T cell metabolism

Question 1

how does the metabolism of T cells change when they engage in an immune response?

Answer 1

1. altered uptake and handling of glucose
2. altered mitochondrial function via glutamine uptake
3. Altered amino acid metabolism

Question 2

how is glucose uptake changed in T cells?

Answer 2

Increased glucose uptake, tendency to convert pyruvate to lactate (increased ECAR)

Question 3

how is glucose metabolism changed in T cells during an immune response?

Answer 3

• 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)

Question 4

how can glucose handling differ in aerobic and anaerobic metabolism?

Answer 4

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

Question 5

what is the more efficient way of using glucose?

Answer 5

aerobic TCA cycle and OXPHOS - 1 molecule of glucose produces 32 ATP

Question 6

how do immune cells use the Warburg effect? why do they do this?

Answer 6

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)

Question 7

do activated T cells undergo a glycolytic switch?

Answer 7

No, T cells still use their mitochondria upon immune responses

Question 8

how does mitochondrial function change in activated T cells?

Answer 8

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

Question 9

what is the overall function of the ETC?

Answer 9

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

Question 10

what substrates can fuel the TCA cycle?

Answer 10

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

Question 11

how to T cells alter their uptake of mitochondrial substrates?

Answer 11

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

Question 12

how is T cell amino acid uptake altered upon activation?

Answer 12

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

Question 13

how does altered glucose metabolism support T cell immune function?

Answer 13

1. Shift in reduction of pyruvate to lactate generate intermediates to support proliferation
2. increased glucose uptake can influence changes in epigenetic landscape of T cells
3. can alter translation of T cell mRNA

Question 14

what is methylation vs acetylation?

Answer 14

• 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

Question 15

how does glucose metabolism alter T cell epigenetic landscape?

Answer 15

Glucose oxidised in mito can form acetyl CoA which can acetylate histones
- Acetylation improves transcription of IFNy – important T cell cytokine – production of mRNA

Question 16

how does increase glucose uptake alter mRNA translation in T cells?

Answer 16

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

Question 17

how does glucose metabolism regulate IFNy?

Answer 17

IFNy expression is regulated at the transcriptional level via metabolic-dependent epigenetic changes and at the translational level via glycolytic enzymes binding mRNA

Question 18

how does increased mito function affect T cell immune function?

Answer 18

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)

Question 19

what happens if ROS isn’t produced in T cells?

Answer 19

Study modified ETC to not produce ROS
- less NFAT entering nucleus
- less IL-2 production

Question 20

how does mito biogenesis change when T cells are in an immune response?

Answer 20

• mito biogenesis increases upon T cell activation

Question 21

how can mito biogenesis in T cells be studied?

Answer 21

Fluorescent label of mito – over time course of T cell stimulation, more mito are produced in T cell

Question 22

Are T cells making more mito similar to original mito in functionality, or are the new mito specialised in function?

Answer 22

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

Question 23

why is amino acid uptake important in T cell immune responses?

Answer 23

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

Question 24

what amino acids promote mTORC1 activity in T cells?

Answer 24

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

Question 25

how can mTORC1 be targeted in graft rejection?

Answer 25

Rapamycin is a potent immune suppressant – it blocks mTOR to prevent immune responses
- treats transplants patients

Question 26

what is the role of serine in T cell immune function?

Answer 26

Serine used to support purine synthesis
- Taken up in larger quantities by activated T cells
- or made via glucose

Question 27

what is the function of methionine in T cell immune responses?

Answer 27

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

Question 28

what is the function of glutamine in T cell immune responses?

Answer 28

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

Question 29

what is rapid recall function of T cells? what facilitates this function?

Answer 29

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

Question 30

how do the mitochondria differ between naive and memory T cells?

Answer 30

• 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

Question 31

why do memory T cells need to undergo metabolic changes?

Answer 31

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

Question 32

how was altered metabolism found to be important for memory T cells?

Answer 32

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

Question 33

what promotes the generation of complex mito in memory T cells?

Answer 33

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

Question 34

how does enforced mitochondrial fusion affect memory T cells?

Answer 34

this induces more memory generation

Question 35

how does mito metabolism differ for different T cell subsets?

Answer 35

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

Question 36

how does classical CD28 co-stimulation drive T cell metabolism?

Answer 36

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

Question 37

how does alternative CD46 co-stimulation drive T cell metabolism?

Answer 37

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

Question 38

how is T cell metabolism altered in tumours?

Answer 38

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

Question 39

how does metabolic competition occur in TME?

Answer 39

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

Question 40

how has metabolic competition in TME been studied?

Answer 40

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

Question 41

how can metabolic competition of TME be targeted?

Answer 41

Checkpoint blockade therapy may modulate this balance
- Tumours take up less glucose, enabling T cells to take up more

Question 42

how is T cell mito function impaired in the TME?

Answer 42

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

Question 43

what are highly glycolytic tumours characterised by?

Answer 43

• depletion of glucose
• accumulation of lactic acid - acidic environment

Question 44

how do highly glycolytic tumours impair T cell function?

Answer 44

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

Question 45

how is the metabolism of T cells affected in autoimmune diseases?

