Innate immunity and memory T cells in transplantation

Question 1

in what ways can the innate immune system modulate rejection?

Answer 1

Innate cells within the graft can trigger adaptive immunity against the transplant

Infection can be recognised by the innate immune system, which then modulates rejection

Complement – chemoattractant for neutrophils, influence mast cell degranulation, forms MAC – complement can also influence adaptive responses

Question 2

are solid organ transplants quiescent?

Answer 2

No:
- Living tissue that has gone through period of stress when removed from donor and plumbed into recipient (ischemia/reperfusion injury (IRI))
- Transplant has interrupted blood supply which starves the tissue of oxygen = induces hypoxia and acidity, as it can’t remove waste products

Question 3

how does the clinic try to slow IRI before transplantation?

Answer 3

Solid organs from cadavers are stored on ice to slow metabolism and reduce damage
Cadaveric graft is flooded with ice cold preservation solution
- Kidneys can be stored for up to 24 hours, heart = 4-5 hours
- Kidney stored for 24 hours in cold = 24 hours of ischemia – how does this affect transplant

Question 4

how does IRI affect grafts?

Answer 4

allografts that suffer prolonged IRI do worse in the long-term

Question 5

how do graft survival rates differ between cadaveric and living donor transplantation?

Answer 5

cadaveric grafts tend to do worse than living organ transplants
- if cadaveric graft lacks urine flow and requires dialysis, the organ is in poor shape and does worse after transplant, with 40% rejected after 3 years, independent of MHC matching

Living donors, no matter being related to the recipient or not, tend to have better graft survival
- with HLA-identical grafts performing the best

history of the graft matters:
- living organ grafts aren’t put on ice and are transplanted immediately
- cadaveric renal graft could be on ice for up to 24 hours
- what happens to graft before transplant impacts its survival

Question 6

why do allografts that suffer prolonged IRI do worse in the long term?

Answer 6

Damage to graft increased which may influence subsequent immune responses to the graft

Question 7

what happens when the organ is removed from the cadaveric donor?

Answer 7

As the organ is no longer supplied by vessels, it goes from normoxia, through hypoxia (insufficient O2) to anoxia as it is stored on ice

Question 8

what happens to the donor organ when it is plumbed into the recipient?

Answer 8

As it is plumbed in, oxygenated blood enters graft tissue through the recipient vessels
- goes from hypoxia to normoxia

Question 9

how does hypoxia in the tissues affect the graft?

Answer 9

hypoxic tissues sense hypoxia:
- Causes initiation of transcriptional changes
- HIF1a becomes stabilised as a TF, leading to upregulation of glucose transport genes (glut1), glycolysis, erythropoiesis, angiogenesis
- there is build-up of calcium which disrupts osmotic balance
- Activation of xanthene oxidase and NADPH oxidase
- overall, initial damage by hypoxia

Question 10

how does oxygen reperfusion to the transplant affect the graft?

Answer 10

When oxygen is reintroduced there are new responses:
- Generation of ROS by xanthene oxidase
- Disruption of mito ETC – further generation of ROS
- known as the superoxide flash
- These ROS and NOS disrupt molecules in the cell = oxygen is added to DNA, lipid, sugars, proteins and disrupst their function
- Overall, this can cause necrosis

Question 11

what are the physiological ligands for innate immune cells in transplantation?

Answer 11

DAMP release is another consequence of ROS buildup, which bind to PRRs:
- e.g. biglycan, HMGB1, heat-shock proteins, uric acid
- uric can’t be removed, so activates TLR2
- ATP is normally inside cell, but necrotic cells release ATP, which can act as DAMP
- Hypoxic cell exposed to ROS becomes activated and express/release pro-inflammatory cytokines and chemokines
- Generates caspase 1 for inflammasome – produces active IL-1B and IL-18 for inflammation

Question 12

Is TLR triggering important for rejection? how was this first shown?

Answer 12

In male-to-female identical twin transplant (H-Y minor antigens stimulate allograft rejection):
- TLR2 and 4 can recognise graft-DAMPs and signal via MYD88 adaptor
- experiment K/O of all of these molecules on both donor and recipient

TLR2 K/O = rejection
TLR4 K/O = rejection
Myd88 K/O = grafts accepted long-term

release and recognition of DAMPs does impact rejection

Question 13

why is the twin model limited?

Answer 13

it is an autograft, so only stimulated by minor antigen and doesn’t recapitulate allografts

Question 14

is DAMP signalling important on donor tissue or resident tissue for rejection?

