T cell metabolism Flashcards
1
Q
how does the metabolism of T cells change when they engage in an immune response?
A
- altered uptake and handling of glucose
- altered mitochondrial function via glutamine uptake
- Altered amino acid metabolism
2
Q
how is glucose uptake changed in T cells?
A
Increased glucose uptake, tendency to convert pyruvate to lactate (increased ECAR)
3
Q
how is glucose metabolism changed in T cells during an immune response?
A
- when T cells are stimulated via TCR and CD28, there is an increase in extracellular acidification rate (ECAR) = increased lactate production from glucose
(released/excreted as lactic acid which acidifies the culture medium)
Immune cells convert glucose to pyruvate to lactate, despite there being enough oxygen for oxidative phosphorylation (OXPHOS)
4
Q
how can glucose handling differ in aerobic and anaerobic metabolism?
A
glucose is broken down to 2x pyruvate in cytoplasm = glycolysis
- anaerobic: pyruvate can be reduced to lactate and excreted
- aerobic: pyruvate can be converted to acetyl CoA to be oxidised in TCA cycle to drive activity of ETC in the mitochondria
5
Q
what is the more efficient way of using glucose?
A
aerobic TCA cycle and OXPHOS - 1 molecule of glucose produces 32 ATP
6
Q
how do immune cells use the Warburg effect? why do they do this?
A
Stimulated immune cells and cancer use anaerobic metabolism only
- Anaerobic glycolysis in presence of oxygen (Warburg effect)
This metabolism enables rapid flux of glucose in glycolysis:
- More intermediates produced e.g. nucleic acids, lipids which can feed into other pathways to drive growth and proliferation (cancers and T cells need this)
7
Q
do activated T cells undergo a glycolytic switch?
A
No, T cells still use their mitochondria upon immune responses
8
Q
how does mitochondrial function change in activated T cells?
A
Increased mito activity:
- mito oxygen consumption rate increases in activated T cells
- Increased activity of TCA and ETC – fuelled by high glucose uptake, so some pyruvate is inevitably oxidised
- Increased uptake and oxidation of glutamine
- glutamine is converted to glutamate, then to alpha-ketoglutarate which can be oxidised in TCA cycle
- TCA generates of NADH and FADH2, to provide electrons and protons to ETC to drive electron pumping
9
Q
what is the overall function of the ETC?
A
Transfer of electrons between complexes of ETC is coupled to the efflux of protons from inner to outer mito membrane
- complex 5 (ATP synthase) pumps protons back to the inner membrane, generating ATP
10
Q
what substrates can fuel the TCA cycle?
A
glucose
fatty acids
glutamine
- immune cells proliferate and make more proteins for effector function, so need more ATP via mito function to fuel these processes
11
Q
how to T cells alter their uptake of mitochondrial substrates?
A
T cells significantly increase their uptake and metabolism of glutamine when activated – particularly in low-glucose environments (metabolic flexibility)
Fatty Acids (FA) are also an important fuel for T cells as they can be oxidised in TCA cycle e.g. acetyl CoA
- This tends to be a feature in resting, ‘quiescent’ T cells (rather than activated) and especially for memory T cells
12
Q
how is T cell amino acid uptake altered upon activation?
A
Uptake of more amino acids when engaging in immune response
- These amino acids have increased uptake during immune response and impact T cell function
- Measured by radiolabelled or fluorescently labelled amino acids to trace their uptake
13
Q
how does altered glucose metabolism support T cell immune function?
A
- Shift in reduction of pyruvate to lactate generate intermediates to support proliferation
- increased glucose uptake can influence changes in epigenetic landscape of T cells
- can alter translation of T cell mRNA
14
Q
what is methylation vs acetylation?
A
- Chromatin is bound to histones
– changes in histone winding can change how accessible DNA is to TFs - Acetylation makes DNA more accessible for TFs to bind and initiate gene transcription
- Methylation makes DNA less accessible
Acetylation is permissive epigenetic modification, whilst methylation is restrictive
15
Q
how does glucose metabolism alter T cell epigenetic landscape?
A
Glucose oxidised in mito can form acetyl CoA which can acetylate histones
- Acetylation improves transcription of IFNy – important T cell cytokine – production of mRNA
16
Q
how does increase glucose uptake alter mRNA translation in T cells?