Answer 45

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

Question 46

how is T cell metabolism altered in chronic, unresolved infection e.g. HBV infection?

Answer 46

• 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

Question 47

how can chronic T cell exhaustion be recapitulated in mice?

Answer 47

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

Question 48

how does HIV affect T cell metabolism?

Answer 48

in CD4+ T cells, mTOR activity links cellular metabolism to the susceptibility of to 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 leads to acetylation of microtubules, which facilitate the transport of these reverse transcription products towards the nucleus

Question 49

how can metabolic competition in TME be targeted?

Answer 49

1. checkpoint blockade
2. supply metabolite of glucolysis with PEP (phospho-enol-pyruvate)
   - Engineering of T cells could make them more metabolically fit to deal with depleting or hostile metabolic environments

Question 50

how can treatment of T cells with PEP overcome metabolic competition in TME?

Answer 50

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

Question 51

how can oncolytic viruses be used to treat T cells in TME?

Answer 51

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

Question 52

how can T cell metabolism be targeted in autoimmunity?

Answer 52

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

Question 53

how can T cell metabolism be targeted in MS?

Answer 53

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

Question 54

what is the seahorse assay used for?

Answer 54

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

Question 55

what are the different compounds that can be injected during the seahorse assay?

Answer 55

• 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

Question 56

what happens when mitochondria are inhibited?

Answer 56

Inhibit mito complex 1 and 5 = increased glycolysis/glucose metabolism to compensate ATP turnover

Question 57

do enlarged mitochondria in memory T cells enable higher ETC complex expression?

Answer 57

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

Question 58

Does increased mito capacity enable memory T cells to survive in metabolically challenged tissues compared to lymph nodes?

Answer 58

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

Question 59

how may increased mito activity support memory T cell circulation in tissues?

Answer 59

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

Question 60

what is a transwell study?

Answer 60

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

Question 61

how well do memory vs naive T cells spontaneously migrate in hypoxic transwell experiment?

Answer 61

Naïve cells have impaired migration

Memory cells had less impairment – fold-inhibition of migration was greater for naïve compared to memory

Question 62

how well do memory vs naive T cells migrate in response to CXCL12 in hypoxic transwell experiment?

Answer 62

Add chemokine at bottom of dish – CXCL12 which naïve and memory cells respond to
- At low oxygen, naïve more inhibited compared to memory

Question 63

how can a microfluidic experiment be used to measure naive vs memory migration?

Answer 63

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

Question 64

how does rapid glycolysis occur in memory T cells for rapid recall?

Answer 64

increased expression of GAPDH in cytoplasm to rapidly engage glycolysis
- this leads to rapid cytokine production

Question 65

how can rapid recall of memory T cells be tested?

Answer 65

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

Question 66

why is rapid recall glycolysis important for memory cells?

Answer 66

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

Question 67

how does competition in TME lead to immune suppression?

Answer 67

Competition for nutrients and metabolite accumulation limit anti-tumour immunity

Tumour-derived cytokines (i.e. TGF-β) suppress immune cell metabolism and interlinked function

Question 68

how does TGFb affect T cell metabolism?

Answer 68

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

Question 69

what happens to TGFb signalling when SMAD2 is K/O in T cells?

Answer 69

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 of 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

Question 70

what is image stream?

Answer 70

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

Question 71

how was image stream used to understand TGFb effects on mito signalling?

Answer 71

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

Question 72

how does TGFb impact ETC complexes?

Answer 72

Complex 5 activity is impaired in presence of TGFb

Question 73

how could TGFb effects on T cell metabolism be modelled in disease?

Answer 73

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

Question 74

how does TGFb effusion fluid affect the effector function of T cells?

Answer 74

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

Question 75

what is succinate?

Answer 75

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

Question 76

how has metabolite build up in T cell environments and their impacts been studied?

Answer 76

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)

Question 77

what is multiple myeloma?

Answer 77

a plasma cell malignancy which develops in bone marrow microenvironment

Question 78

what metabolites can build up in the bone marrow in multiple myeloma?

Answer 78

Succinate is enriched in myeloma BM, and not in healthy controls

Question 79

what are the roles of succinate?

Answer 79

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

Question 80

what happens to T cells when activated in the presence of succinate?

Answer 80

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

Question 81

how can succinate uptake into T cells be studied?

Answer 81

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

Question 82

did succinate have an effect on HIF in T cells?

Answer 82

Expose cells to hypoxia
- No effect of succinate on HIF

succinate doesn’t seem to be stabilising HIF in this case

Question 83

how does succinate effect mitochondrial membrane potential in T cells?

Answer 83

Metabolism impaired in presence of succinate – impaired mitochondrial function
- mitochondrial membrane potential was decreased in cells cultured with succinate

Question 84

how does succinate affect glucose uptake and metabolism in T cells?

Answer 84

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

Question 85

how does succinate inhibit mito function?

Answer 85

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

Question 86

could T cell immune function be rescued from succinate inhibition?

Answer 86

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

Question 87

in what other disease can succinate accumulate in really high levels?

Answer 87

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

Question 88

how are T cells affected in neuroendocrine tumours in succinate accumulation?

Answer 88

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 impairsT cell function in this context