Answer 14

MYD88 K/O on donor or recipient:
- WT = rejection
- MYD88 K/O on both = survival of graft
- MYD88 K/O on donor = some increase in survival
- MYD88 K/O on recipient = some increase in survival

DAMP signalling is important on both donor and recipient for rejection

Question 15

how does TLR triggering affect dendritic cell activation?

Answer 15

looked at no. DCs in draining lymph node following skin transplant where MYD88 is K/O:
- WT = increased DC count in lymph node
- K/O of MYD88 on both donor and recipient = drastic reduction of DCs in lymph node
- K/O of MYD88 in recipient = smaller reduction in DCs
- K/O of MYD88 in donor = smaller reduction in DCs

DAMP signalling impacts DC function in draining lymph node, whether from donor or recipient

Question 16

what does IRI, hypoxia and DAMPs induce?

Answer 16

Hypoxia and DAMPs induce activation of stromal cells, vascular cells of the graft and also DCs
- DCs drive the alloreactive T cell responses
- Immature DCs in donor tissue or lymph nodes are activated by DAMPs: fragmented ECM, necrotic cells, stressed cells e.g. HMGB1 secretion
- DAMPs trigger PRRs on immature DCs, converting them to mature DCs to generate T cell response

both resident donor DCs and recipient DCs require PRRs for activation

Question 17

what model shows the importance of TLRs in driving rejection?

Answer 17

an allograft model
e.g. transplant across full MHC plus minor antigen mismatch/barrier
- skin graft from BALBc mouse into B6 mouse

Question 18

is TLR signalling important in full MHC mismatch allograft rejection?

Answer 18

K/O of MYD88 on both donor and recipient has no effect on graft rejection and the speed of graft rejection
- TLR signalling may be less important for allograft rejection compared to autograft

Question 19

does TLR signalling affect alloreactive T cell responses in full MHC mismatch rejection?

Answer 19

Elispot for IFNY, IL-2 and IL-4:
- shows frequency on donor-specific T cells that produce these cytokines rapidly following in vitro reactivation
- Myd88+ donor and recipient = lots of IFNy, IL-2 and IL-4 production
- even though there is no difference in graft rejection, loss of Myd88 reduces IFNy and IL-2 production by T cells in the lymph node, but IL-4 secretion isn’t affected

Although TLR loss doesn’t affect graft rejection, it does cause a reduced alloreactive T cell response

Question 20

how does TLR4 signal?

Answer 20

Myd88 is adaptor for most TLRs, but TLR4 also has a TRIF adaptor when expressed on endosomes to recognise DAMPs
When myd88 is K/O in these experiments, TRIF is still intact – so the TLR4 signal may still occur

Question 21

what happens when both myd88 and TRIF are knocked out, with addition of rapamycin in a full MHC mismatch cardiac allograft model?

Answer 21

siRNA to K/D Myd88 or TRIF or both:
- scramble + rapamycin control = rapid rejection
- K/O of both (DKO) = some graft prolongation
- if rapamycin is added with DKO (inhibits mTOR) = some grafts survive long-term

loss of TLR signalling can enhance the role of the immunosuppressant

Question 22

overall, is TLR triggering important for rejection?

Answer 22

Yes:
- Contributes to maturation of DCs, both donor-derived DCs from graft, and DAMP-activated recipient DCs
- Contributes to the priming of Th1 cells via presentation of alloantigen

BUT
- Conditional on model – subtle, and may need other immunosuppressive agents to see a marked effect

Question 23

summary of DAMPs and TLR signalling in rejection:

Answer 23

ROS release and DAMPs signal into DC via PRRs to become activated:
- Change in TFs, co-stimulatory molecules to activate T cells
- Loss of TLR signalling can lead to reduced DC activation, and therefore reduced alloreactive T cell responses

Question 24

how can infection amplify innate alloreactive responses compared to DAMPs?

Answer 24

There are a lmited no. of PRRs that recognise DAMPs induced by IRI
-only TLR2, TLR4, and RAGE

In infection, there are DAMP and PAMP signals for more PRR activation – these may enhance rejection

Question 25

how is infection implicated in transplant rejection?

Answer 25

at point of transplant, chance of infection increased due to:
- severe induction of immunosuppression via drugs
- pathogens derived from donor or recipient
- surgical procedure taking place

following transplant:
- 0-4 weeks, susceptible to pathogenic bacteria and candida
- 6-12 months, susceptible to opportunistic infections and viral reactivation e.g. EBV due to long term immunosuppression

Question 26

how likely is it that transplant patients suffer severe infections?

Answer 26

80% patients will have infection within first couple years
- Viral e.g. CMV reactivation, herpes
- Fungal e.g. oral candidiasis
- Bacterial e.g. TB

Question 27

how were infections first shown to impact allograft rejection?