A
Glycolytic enzymes can have moonlighting functions:
- GAPDH sits in middle of glycolysis pathway
- When GAPDH is not engaged in glucose metabolism, it can bind mRNA and prevents their translation into protein at ribosomes
- When more engaged in glucose metabolism, GAPDH can no longer bind mRNA, so mRNA can associate with ribosome and translate into protein
17
Q
how does glucose metabolism regulate IFNy?
A
IFNy expression is regulated at the transcriptional level via metabolic-dependent epigenetic changes and at the translational level via glycolytic enzymes binding mRNA
18
Q
how does increased mito function affect T cell immune function?
A
Increased ETC activity generates more ATP, and also more ROS produced at complex 1 and complex 3:
- Increased ROS can disseminate out of mito and have signalling function
- ROS can stabilise TF NFAT in cytoplasm so that there is more of it, so more likely to translocate into nucleus and regulate transcription of IL-2 (T cell growth factor)
19
Q
what happens if ROS isn’t produced in T cells?
A
Study modified ETC to not produce ROS
- less NFAT entering nucleus
- less IL-2 production
20
Q
how does mito biogenesis change when T cells are in an immune response?
A
- mito biogenesis increases upon T cell activation
21
Q
how can mito biogenesis in T cells be studied?
A
Fluorescent label of mito – over time course of T cell stimulation, more mito are produced in T cell
22
Q
Are T cells making more mito similar to original mito in functionality, or are the new mito specialised in function?
A
Used un-biased proteome analysis of abundance of mito proteins:
- Mito proteins increased in the cell, but proportionally with amount of mito
- A proportion of proteins increased in abundance above expected for mito content – specific increased expression of certain enzymes in metabolic pathways
These enzymes are involved in 1 carbon metabolism
- forms purines and thymidine to support T cell proliferation via nucleic acids for DNA/RNA production
23
Q
why is amino acid uptake important in T cell immune responses?
A
Amino acids are used for protein production – cytokines, granzymes, cell surface proteins
- e.g. Alanine is taken up more during T cell activation – directly incorporated into effector protein synthesis
24
Q
what amino acids promote mTORC1 activity in T cells?
A
Amino acid sensing of abundance of leucine and arginine
- increased presence of these amino acids drives mTOR activation
- mTOR is a key regulator of immune cell metabolism and function
25
how can mTORC1 be targeted in graft rejection?
Rapamycin is a potent immune suppressant – it blocks mTOR to prevent immune responses
- treats transplants patients
26
what is the role of serine in T cell immune function?
Serine used to support purine synthesis
- Taken up in larger quantities by activated T cells
- or made via glucose
27
what is the function of methionine in T cell immune responses?
Methionine contains methyl groups which can be donated and post-translationally modify proteins or RNA – influence stability of proteins or mRNA
– increase stability for more protein production
Active T cells take up more methionine - donation of methyl group regulates protein synthesis
28
what is the function of glutamine in T cell immune responses?
Taken up in high quantities by activated T cells
- most glucose- derived pyruvate is reduced to lactate, so glutamine becomes main oxidative substrate of mito
29
what is rapid recall function of T cells? what facilitates this function?
Memory T cells protect from secondary encounter due to rapid recall function – rapid proliferation and engagement of effector functions:
- Rapid recall is supported by their different metabolism – they are metabolically primed to support rapid recall
30
how do the mitochondria differ between naive and memory T cells?
- memory CD8 T cells have large, complex mito compared to naïve
- More internal cristae projections of inner mito membrane of memory T cells – more SA – increased levels of mito enzymes and ETC complexes
- mito are more efficient in function - greater capacity
- Memory cells have abundant GAPDH expression in cytoplasm – more glycolytic enzymes – more glycolysis as soon as immune signal is perceived
31
why do memory T cells need to undergo metabolic changes?
Memory T cells tolerate different tissue environments better – circulate through variety of peripheral tissues
- Different oxygen states in different tissues
also altered metabolism is required for the generation of memory
32
how was altered metabolism found to be important for memory T cells?
Memory T cells have more mito content than short-lived effector cells
- generation of memory T cells in vitro from mouse T cells have more mito content
complex mitochondria is crucial for memory
33
what promotes the generation of complex mito in memory T cells?
Mitochondrial fusion via OPA1
- Deletion of OPA1 from T cells
- Modelled immune response to LCMV virus which expresses OVA peptide to track antigen-specific T cells
- OPA1 expressing and deficient T cells expanded normally to first encounter
- To second encounter, only OPA1 competent T cells could form memory cells
- lacking OPA1 meant that memory cells couldn’t proliferate
- Metabolic changes crucial for memory
34
how does enforced mitochondrial fusion affect memory T cells?
this induces more memory generation
35
how does mito metabolism differ for different T cell subsets?
