what happens when twin cows shared a blood supply?

A

Twins are dizygotic (different dads), so the different dads provide different blood group antigens to the twins
- Bled the twin animals at birth – if they contain cells with blood group antigen of opposite cow, they shared the same blood supply and are chimeric
- cows which aren’t chimeric lacked the anastamoses
- Chimeric cows that grow up can accept skin grafts between each other, despite being dizygotic due to different paternal inheritence – tolerance of each other

65
Q

how was acquired transplant tolerance first demonstrated?

A

Took CBA pregnant mice:
- Exposed foetuses in utero to strain A mice cells via injection
- Challenged the born mice with skin graft from strain A mice = the born mice accepted, so were tolerant to antigens of strain A mice
- If challenged with skin graft from a different strain, this caused rejection
- forms CBA mouse with strain A skin graft, with no rejection, and vice versa
- can also be done to chickens - become tolerant to what they are exposed to in oval (in utero)

66
Q

how should transplant tolerance be translated into adult humans?

A

Rejection is dictated by the balance of the immune effector arm (Th1) and regulatory arm (Tregs)
- during rejection, the effector outweights the regulatory arm
- Aim of tolerance induction is to reduce the effector arm whilst increasing the number/function of Tregs – tip balance to tolerance, not rejection

67
Q

in what ways can transplant tolerance be induced?

A

centrally: bone marrow or thymus

peripherally: manipulating Tregs

68
Q

how can we induce central thymic tolerance?

A

K/O CD4 T cells from periphery using anti-CD4 and put cells of donor into thymus (intrathymic injection):
- Newly generated CD4 T cells have now been exposed to donor cells in the thymus and should be tolerant
- These animals now accept donor heart graft long-term
- Deplete CD4 cells and inject cells from different mouse strain = rejection

Manipulation of central tolerance

69
Q

how was haematopoietic chimerism first demonstrated?

A

Lethally irradiated BALBc mice:
- depletion of T cells to avoid graft vs host disease
- gave BALBc mice a B6 mouse strain BM transplant – these two strains are completely MHC mismatched
- Allow T and B cells to reform in the BALBc mice, but these cells are derived from the donor, not the recipient = tolerance is induced
- the BALBc mice can now accept a B6 heart graft, as they have donor-specific tolerance

70
Q

how has haematopoietic chimerism been limited?

A

T cells are positively selected by cTECsin the thymus, and these are radio-resistant:
- T cells produced by B6 donor are selected by recipient MHC
- but the APCs are also B6-derived, so have donor MHC also - there is MHC mismatch - semi/non-specific immunosuppression
- recipient APCs are required for immunocompetence

Lethal irradiation is fully myeloablative:
- Lethal irradiation causes K/O of neutrophils
- Short-term neutropenia can cause problems with infection

71
Q

how has haematopoietic chimerism been improved?

A

switched to MIXED haematopoietic chimerism

Also replace myeloablative therapy to lymphoablative
- small dose of irradiation to not completely knockout recipient bone marrow, but enables donor BM to engraft
- means neutropenia will not occur
- Produce both donor and recipient haematopoetic cells =
chimeric, tolerant animals

72
Q

has haematopoietic chimerism worked in large animal models?

A

this approach worked in non-human primates but did not
require long-term chimerism
- enables organ graft from donor to survive long term without immunosuppression
- some of these primates were not chimeric but still had tolerance - role for immunoregulation?

73
Q

has mixed haematopoietic chimerism worked in humans?

A

5 patients – received HLA mismatched kidney and BM from living-related donor:
- initial immunosuppressive treatment: cyclophosphamide, anti-CD2, cyclosporine, thymic irradiation

1 patient rejected as they had a B cell response - antibody-mediated rejection
4 patients – immunosuppression stopped ~ 12 months and remained rejection-free for 2-5 years without immunosuppression

74
Q

does mixed haematopoietic chimerism work long term against chronic rejection?

A

11 years post renal transplant, analysed serum creatinine for renal function:
Patient 1 = no-immunosuppression post year 1, still year 11 with no rejection = tolerant
Patient 8 = full rejection even on immunosuppression
Patient 10 = reduced doses of tacrolimus, caused rebound rejection – tried to save with immunosuppression but lost graft after 3 years
Patient 4: maintained graft after removal of tacrolimus – but 5 years after transplant, still some rejection – not 100% tolerant to graft – something re-initiated the response

Can it ever cause full, maintained tolerance?
Can infection break tolerance?

75
Q

how could peripheral transplant tolerance be induced?

A

downregulate effector cells:
- e.g. block co-stimulation, cytokines, adhesion molecules, chemokines, signalling pathways
- block th1 receptors or neutralise IFNy

enhance regulatory cells:
- e.g. nTregs, iTregs, NKT, M2/MDSC, tolDC, MSC, Breg
- Increase regulation to hold grafts in place

76
Q

what is the rationale of co-stimulatory blockade?

A

T cell needs TCR-MHC+peptide, plus co-stim signal 2 and signal 3 for optimal activation
- this is what drives alloreactive T cell activation, leading to rejection

Drugs tend to interfere with TCR signal – calcineurin inhibitors block TCR signal to stop rejection
- But as soon as the inhibitor is stopped being given, the TCR signal can be reactivated for rejection

77
Q

How can alloreactive T cell co-stimulation be blocked?