Answer 27

Full MHC-mismatch cardiac allograft
- animals infected with mouse-CMV
- normal rejection without infection is already rapid, within 8 days
- when infected with MCMV, rejection is significantly sped up to within 7 days, without T cell cross-reactivity between MCMV and graft

Question 28

how can infections impairing the control of rejection and tolerance induction be studied?

Answer 28

Cardiac full-MHC mismatch allograft, treated with co-stimulatory CD40L blockade and donor-specific blood transfusion
- this is enough to enable full graft survival in these mice as they are tolerised
- infected mice with 3 different bacteria at the time of transplant to see how it affected tolerance induction

Question 29

how do different bacteria affect tolerance induction in the allograft mouse model?

Answer 29

• S. aureus infection = induces break of tolerance – 75% animals reject grafts
• L. monocytogenes = also reject quickly – induces break of tolerance
• P. aeruginosa = still tolerant and grafts survive

Certain infections break induction of tolerance, whereas others don’t

Question 30

what different innate responses do different bacteria induce against the allograft?

Answer 30

analysed serum IL-6 in allograft mice:
S. aureus increases IL-6 and breaks tolerance
L. monocytogenes increases IL-6 and breaks tolerance
P. aeruginosa has no IL-6 response and doesn’t break tolerance

Correlation between innate cell activation, production of IL-6 and prevention of tolerance induction

Question 31

summary of infection and IL-6-dependent prevention of tolerance induction

Answer 31

Bacteria activates an innate cell, causes IL-6 secretion, and this somehow switches tolerogenic T cells to effector alloreactive T cells, without co-stimulatory interactions

Question 32

what other cytokine induces break of tolerance following infection?

Answer 32

Infect heart and skin allograft mice with L. monocytogenes at time of transplantation:
- leads to break of tolerance, with type 1 interferons being released by innate cells

Question 33

how does infection impact established transplant tolerance, even after many yeas of graft survival?

Answer 33

used co-stimulatory blockade to induce long-term tolerance and graft survival
- after 60 days, infected mice with L. monocytogenes = 70% of grafts were rejected within 10 days

Late infection can perturb established tolerance

But are animals truly tolerant after 60 days?

Question 34

how do cytokines impact established transplant tolerance, even after many years of graft survival?

Answer 34

Replacement of L. monocytogenes with IL-6 and IFNb:
- Injection of plasmids that produce these cytokines into long-term tolerant animals leads to rapid rejection
- innate cytokines can perturb established tolerance

But are animals truly tolerant after 60 days?

Question 35

how does the duration of T cell stimulation by graft alloantigen affect rejection?

Answer 35

rejection is carried out by both MHCII-CD4 T cells and MHCI-CD8 T cells
- MHCII-CD4 responses are transient, as MHCII is expressed by donor DCs which die over time - short-term direct pathway
- when these animals are infected with L. monocytogenes, CD4 T cells are easily reactivated and tolerance is broken

Persistent alloantigen stimulation via MHCI, which is always expressed on all cells of the graft persistently, leads to more T cell dysfunction, so T cells are not reinvigorated upon L. monocytogenes infection

suggests that alloantigen is constantly needed to keep T cells in a tolerant state
- loss of antigen means T cells lose tolerance, so can be reactivated

Question 36

how was prolonged alloantigen signalling shown to cause resistance to rejection?

Answer 36

Following loss of donor DCs expressing MHCII over time, mice are given multiple donor-specific blood transfusions repetitively
- this provides further MHCII donor antigens to T cells
- showed that upon L. monocytogenes infection, tolerance is no longer broken, as MHCII alloantigens are available long-term for the T cells, so animals become resistant to rejection

Infection can break T cell tolerance, but not if the T cells have been driven by repetitive MHCII donor antigen

Question 37

what are the key functions of complement?

Answer 37

C3b induces opsonisation

C5b-C9 induces membrane attack complex

C3a and C5a can act as chemoattractants for phagocytes, mediate inflammation and induce mast cell degranulation

Question 38

how has C5a been shown to enhance T cell activation in vitro?

Answer 38

Used CFSE to label dividing T cells and annexin 5 to stain those T cells which undergo proliferation-induced cell death
- Activate T cells in vitro to proliferate, and some effectors become apoptotic
-Addition of C5a in vitro leads to less T cell proliferation-induced cell death - saving effector T cells from apoptosis, so maintains the increased T cell population

Question 39

how has C5a been shown to enhance T cell alloreactive responses in vivo?