Th1 and Th17 cells are reported to be highly glycolytic both in vitro and when isolated from disease models.
- Inhibiting glycolytic metabolism has been beneficial in autoimmune and allograft models.
Treg reported to both favour OXPHOS and be glycolytic, depending on in vitro culture conditions
- However, it appears that fatty acid metabolism is critical to Treg function – offering potential to modulate CD4 immunity and inflammatory/regulatory balance
- Can target proinflammatory cells specifically whilst sparing Tregs in autoimmunity treatment
36
how does classical CD28 co-stimulation drive T cell metabolism?
Classical CD28-mediated co-stimulation for full metabolic reprogramming:
- downstream of CD28 is PI3K pathway
- this phosphorylates AKT which activates mTOR
- Increased mTOR drives reprogramming – increases glucose uptake and metabolism, protein synthesis, lipid metabolism
37
how does alternative CD46 co-stimulation drive T cell metabolism?
Binding of C3b to complement receptor, CD46, on T cell surface stimulates metabolic reprogramming:
- CD46 signals via CYT1 to drive transcription of glucose and amino acid transporters (Glut1, LAT1)
- this promotes LAMTOR5 amino acid sensing machinery to activate mTOR
- activated T cells produce C3b to act back on cell in autocrine manner – enhance metabolic changes
38
how is T cell metabolism altered in tumours?
T cells that enter TME of solid tumours demonstrate impaired function:
- TILs in TME look functionally impaired
- Cancer upregulates immune checkpoint inhibitors
- Altered metabolic TME also plays a role in dampening TILs
- Highly glycolytic tumours deplete glucose from the tumour microenvironment leading to anti-tumour T cell hyporesponsiveness
39
how does metabolic competition occur in TME?
Activated immune cells and cancer excrete high quantities of lactate – acidification
- Tumour consumes lots of glucose – competition for glucose uptake with T cells
- T cell can't get enough glucose to support function
- Tumour-associated macrophages also consume glucose and limit T cell access
- also true for amino acids
40
how has metabolic competition in TME been studied?
Engineered cancer cells to have more/less capacity for glucose uptake and transferred into mice skin to generate localised tumour:
- Low glycolytic tumours = improved T cell function and tumour clearance
- Standard tumours with more glucose uptake – functional impairment of T cells due to glucose competition
41
how can metabolic competition of TME be targeted?
Checkpoint blockade therapy may modulate this balance
- Tumours take up less glucose, enabling T cells to take up more
42
how is T cell mito function impaired in the TME?
Electron microscopy:
- effector T cell from spleen/lymph node = large, complex mito with lots of cristae.
- TILs form tumour = dysmorphic mito, loss of cristae in IMM, dysfunctional mito
- This is driven by chronic antigen stimulation by tumour antigens, combined with hypoxia, leading to mito dysfunction
T cell has hypometabolism
43
what are highly glycolytic tumours characterised by?
- depletion of glucose
- accumulation of lactic acid - acidic environment
44
how do highly glycolytic tumours impair T cell function?
Tumours are highly glycolytic – build-up of lactic acid and waste products:
- Downstream waste may impair function of TILs
- Lactate can suppress T cells – alter intracellular pH which can affect NFAT signalling and cytokine secretion
- but in other contexts, lactate can drive T cell metabolism and be taken up to be used as substrate – depends on pro/anti-inflammatory environments
45
how is the metabolism of T cells affected in autoimmune diseases?
In autoimmunity, T cells demonstrate hypermetabolic state – dysregulated, aberrant activity
T cells from autoimmune mouse models or patients – heightened mito OXPHOS function and glycolysis in SLE and MS
- also seen in type 1 diabetes
- when looking at autoantigen-specific T cells, this is seen to a greater extent – these cells are activated and drive disease
46
how is T cell metabolism altered in chronic, unresolved infection e.g. HBV infection?
- HBV-specific T cells demonstrate dysregulated cellular metabolism, accompanied by high inhibitory receptor (PD-1) expression
- HBV-specific cells demonstrate high glucose uptake (elevated Glut1) but low mitochondrial membrane potential and a dependency on glucose for cytokine production (metabolic inflexibility)
- T cells become metabolically dysfunctional – had many glucose transporters, but mito couldn’t oxidise the pyruvate under chronic antigen exposure
47
how can chronic T cell exhaustion be recapitulated in mice?
