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MICB 402 > Paper 4: TLR4, HMGB1, Anticancer Chemotherapy > Flashcards

Paper 4: TLR4, HMGB1, Anticancer Chemotherapy Flashcards

1
Q

what evidence is present for the immune system having a major role in protection against cancer

A
  • cancer is more common in immunosuppressed individuals
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2
Q

what evidence is present for the immune system having a major role in supporting cancer (2)

A
  • inflammation is often necessary for tumour growth and metastasis
  • chronic inflammation is firmly linked to cancer
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3
Q

why do immune responses against tumours fail or not develop (4)

A
  • tumours may induce tolerance by manipulating immune system
  • tumours or immune cells infiltrating tumour may produce immunosuppressive cytokines that block CTLs and NK cells
  • some tumours may kill infiltrating leukocytes by expressing death ligand receptors
  • some tumours may not induce activation of immune system
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4
Q

describe the discovery of the ability to fight cancer by prodding the immune system (2)

A
  • evidence of tumour specific antigen T cells that could response to tumour cells
  • inhibitory receptors were previously blocking T cell activity, so by blocking these inhibitors, treatment of severe cancers were successful
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5
Q

elimination phase of cancer: phase 1 (2)

A
  • lymphocytes that participate in innate immunity recognize transformed cells that have accumulated above threshold
  • cells stimulated to produce IFN-g
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6
Q

elimination phase of cancer: phase 2 (innate details) (4)

A
  • initial IFN-g starts a cascade of innate immune reactions
  • induction of chemokines that block new vascularization in the tumour and affect NK cell, DC, and macrophage recruitment to the tumour site
  • antiproliferative action of IFN-g on developing tumour
  • activation of cytocidal activity in macrophages and NK cells entering tumour
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7
Q

elimination phase of cancer: phase 2 (adaptive details) (4)

A
  • tumour cell death is caused by innate mechanisms
  • dead tumour cells or debris are ingested by DCs and trafficked to draining lymph nodes
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8
Q

elimination of cancer: phase 3

A
  • tumour growth is kept in check by cytocidal activities of NK cells and activated macrophages
  • CD4+ and CD8+ T cells specific for tumour antigens develop in draining lymph nodes
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9
Q

elimination of cancer: phase 4

A
  • tumour-specific CD4+ and CD8+ T cells travel to tumour along chemokine gradients where they recognize and destroy tumour cells expressing distinctive tumour antigens
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10
Q

what are the 3 possible interactions between our tumours and our immune system (3)

A
  • elimination
  • equilibrium
  • escape
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11
Q

tumours and the immune system: elimination

A
  • immune system successfully eliminates the tumour
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12
Q

tumours and the immune system: equilibrium (3)

A
  • constant action of immune system to keep tumour at bay so that a small population of tumours are always present
  • thought to be always occurring
  • tumours are not noticeable to us as they are under-developed due to immune control
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13
Q

tumours and the immune system: escape (2)

A
  • where tumour growth starts to become apparent, evade immune system, and affect health
  • this is the cancer that we are aware of
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14
Q

what are some examples of the immune system supporting tumours (3)

A
  • use of immunosuppressive cytokines
  • inhibitory/death receptor ligands that cause incoming leukocytes to undergo apoptosis
  • use of cytokines that increase vascularization to the tumour
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15
Q

what are the main cell types involved in tumour immunity (3)

A
  • CD4+ T cells
  • CD8+ T cells and CTL
  • mature DC
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16
Q

what is a B3Z cell

A
  • a type of T cell that can recognize the OVA antigen
  • MHC class I; related to CD8 T cells
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17
Q

what is a B09710 cell

A
  • a type of T cell that can recognize the OVA protein
  • MHC class II; related to CD4 T cells
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18
Q

what is SINFEKL (3)

A
  • a sequence of amino acids found within the OVA protein and the dominant epitope recognized by T cells
  • acts as a positive control for experiments as DC’s don’t need to process this protein to activate T cells
  • as a positive control, it tells us if the DC is competent of presenting antigen in general
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19
Q

what is IL-2 production indicative of

A
  • the successful MHC-antigen presentation occurring between DCs and T cells
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20
Q

what was the purpose of figure 1

A
  • to determine which TLR might control immune response against dying tumours using IL-2 production as a marker of successful DC antigen presentation
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21
Q

what was observed in figure 1 (3)