Answer 39

• allogeneic response: T cells are added into a mouse that are from a different strain - like GVHD response
• Label T cells with CFSE to trace divisions
• WT recipient and WT donor T cells = 10% T cells recognise alloantigen and divide, with 6% of those dying
• DAF-/- (complement inhibitor protein) induces more C5a in the recipient, so when recipient is injected with WT donor T cells, this induces an enhanced allogenic T cell response: more expansion with reduced apoptotic T cells

In increased presence of complement = more T cells available to react to alloantigen

Question 40

what happens when T cells are unresponsive to C5a receptor?

Answer 40

If T cells are unresponsive to C5a with receptor K/O, there is less alloreactive T cell proliferation and more apoptosis of those T cells

overall, C5A can co-stimulate T cells, leading more proliferation and less T cell death

Question 41

how does C5a co-stimulate T cell responses?

Answer 41

Normally, there TCR-MHC-peptide interaction with co-stimulation
- additionally, C3a and C5a can co-stimulate T cells further by increasing BCL2 (anti-apoptotic) and decreasing caspase 3 (central apoptotic caspase)

Question 42

why are Tregs important in allograft rejection?

Answer 42

To induce tolerance, we need Tregs to form a regulatory network to keep the graft in place:

Tregs come from:
- Naïve T cells, which are activated in the presence of TGFb, convert to iTregs
- nTregs from thymus

10% Tregs can recognise alloantigen via direct pathway – need to expand this population and function for tolerance to the graft

Question 43

do Tregs express complement receptors?

Answer 43

resting Tregs lack C3a/C5a receptors, but upregulate these receptors following activation in vitro, specifically C5aR

Question 44

how have nTregs been shown to be impacted by complement in vitro?

Answer 44

Used a T cell suppression assay, where effector conventional T cells have an increasing titration of nTregs into the media
- Titrate in increasing WT nTreg conc = effector T cell proliferation is suppressed over time, from 5:1 ratio
- nTregs which lack C5aR or C3aR are more suppressive to effector T cells – suppress earlier and at a lower ratio compared to WT

Without C5a/C3a signals, nTregs are more suppressive of effector T cells

Question 45

how have nTregs been shown to be impacted by complement in vivo?

Answer 45

Skin graft model of RAG-/- mouse, so no rejection as they lack adaptive immune cells
- Add defined conventional no. T cells to reject skin graft
- Add WT nTregs in and this suppresses rejection to an extent – graft has prolonged survival, but most grafts eventually is rejected - nTregs can’t completely suppress rejection
- Add C3aR/C5aR-/- nTregs – 70% grafts are accepted long term

Complement reduces suppressive nTregs

Question 46

how does complement affect iTreg conversion?

Answer 46

In vitro conversion of conventional T cells to express foxp3 and become iTregs
- WT T cells = no conversion, effector T cells remain
- K/O C3aR or C5aR T cells = some conversion to iTreg
K/O of both in T cells = 30% conventional T cells become iTreg

C3a and c5a impact ability of conventional T cells to become iTreg

Question 47

is iTreg differentiation dependent on TGFb?

Answer 47

Conventional T cells convert to iTregs when deficient in both C3aR and C5aR:
- if TGFb is inhibited = no conversion
- if TGFbR is inhibited = no conversion
- if downstream SMAD3 is inhibited = no conversion

TGFb presence is required for conversion to iTregs

Question 48

are human iTregs impacted by complement?

Answer 48

• anti-C3a and anti-c5a treatment cause iTregs to function better
• Block complement receptors = iTreg works better to inhibit T cell proliferation

C5a and c3a impact function and induction of Tregs in human and in mouse
- informs tolerance induction strategies

Question 49

what is C5aR2 and how does it affect iTreg conversion?

Answer 49

C5aR2 is a competitor of C5a binding C5aR1 on T cells, so increases iTreg conversion:
- K/O of C5aR2 increases C5a activity
- Put conventional cells into RAG-/- hosts and look for conversion after few days
- WT T cells = more iTreg conversion in vivo
-In C5aR2 K/O T cells = less conversion of T cells to iTregs in vivo, as C5a is more available

Increased C5a inhibits iTreg conversion

Question 50

summary of allografts and innate immunity:

Answer 50

T cells are most important in rejection, but innate immunity can affect T cell responses early on and in long-term
- Graft DCs important for initial rejection via DAMPs and TLR signals
- Infection can impact at time of transplant and long-term
- proinflammatory cytokines such as IL-6 and IFNb can enhance rejection
- Complement C5a and C3a negatively affect tolerance induction, promoting effector T cells whilst diminishing n/iTregs

Question 51

how may alloreactive memory T cells arise post-transplant?