In mouse models of short resolved viral infections vs chronic, unresolved infections via LCMV in different forms:
- Armstrong strain of virus = controlled
- CL13 - not controlled
- T cells specific for Armstrong – mito in tact
- In T cells specific for chronic version that is less controlled – mito are dysmorphic and dysfunctional
48
how does HIV affect T cell metabolism?
in CD4+ T cells, mTOR activity links cellular metabolism to the susceptibility of HIV-1 infection.
- T cells has heightened metabolic activity – supports viral replication
- Increased mTOR activity, downstream of TCR ligation or IL-7/IL-15 signalling, expands the pool of dNTPs, thereby facilitating the
synthesis of reverse transcription products
- It also increases acetylation of microtubules, which facilitate the transport of these reverse transcription products towards the nucleus
49
how can metabolic competition in TME be targeted?
1. checkpoint blockade
2. supply metabolite of glycolysis with PEP (phospho-enol-pyruvate)
- Engineering of T cells could make them more metabolically fit to deal with depleting or hostile metabolic environments
50
how can treatment of T cells with PEP overcome metabolic competition in TME?
Identified that T cell impairment was due to need for specific glycolytic metabolite – phospho-enol-pyruvate (PEP) to aid calcium flux for signalling
engineer T cells to generate PEP independently of glucose, rendered tolerant to TME environment
– overexpressed PCK1 to generate PEP metabolite from TCA cycle via glutamine – T cell may be less dependent on glucose in TME
Mice with T cells overexpressing PCK1 improved mouse survival compared to normal PCK1 levels
51
how can oncolytic viruses be used to treat T cells in TME?
Engineered oncolytic virus have been used to infect tumour and replicate to induce lysis
- Can we engineer the virus to deliver cargo in TME to improve environment for T cell?
- Engineered virus to express leptin hormone to improve T cell metabolic capacity
Mice with leptin oncolytic virus had improved T cell metabolism and tumour clearance
52
how can T cell metabolism be targeted in SLE autoimmunity?
Mouse model which spontaneously develops lupus-like disease:
- Treated with metformin – inhibits ETC at complex 1 in OXPHOS
- 2-DG which inhibits glycolysis
enlarged spleen in lupus compared to WT mice
- Treatment with metabolic inhibitors reduces spleen size in lupus mice
Measured dsDNA autoantibodies
- In untreated mice – more autoantibodies
- In treated mice – reduction in autoantibodies
target hypermetabolism in T cell autoimmunity
53
how can T cell metabolism be targeted in MS?
Interestingly, it was recently found that a drug which is already used for the treatment of MS – dimethyl fumarate – may in fact work by suppressing T cell glycolysis and related inflammatory cytokine production
54
what is the seahorse assay used for?
in vitro extracellular flux analysis:
- Measures oxygen consumption (mito function) and ECAR (glucose uptake and metabolism) in parallel
Can inject different compounds during assay and measure cell responses
55
what are the different compounds that can be injected during the seahorse assay?
- Oligomycin – blocks complex 5 – what proportion of oxygen consumption is related to mito ATP generation
- FCCP – uncoupling agent, stimulates respiration in mito by uncoupling ATP synthesis from ETC - induces maximal respiration
- Rotenone – ETC inhibitor - blocks all mito respiration
56
what happens when mitochondria are inhibited?
Inhibit mito complex 1 and 5 = increased glycolysis/glucose metabolism to compensate ATP turnover
57
do enlarged mitochondria in memory T cells enable higher ETC complex expression?
Effector memory cells have increased ETC complex subunit expression – measured by flow cytometry
in seahorse assay, this translates to increased mito capacity in memory T cells
58
Does increased mito capacity enable memory T cells to survive in metabolically challenged tissues compared to lymph nodes?
Sort naïve and memory cells with different metabolic activities in
normoxia or hypoxia:
- Can memory cells maintain ATP levels better in hypoxia?
- Naïve cells in low oxygen lose ATP generation
- Memory cells in low oxygen have less loss of ATP generation
- Caspase 9 activity – naïve cells have more caspase 9 (apoptosis), whilst memory cells do not express caspase 9 – improved survival at low oxygen
- Annexin 5 = late apoptosis – binds phosphatidylserine – apoptotic cells flip membrane so that phosphatidylserine is exposed
- Naïve cells at low oxygen have increased phosphatidylserine, whilst memory cells don’t – so survive longer
59
how may increased mito activity support memory T cell circulation in tissues?