A

Co-stimulatory blockade allows specific TCR and donor MHC interaction, but prevents co-stimulation
- T cell gets signal, but not enough for response
- Incorrect activation, leading to tolerance
- even if the blockade therapy is stopped, there is still no rejection = anergy and tolerance are induced as they have incorrect response

78
Q

what are the possible results of blocking T cell co-stimulation?

A
  1. Can induce anergy and inactivation
    - Altered TF programme – NFAT but without AP-1 complex, making the T cell unresponsive to the TCR signal
  2. activation-induced apoptosis:
    - TCR interactions cause T cells to proliferate rapidly and then die
    - Loss of anti-apoptotic molecules e.g. BCL-XL, and loss of IL-2 due to lack of co-stimulation
  3. Conversion to iTregs in high TGFb environments
79
Q

why is co-stimulatory blockade limited?

A

Co-stimulation is not a single interaction:
- Many different molecules have been identified to have co-stimulatory function
- There are also co-inhibitory molecules which share ligands with co-stimulatory molecules e.g. CTLA-4 and CD28 bind CD80/86 - complex interactions

80
Q

what was the first demonstration that blocking co-stimulation could induce tolerance?

A

Mouse model of skin graft with full MHC mismatch:
- Acute rejection within 12 days without treatment
- CTLA-4 Ig treatment: binds CD80/86 and blocks CD28 co-stimulation – still rejection
- Antibody to CD40L (co-stim molecule) – still rejection
- If both CD28 and CD40L are blocked = mice become tolerant and maintain their graft long-term, even with therapy withdrawal after 6 days - operational transplant tolerance induced
- short-term delivery results in long-term acceptance without chronic immunosuppression

81
Q

how has co-stimulatory molecule blockade translated into the clinic?

A

CTLA-4 Ig e.g. abatacept in RA:
- Belatacept is a modified version of CTLA-4 Ig for transplant – currently in clinical trial:
- medium intensity, low intensity belatacept, and control is calcineurin inhibitor plus MMF and corticosteroids

Kidney graft functinn:
- Cyclosporin = nephrotoxic
- Belatacept = grafts in better condition as CD28 is blocked by CTLA-4 Ig

82
Q

overall, is belatacept a good therapy for transplants?

A

Belatacept is as good as a calcineurin inhibitor
Kidney transplant is in better shape – increased glomerular function
Side-effect profile good
Increased acute rejection episodes but none of the grafts were lost

But not meant to be used to induce tolerance in this setting

83
Q

what other co-stimulatory blockade therapies are undergoing trials?

A
  • CTLA-4 Ig can have issues, so may use molecules to target CD28 directly
  • May start to use anti-CD40/CD40L
  • cGMP agents to prevent co-stimulation e.g. anti-ICOS, anti-OX40

many available agents at clinical standard in autoimmunity and transplant

84
Q

what is the issue with identifying tolerant patients after a tolerance-inducing therapy?

A

for ethical reasons, transplant patients must be on conventional immunosuppressants, even if they are in a trial
- so how do we know that the tolerance therapy is causing these tolerance, and not the
immunosuppression holding the graft in place
- Some patients can have gradual removal of immunosuppression to identify truly tolerant patients to inform stopping conventional immunosuppression

85
Q

how can we identify tolerant patients?

A

Biomarker discovery:
- 30 renal transplant recipients became naturally tolerant and didn’t reject - looked at the blood of these patients

86
Q

what biomarkers are observed in tolerant kidney transplant patients?

A

Tolerant patients have a similar PBMC gene expression profile to healthy non-transplanted controls
- But was different from patients with stable graft function who had had a transplant

These tolerant patients displayed a B cell gene expression signature, not T cell!
- IGKV4-1, IGLL1, and IGKV1D-13 increased, CD20 increased, number of circulating B cells increased
- Population of B cells identified = transitional B cells which produce IL-10 when activated - Bregs

87
Q

what biomarkers are observed in tolerant liver transplant patients?

A

Liver transplants are more tolerogenic - 20-30% patients are tolerant and can come off immunosuppression:

Tolerant patients displayed a NK, NKT and gamma-delta T cell gene expression signature pattern
- No B cell signature unlike renal transplant

88
Q

how must biomarker discovery be further developed to identify tolerant patients?

A

Tolerant patients that receive different transplants may need identification by a different set of biomarkers
- Biomarker assays need to be on organ-specific basis
- Different organs will have different tolerant effector mechanisms

Are we looking in the right place? - PBMCs vs Transplant – transplant is where mechanisms are happening
- Difficult to do as we don’t want to mess up the tolerance by testing the graft

Are we using the right assays/tools and are they sensitive enough?
- are the PBMC T cells actually affecting the transplant, because only 10% of those actually are alloreactive

89
Q

summary of transplant therapies:

A

Current immunosuppression is suboptimal at enabling long-term survival of allografts
- Good for renal, less good for heart, small bowel
- Current evidence suggests that tolerance would be preferable and that it is achievable in humans – but intense protocol of immunosuppression is needed
- We need further experimental studies to figure out how to induce robust tolerance consistently
- Need to develop better biomarkers to define tolerance to know if patients can withdraw from immunosuppression or not

Molecular Mechanisms (9 decks)

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