Answer 51

After transplant – an acute/chronic ejection episode is driven by T cell responses
- Immunosuppressive agents may be suboptimal, leading to rejection (acute or chronic) – leads to breakthrough responses that form memory

Question 52

how may alloreactive memory T cells arise pre-transplant?

Answer 52

Before transplant: prior sensitisation
- donor-specific antibody generated, as well as alloreacitve memory T cells from previous transplant, transfusion, or pregnancy

Question 53

how are memory responses generated?

Answer 53

Naïve T cells encounter antigen A
- Period for differentiation and clonal expansion
- Once pathogen is cleared, most effector T cells die, leaving small population of antigen-specific memory T cells
- Re-encounter of antigen A = better secondary response by memory T cells – rapid effector function and clonal expansion
to remove the infection rapidly

Question 54

what is the problem with studying memory T cell responses to allografts?

Answer 54

comparing naive and memory cell responses are limited due to:
- different frequency of memory and naive T cells
- clonal distinction between memory and naive T cells (memory T cells have higher affinity for antigen)

these may result in different immune responses which can’t be compared as they are independent of memory, and may be to do with differences in frequencies or TCR affinity

Question 55

how does different naive and memory frequencies affect study of memory to allografts?

Answer 55

memory T cells are activated and clonally expand
- not all alloreactive T cells will be equally competitive
- there is competition for specificity/affinity to antigen during development of memory
- in the T cell pool, the frequency of memory T cells that respond to an antigen is different to the naive pool
- memory T cells tend to be enriched in certain specificities, so there are more of them compared to naive

Question 56

how does clonal distinction between naive and memory T cells affect study of memory to allografts?

Answer 56

Not every alloreactive T cell specificity/affinity within the naive pool after antigen challenge will form memory T cells due to competition
- naive and memory therefore may have different TCRs of different affinities

Question 57

how can the limitations of studying memory vs naive T cells in allografts be overcome?

Answer 57

Generated mice that have TCR knock-ins that we know are alloreactive in RAG-/- mice:
- when they breed the only T cells that these mice produce are BM3-transgenic T cells with the exact same TCR, reactive to an MHCI alloantigen (H2Kb)
- can inject these BM3 CD8 T cells into the RAG-/- mice and challenge them with a skin graft, leading to rejection
- following rejection, naïve BM3 T cells cells are converted to memory T cells, with the same TCR
- this generates a population of memory and naïve cells with same alloreactive TCR to HK2b

Question 58

how is it shown that BM3 memory T cells are generated during rejection?

Answer 58

• Naïve BM3 T cells are CD44 low
• Following skin graft rejection, there is conversion of these cells to CD44hi = marker of mouse CD8 memory T cells

Question 59

how can BM3 memory T cell alloreactive responses be analysed?

Answer 59

Isolate the BM3 memory T cells from the animal and challenge in vitro with alloantigen:
- There is high and rapid IFNy secretion by BM3 memory T cells within the first 24 hrs
- whereas naïve T cells take a much longer time for IFNy secretion
- Functionally look like memory T cells, as well as phenotypically

Put naive or memory T cells into another animal and track clonal expansion following alloantigen challenge:
- naïve cells challenged with donor cells = some clonal expansion
- memory cells challenged with donor cells = clonal expansion is more rapid

These BM3 cells have phenotype and function of memory T cells

Question 60

how can we directly compare naive and memory T cell responses to alloantigen?

Answer 60

Put naive or memory cells into the mouse at the same frequency e.g. 1x10^5 cells)
- TCR is transgenic in BM3 T cells, so memory and naive have the same TCR
- Avoided differences in specificity and frequency to solely look at function

Challenge these naive vs memory mice with skin graft:
- Naïve cells: acute rejection in 27 days
- Memory cells: acute rejection in 17 days – much faster

Question 61

how does memory T cell rejection affect tolerance induction?

Answer 61

Co-stim mAb CD40L blockade to induce tolerance – mice accept cardiac allograft and are tolerant
- A separate mouse is given skin graft which is rejected, forming memory cells
- Then inject these memory T cells into the cardiac recipient mouse – these mice are now resistant to tolerance and reject their allograft

Memory T cells formed to alloantigen due to prior transplant exposure can form a barrier to tolerance

Question 62

can alloreactive memory T cells arise in patients that have not been sensitised to alloantigen pre-transplant?

Answer 62

yes: non-allo-sensitised blood contains alloreactive memory T cells

Question 63

how was it shown that non-sensitised people have alloreactive memory T cells?