Memory T cells migrate through tissue environments to encounter pathogens
- this requires motility which depends on ATP
– increased mito activity supports migration
Increased memory cell metabolic capacity supports survival and migration in low oxygen
60
what is a transwell study?
Put cells in well which contains membrane with pores small enough for cells to pass through
- Sit cells on membrane on top of culture dish
- Cells can pass through membrane to bottom half of cell culture dish
- Transwell inserts can have different pore size – can be just smaller than lymphocyte to test if cell can remodel cytoskeleton with ATP to pass through pores
- Flow cytometry can then analyse cells with surface marker antibodies at the bottom to see if they are more memory or naïve
61
how well do memory vs naive T cells spontaneously migrate in hypoxic transwell experiment?
Naïve cells have impaired migration
Memory cells had less impairment – fold-inhibition of migration was greater for naïve compared to memory
62
how well do memory vs naive T cells migrate in response to CXCL12 in hypoxic transwell experiment?
Add chemokine at bottom of dish – CXCL12 which naïve and memory cells respond to
- At low oxygen, naïve more inhibited compared to memory
63
how can a microfluidic experiment be used to measure naive vs memory migration?
Track migration of individual cells towards chemokine gradient
- Each line = track of individual cell
- Without chemokine – no direction of movement
- with chemokine – tracks towards gradient
- Couldn’t use this in hypoxia, so mimicked hypoxia with oligomycin to inhibit ETC
- Migration of naïve was impaired, while memory cells had unaffected migration
64
how does rapid glycolysis occur in memory T cells for rapid recall?
increased expression of GAPDH in cytoplasm to rapidly engage glycolysis
- this leads to rapid cytokine production
65
how can rapid recall of memory T cells be tested?
seahorse:
- Inject anti-CD3 and anti-CD28 to stimulate T cells and see how they metabolically respond
- Memory T cells upregulate glycolysis within seconds – increased ECAR – this doesn’t happen if no glucose is present, indicating reliance on glycolysis
- Naïve cells don’t really upregulate glycolysis until 10-12 hours
- Supports rapid production of cytokines
66
why is rapid recall glycolysis important for memory cells?
it enables rapid cytokine production
at 12 hours:
- Naïve cells haven’t made IFNy
- Effector memory cells rapidly made IFNy in high quantities
- Inhibition of glucose metabolism with 2-DG causes impaired IFNy in memory cells – glycolysis dependent
67
how does competition in TME lead to immune suppression?
Competition for nutrients and metabolite accumulation limit anti-tumour immunity
Tumour-derived cytokines (i.e. TGF-β) suppress immune cell metabolism and interlinked function
68
how does TGFb affect T cell metabolism?
Activate T cells in presence of TGFb for 2 days – metabolism is suppressed
- Basal and maximal oxygen consumption is impaired
- Small decrease in glycolysis (basal ECAR), mito impairment is more significant (basal OCR)
TGFb impacts mito function in T cells
- highest effect on effector memory cells, then central memory, then naive
- also more of an effect on activated T cells rather than resting cells
– correlated with TGFbR expression which increases upon stimulation
69
what happens to TGFb signalling when SMAD2 is K/O in T cells?
SMAD2 is a T cell signalling protein induced by TGFb
- K/O of smad2 reduced TGFb affects - T cell metabolism was less impaired
BUT no change in mitochrondrial mass or ETC expression
- western blot: TGFb doesn’t affect expression of mito ETC subunit machinery
TGFb must effect mito function, not expression
- K/O of signalling proteins stopped TGFb effects
70
what is image stream?
Image stream – combo of flow cytometry and microscopy
- Run cells through fluid in machine
- Cells in single file excited by lasers – fluoresce
- Also takes image of every cell – can see where the molecules that were stained for are in the cell
71
how was image stream used to understand TGFb effects on mito signalling?
Stain for MTR – marker of mito, DAPI = nucleus, stain for phosphorylated SMAD
- MTR red = high in mito
- DAPI high in nucleus
- SMAD associates with both nucleus and mito, which increases with TGFb treatment
SMAD associates with mito
- signalling proteins induced by TGFb may impact mito function
72
how does TGFb impact ETC complexes?
Complex 5 activity is impaired in presence of TGFb
73
how could TGFb effects on T cell metabolism be modelled in disease?