A
  • all DCs could present antigen from dying tumour cells expect for TLR4-/- and MyD88-/- DCs, judging from their ability to induce IL-2 expression in B3Z and B09710 cells
  • wt mice with TLR4 inhibitory peptides or antibodies were also defective at inducing a MHC class I-restricted OVA-specific response
  • live cells induce NO reaction as they don’t contain “alarming” antigens
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22
Q

what was the purpose of figure 2 (2)

A
  • to determine which TLR might control immune response against dying tumours using IFN-g production as a marker of successful antigen presentation by DCs
  • to investigate if DC induction of T cell activation by irradiated tumour cells also occurs in vivo (fig. 1 was in vitro)
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23
Q

what is IFN-g production indicative of (2)

A
  • DCs are activated and tumour antigen is presented to T cells
  • T cells that are presented with antigen will make the IFN-g
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24
Q

how is figure 2 done in vivo (3)

A
  • live or dead tumour cells are injected into the footpad of mice
  • draining lymph node fluids are harvested
  • OVA or PBS is added before measuring IFN-g production
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25
Q

why is OVA/PBS added again after lymph fluid is harvested

A
  • re-stimulation is needed for T cells to produce IFN-g, stimulating T cells going back out to tissues and responding to pathogen
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26
Q

what was observed in figure 2a

A
  • IFN-g production was induced in wt and all TLR-/- mice except for the TLR4-/- mice
  • live cells induce NO reaction as they don’t contain “alarming antigens”
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27
Q

doxorubicin (DXR)

A
  • another drug that can be used to kill tumour cells (an alternative to X-ray or oxaliplatin in this study)
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28
Q

CT26 cells and MCA205 cells (2)

A
  • colon cancer cells and sarcoma cells
  • these cells don’t express OVA, which is known to induce a powerful T cell response
29
Q

what was observed in figure 2b (2)

A
  • T cell response was TLR4-dependent and required treating tumour cells with drug to induce cell death
  • wt and TLR4-/- mice could elicit T cell response in both cell types, but TLR4-/- mice couldn’t
30
Q

what was observed in figure 2c (2)

A
  • TLR4-/- mice mount a normal response to OVA with the adjuvant CpG (TLR-9 agonist)
  • both CD4 and CD8 T cells produce IFN-g
31
Q

what were the findings from figure 2c

A
  • absence of TLR4 selectively compromised immune response against dying cells, not soluble antigen
32
Q

what was observed in figure 2d

A
  • mice that were DC depleted could not mount an anti-tumour response, measured by IFN-g production
33
Q

what were the findings from figure 2d

A
  • DCS are required for the TLR4-dependent anti-tumour response
34
Q

how were the results from figure 2e obtained (4)

A
  • tumour cells were injected into mice thigh and allowed to grow
  • tumour site was either untreated or X-ray irradiated
  • lymph-nodes were harvested and incubated with PBS, OVA peptide, or OVA protein
  • T cell activated was detected with IFN-g secretion
35
Q

what was observed in figure 2e (3)

A
  • untreated tumours do not elicit successful DC antigen presentation
  • DC antigen presentation was successful only if tumour cells were treated with X-ray
  • DC antigen presentation was not successful in TLR4-deficient mice
36
Q

what were the findings from figure 1 and 2

A
  • TLR4 must be present in DCs for the optimal presentation of antigen derived from dying tumour cells
37
Q

what is the suspected role of TLR in DCS

A
  • regulates the processing and presentation of tumour cell antigens by DCs, presumably by inhibiting the lysosomal destruction of antigens
38
Q

what is the purpose of figure 3

A
  • to determine what tumour cells are expressing (ie. some sort of PAMP) that is detected by DC TLR4s and used to prime T cell responses against tumours
39
Q

what was observed in figure 3a

A
  • after killing tumour cells, only the HMGB1 protein that is known to be detected by TLR4 was released/exposed
40
Q

what method was used to determine figure 3a

A
  • western blot
41
Q

what was observed in figure 3b

A
  • HMGB1 secretion is observed in multiple tumour types in response to various cytotoxic agents, but not in live cells
42
Q

what method was used to determine figure 3b

A
  • ELISA
43
Q

what method was used for figure 3c (2)