Answer 63

Used healthy, non-sensitised volunteers:
- Split T cells from volunteers into naïve and memory
- looked precursor frequency of naïve compared to memory pool that were responsive to alloantigen
- Despite lack of sensitisation in these volunteers, there were similar precursor frequencies of alloreactive T cells in both naive and memory pools

Question 64

what is the clinical evidence for non-sensitised patients having alloreactive memory T cells prior to tranplant?

Answer 64

Living renal transplant recipients:
- used IFNy Elispot to look at precursor frequency of T cells that release IFNy within 24 hours before and after transplant
- Patients with low precursor frequency of memory T cells specific to donor had no acute rejection
- Patients with high precursor frequency of donor-reactive memory T cells = more likely to suffer acute rejection

Memory T cells pose barrier to graft survival:
- Even non-sensitised patients have alloreactive memory T cells
- Correlates with likelihood of an acute rejection episode

Question 65

overall, how do memory T cells affect transplant outcomes?

Answer 65

They induce a barrier to tolerance

They induce a barrier to graft survival

Question 66

how can viruses affect tolerance induction?

Answer 66

Tolerance induction with CD40L blockade and CTLA-4 Ig and donor BM transplant – mice accept skin allograft
- If they infect mice with viruses prior to transplant and tolerance induction and enabled to clear the virus, then tolerance failed
- Researchers kept giving mice viral infection and allowing them to clear, and then tried to induce tolerance

Infection with a single virus = some mice are now resistant to tolerance induction

Multiple, repetitive infections = most mice reject tolerance
- representative of humans in winter with lots of infection

Question 67

how do alloreactive memory T cells form following a skin graft?

Answer 67

Give animal skin graft, isolate the T cells and re-stimulate with donor cells in vitro, looking at IFNy secretion
- many CD8 T cells secreting IFNy in vitro = generation of effector CD8s
- Once graft is rejected, there death is of most effectors, leaving a memory pool specific for alloantigen that secretes IFNy in re-exposure

Question 68

how do anti-viral responses induce the formation of alloreactive memory T cells?

Answer 68

Looked at IFNy-secretion after viral infection and re-stimulation in vitro with viral antigen:
- at first, forms effector response during peak infection of LCMV, VV, VSV (murine viruses)
- peak response semi-collapses and forms a memory population

If previously infected with virus and restimulated with alloantigen:
- small population of effector cells that were generated from viral responses can cross-react and are also stimulated against alloantigen
- New memory pool is generated to viral responses, where some now have a recall response to alloantigen

Question 69

overall, how can alloreactive memory T cells form without prior sensitisation?

Answer 69

Alloreactive memory T cells without sensitisation to alloantigen can be generated by normal anti-viral responses
- a proportion of T cells induced by vaccination become alloreactive, forming crossreactive, donor-reactive memory T cells
- Proportion of alloreactive memory T cells is unique to the individual due to their history of infection

Question 70

how difficult is it to induce tolerance in humans?

Answer 70

Many ways to induce tolerance in mice
Tolerance has proved more difficult to induce in non-human primates, due to memory T cells (in part)

Question 71

what study was used to determine if alloreactive memory T cells prevent tolerance induction in higher animals?

Answer 71

Non-human primate renal transplant study:
- Tolerance induction with co-stimulatory blockade
- wanted to see if there is rejection or tolerance, looking at graft-reactive memory T cell frequency before and after Tx

Question 72

Do alloreactive Tm prevent tolerance induction in higher animals?

Answer 72

3 groups:
- low frequency of alloreactive memory T cells = accepted Tx after tolerance induction
- rejecting control (without tolerance induction) = big spike in donor-specific memory T cells and rejection
- 2 animals had the same tolerance induction, but rejected their grafts – these NHPs, pre-transplant, had a higher frequency of alloreactive memory T cells at the time of tolerance induction - these memory T cells acted as barrier to tolerance and led to rejection

Question 73

what generates memory T cells in clinical transplantation?

Answer 73

• acute rejection - generates memory
• chronic rejection - generates memory
• lymphodepletion
• cross-reactivity - due to infection, vaccination
• prior sensitisation

Question 74

what is lymphodepletion?

Answer 74

T cells in a low T cell environment clonally expand to fill the space
- this forms memory T cells via homeostatic proliferation

Question 75

what generates memory T cells in experimental transplantation?

Answer 75

• Generate memory T cells via exposure to alloantigen
• Heterologous immunity by prior infections
• Lymphopenia – low T cell population, so some naive convert to memory cells

These all form memory T cells which induce barrier to tolerance

Question 76

how might memory T cells disrupt tolerance induction and cause quicker graft rejection?