Use effusion fluid of ovarian cancer oedema, which is enriched in products from tumour and may contain TGFb
Test if effusion fluid suppresses T cell function, and if it depended on TGFb:
- used antibody to neutralise TGFb
- Compared with isotype antibody which doesn’t block TGFb
- Anti-TGFb – T cells had higher mito function
- TGFb in fluid – T cells had impaired mito function
Effusion fluid has sufficient TGFb to impair T cell metabolism
74
how does TGFb effusion fluid affect the effector function of T cells?
In T cells cultured with effusion fluid where TGFb is active, there is less IFNy production
In T cells cultured with effusion fluid where TGFb is blocked by antibody, more IFNy production
Inhibition of SMAD phosphorylation - IFNy production is no longer impaired
Inhibition of SMAD nuclear translocation causes no effect on IFNy
- SMAD must not enter nucleus and instead localise at mito
Inhibition of mito with oligomycin impaired IFNy production
75
what is succinate?
a TCA cycle intermediate
- this can build up in TME of glioma, multiple myeloma, and in inflammatory contexts e.g. MS, RA
not understood how this affects immune cell funciton
76
how has metabolite build up in T cell environments and their impacts been studied?
Metabolic tracer studies using stable isotope enriched metabolic precursors
Provide high-resolution information on the metabolic fate of labelled nutrients
(i.e. glucose, glutamine, fatty acids)
77
what is multiple myeloma?
a plasma cell malignancy which develops in bone marrow microenvironment
78
what metabolites can build up in the bone marrow in multiple myeloma?
Succinate is enriched in myeloma BM, and not in healthy controls
79
what are the roles of succinate?
intermediate of TCA
Succinate can stabilise HIF-1a
influence epigenetic remodelling - impacts chromatin enzymes
can post-translationally modify proteins
has GPCR (SucNR1) which it can signal through
80
what happens to T cells when activated in the presence of succinate?
Looked at IFNy expression and ability of cells to degranulate
T cells activated in presence of succinate demonstrate impaired IFNy production and degranulation
- also affected production of other cytokines such as IL-2, IL-6, IL-4 etc
81
how can succinate uptake into T cells be studied?
tracing study where T cells were incubated with carbon 13 labelled succinate
- in T cells cultured with C-13 succinate, almost all of their intracellular succinate levels had a mass shift of 4 heavy carbons – indicates that all succinate came from outside of cell
- some succinate is being metabolised – heavy M4 carbons seen in fumarate and malate
succinate may effect intracellular T cell mechanisms
82
did succinate have an effect on HIF in T cells?
Expose cells to hypoxia
- No effect of succinate on HIF
succinate doesn't seem to be stabilising HIF in this case
83
how does succinate effect mitochondrial membrane potential in T cells?
Metabolism impaired in presence of succinate – impaired mitochondrial function
- mitochondrial membrane potential was decreased in cells cultured with succinate
84
how does succinate affect glucose uptake and metabolism in T cells?
Glucose tracing:
- Cells incorporated more glucose carbons into lactate in presence of succinate
- more glycolysis, less TCA
- Also the same for alanine which can be made from pyruvate
- Less oxidation of pyruvate in mito in presence of succinate
In presence of succinate
- Less mito oxidation of pyruvate, and more glycolytic lactate/alanine
- inhibition of mito function
85
how does succinate inhibit mito function?
succinate can inhibit its upstream enzyme succinyl-CoA synthetase
T cell uptakes succinate, which then inhibits its upstream enzyme, which limits TCA cycle and therefore mito function
86
could T cell immune function be rescued from succinate inhibition?
K/D of enzyme to prevent pyruvate conversion to alanine with siRNA or with BLCA (inhibitor drug) to deliver more pyruvate to the TCA cycle
- succinate no longer had suppressive effect on IFNy – rescued T cell function
or inhibited conversion of pyruvate to lactate with DCA - also rescued inhibitory effect of succinate
or just added more pyruvate into the system to force glucose flux through TCA to rescue inhibitory effect of succinate on mito function
87
in what other disease can succinate accumulate in really high levels?
neuroendocrine tumour - mutations in succinate metabolising enzymes where succinate builds up
RNA seq of neuro tumours which had buildup of succinate:
- Some have mutation in succinate-metabolism enzymes = succinate accumulation
- Others do not have these mutations, so don’t have succinate accumulation
88
how are T cells affected in neuroendocrine tumours in succinate accumulation?
Looked for IFNy as a indication of T cell activity in the tumour
Gamma-responsive genes are normally switched on by IFNy e.g. HLA - can look for these genes in the tumour cells
- indirect indication of T cell activation
Gamma-responsive genes are reduced in tumours where succinate accumulates
- Succinate impairs T cell function in this context