Answer 76

1. Differential homing – naïve T cells stay in lymph and blood circulation, memory T cells can enter tissues in normal migration – memory could enter graft instantly
2. Memory T cells can activate outside of lymphoid tissues, enabling a faster response within graft – naïve T cells can only be activated in lymph nodes
3. Memory cells have rapid reactivation, higher affinity/avidity during prior sensitisation (not prior infection), lower co-stimulatory requirements via CD40L and CD28 as these are dispensable – hence why co-stim blockade is futile for memory
4. Memory T cells can rapidly enter transplant as they are rapidly activated
5. May have more potent or different effector functions to naive T cells

Question 77

what are ALI mice?

Answer 77

these mice lack lymph nodes:
- their naive T cells can’t be activated, but their memory T cells can

Question 78

how does the clonal expansion of memory T cells compare to naive in skin allografts?

Answer 78

labelled BM3 memory and naive cells with CSFE in RAG-/-, challenged with skin graft:
- no difference in proliferation after 5 days
- no difference in proliferation after 10 days

both T cell populations respond to the transplant at the same proliferative rate – no clonal expansion differences between memory and naive in rejection

Question 79

do memory T cells have increased infiltration into skin allografts compared to naive?

Answer 79

rt-PCR for CD3 as marker for T cell infiltration:
- at day 5, similar number of memory T cells in graft as naive, very low infiltration
- at day 10, still no statistical difference between infiltration numbers of naive and memory T cells

Question 80

do memory T cells have enhanced effector responses to skin allografts compared to naive?

Answer 80

Effector response with rt-PCR for IFNy and perforin
- both IFNy and perforin induced, but no difference between memory and naïve cells at day 10
- Induction of CCL5, XC1, CXCL9, CXCL10 (important for T cell migration via CCR3) – but no difference between naïve and memory cells

Similar induction of intragraft pro-inflammatory genes
during rejection mediated by memory and naïve T cells

Question 81

do memory T cells induce increased immune activation in skin allografts compared to naive?

Answer 81

Stain graft with GR1 antibody for neutrophils:
- Rejection by naïve at day 10: few neutrophils in graft
- Rejection by memory at day 10: loads of neutrophils in graft

No difference directly between naïve and memory mechanisms in alloreactivity, but difference between what is recruited in, as mems cause rapid neutrophil recruitment

Question 82

does increased neutrophil recruitment explain how memory T cells cause quicker graft rejection?

Answer 82

yes:
- usedAnti-GR1 antibody to deplete neutrophils and tranfer in memory T cells
- this causes graft rejection to be slowed further than naive
- Transfer in naïve cells and deplete neutrophils causes same slowed rejection

Both naïve and memory T cells use neutrophils for effector mechanism, but memory cells pull them in faster, hence the speedier rejection

Question 83

summarise how memory responses to transplants differ to naive responses:

Answer 83

no difference in clonal expansion, graft infiltration and effector functions in alloreactive naive and memory T cells

BUT memory T cells induce rapid neutrophil recruitment into the graft compared to naive

Question 84

how can Tregs be studied in suppressed alloreactive memory responses?

Answer 84

BM3 naïve or memory cells injected into RAG-/- mice
- add in Tregs generated from a different animal
- challenge the mice with a allo-matched skin graft and look at survival

Question 85

do Tregs suppress alloreactive memory T cells as well as they do naive T cells?

Answer 85

Naïve = rejection
Memory = quicker rejection
Co-transfer Treg with naive = most animals have graft survival – Tregs can suppress naïve
Co-transfer Treg with memory = most animals reject at naïve control rates, with no graft prolongation

Tregs can control naïve rejection, but not memory-mediated rejection

Question 86

how can Treg suppression of alloreactive memory T cells be enhanced?

Answer 86

Memory rejection is mediated by faster neutrophil recruitment,
- if neutrophils are removed, can Tregs suppress memory cells?:

Tregs make no difference on normal memory-mediated rejection
Memory cells with neutrophil depletion have slowed rejection, but still eventually reject
Memory cells with neutrophil depletion and Tregs injected lead to survival and prolongation of most grafts

Memory cells are more difficult to control due to rapid rejection but can be controlled by Tregs when memory effector function is limited

Question 87

how do memory cells affect transplant tolerance?

Answer 87

In rejection, the effector arm outweighs regulatory arm

In tolerance, we want to increase the regulatory arm and reduce the effector arm:
- but memory cells tip balance to rejection, so we need to target these to tip the balance towards regulation and tolerance

Question 88

how might we control memory T cell alloresponses?

Answer 88

• memory T cell depletion
• use conventional immunosuppressive agents at increased dose
• co-stimulatory molecule blockade specific for memory cells (there are 30 co-stims available, yet we only block CD40L and CD28)
• increased ratio of immunoregulatory cells to memory cells

Question 89

what is the aim of memory T cell depletion?

Answer 89

Reduce the number of graft-reactive memory T cells to reduce impact on tolerance induction

Question 90

what are the available T cell-depleting agents?

Answer 90

anti-CD52 (CAMPATH), anti-thymocyte globulin (ATG) – depletes 99% of naïve cells, but memory cells are resistant to this

Question 91

what are the limitations of memory T cell depletion?

Answer 91

• memory cells are resistant
• Depletion causes a lymphopenic environment, so more memory cells are generated by homeostatic proliferation to compensate
• Depletion may enhance the risk of infections during transplant surgery as pre-existing memory cells are removed, leading to enhanced opportunistic infections
• Memory cells recognise antigens that we are vaccinated against – so depleting these results in the need for re-immunization, which generates more memory - vicious cycle

depletion shouldn’t be used

Question 92

what are the aims of conventional immunosuppression on memory T cells and how is it done?

Answer 92

aim: decrease number of actively responding memory T cells
- may be drug-specific
- may require increased doses to be effective

Question 93

what are the key immunosuppressants used in transplantation?

Answer 93

tacrolimus: calcineurin inhibitor
sirolimus: rapamycin - inhibits mTOR
MMF: anti-proliferative
steroids e.g. methyl prednisolone

Question 94

how effective are conventional immunosuppressants for naive vs memory T cells?

Answer 94

TCR transgenic CD4s recognise alloantigen in vitro, treated with tacrolimus, sirolimus, MMF and steroids:
- Naive cell proliferation: all drugs inhibit
- naive IFN secretion: all drugs inhibit

For memory cells:
- tacrolimus does not affect proliferation, some effect on IFNy production, but less than naive
- sirolimus does not affect proliferation or IFNy production
- MMF inhibits proliferation but no effect on IFNy production
- steroid lowers proliferation and can reduce IFNy production, but not to same extent as naive

overall immunosuppressives have less effect on proliferation and cytokine secretion of memory cells compared to naive

Question 95

are immunosuppressants alone effective in suppressing memory-mediated rejection?

Answer 95

Skin graft model in vivo: CD4-mediated rejection via naïve and memory cells
- MMF treatment of naïve cells maintain graft for longer until drug removed
- MMF treatment of memory cells: all grafts rejected whilst on drug – drug doesn’t work for memory cells

Sarolimus:
Good for preventing naïve graft rejection, and when drug is removed, mice then reject
- Memory cells reject whilst on sirolimus

Question 96

are immunosuppressives against memory-mediated rejection better when combined?

Answer 96

yes, Sarolimus + MMF can inhibit memory-mediated rejection, until thedrug is stopped

Some combinations may be able to control memory-mediated rejection

Question 97

what are the limitations of conventional immunosuppression for treating memory-mediated rejection?

Answer 97

Increased doses of immunosuppressives may not be practical
- Over-immunosuppression = increased side-effects

Certain immunosuppressive drugs may interfere with the
generation/function of Treg and actually block tolerance
- Cyclosporine can impair tolerance

Question 98

is co-stimulatory blockade feasible against alloreactive memory T cells?

Answer 98

Co-stimulatory blockade so far isn’t good for memory cells
- they are not reliant on CD28 and CD40 for stimulation

Do memory cells not need co-stimulation at all, or do they use different co-stimulatory molecules?
- Maybe uses OX40 and OX40L, or may use VLA-4 (adhesion molecule but can be co-stimulatory)

Antibody against CD28 may work better than CTLA-4 Ig (CTLA-4 Ig blocks both CD28 and CTLA-4 on Tregs, so is limited in inducing tolerance)

Question 99

how might we increase the ratio of immunoregulatory cells to memory cells?

Answer 99

Bregs that produce IL-10 may be important in tolerance induction
- Invariant NKT cells are immunomodulatory, as well as MDSCs
- Boost Treg population and function in vivo using IL-2 receptor antibodies conjugated to IL-2 to preferentially stimulate Tregs over naïve and effector cells
- Or expand Tregs in vitro and give back to patient

Question 100

summary of memory T cells in rejection:

Answer 100

Tm are a significant barrier to the induction of tolerance and graft survival
- but they can be tolerised by manipulating the system e.g. neutrophils
- Tm responses may be attenuated by new co-stimulatory molecule blockade
- Tm responses can be suppressed by Treg