A
  • co-immunoprecipitation
  • western blot
44
Q

what were the steps to obtain figure 3c (3)

A
  • supernatants from killed or live tumour cells were incubated with macrophages expressing TLR4
  • HMGB1 was IP after cell lysis and then examined for TLR4
  • HMGB1 and TLR4 were detected using western blot
45
Q

what was observed in figure 3c

A
  • HMGB1 secreted by dying tumour cells was bound to TLR4
46
Q

what was observed in figure 3d

A
  • injection of antibody against HMGB1 blocks T cell activation induced by irradiated tumour injection but use of antibodies to other proteins did not affect T cell activation
47
Q

what was observed in figure 3e

A
  • use of an siRNA that depleted HMGB1 protein blocked T cell activated induced by irradiated tumour injection in all different tumour types
48
Q

what were the findings from figure 3

A
  • HMBG1 represents the principal damage-associated molecular pattern that dictates the TLR4-dependent immune response to dying tumour cells
49
Q

what was the purpose of figure 4

A
  • to investigate the efficacy of using the HMGB1-TLR4 dependent immune response to dying tumour cells in anticancer drugs
50
Q

what were the steps for figure 4a (3)

A
  • injection of saline or varying dead tumour cells (killed with different methods/toxins) to investigate if induction of immune response occurs
  • a week later, live tumour cells were injected
  • number of tumours that subsequently developed was measured
51
Q

what was observed in figure 4a (2)

A
  • injection of toxin-treated cancer cells into mice was highly efficient in inducing an immune response that prevents growth of subsequent live injected cells in wt mice
  • no tumour vaccination could be achieved in TLR4-/- mice
52
Q

what was observed in figure 4b

A
  • blocking of HMBG1-TLR4 pathway with an inhibitory peptide blocking TLR4 injected with the initial dying tumour cell injection impaired future anti-tumour immunity
53
Q

what was observed in figure 4c

A
  • blocking of HMBG1-TLR4 pathway with a neutralizing antibody against HMBG1 injected with the initial dying tumour cell injection impaired future anti-tumour immunity
54
Q

what was observed in figure 4d

A
  • blocking of HMBG1-TLR4 pathway with a HMBG1-specific siRNA injected with the initial dying tumour cell injection impaired future anti-tumour immunity
55
Q

what was observed in figure 5 (2)

A
  • chemotherapy with appropriate cytotoxic agents or local radiotherapy reduced tumour growth and prolonged survival of tumour-bearing wt mice, but not TLR4-/-, nu/nu, and MyD88-/- mice
  • use of chloroquine in TLR4-/- mice enhanced the efficacy of chemotherapy
56
Q

what are nu/nu mice

A
  • mice lacking a thymus
57
Q

what is the difference in results between figure 4 and 5 (2)

A
  • the vaccination-like method used in figure 4 acted as a preventative factor, ensuring that mice could be tumour free in subsequent tumour injections
  • the treatment-like method used in figure 5 was not preventative and was acting after tumours were already bad; TLR4 pathway could only stabilize tumours and not results in tumour-free mice
58
Q

how do we know that TLR4 controls tumour Ag processing and presentation (4)

A
  • wt and TLR4-/- DCS phagocytosed tumours equally well
  • killed tumour cells activated wt and TLR4-/- DCs equally well in terms of cytokine secretion and DC maturation
  • both DC types equally effective at activating T-cells when loaded with exogenous OVA
  • only the presentation of OVA peptide from dying tumour cells to T cells was significantly impaired inTLR4-/- DCs
59
Q

chloroquine and bafilomycin

A
  • compounds that block normal lysosome function
60
Q

what is the proposed mechanism that TLR4 is controlling antigen processing and presentation by

A
  • TLR4 may prevent degradation of antigens in phagocytic vesicles and thus enhance the presentation of tumour antigens to T cells
  • chloroquine and bafilomycin enhanced tumour antigen presentation in TLR4-/- DCs
61
Q

what were the findings from figure 5

A
  • HMGB1/TLR4/MyD88-dependent immunity contribute to chemotherapeutic regimens
62
Q

what is the purpose of figure 6

A
  • to investigate the relevance of TLR4 mutants in chemotherapy efficacy
63
Q

what was observed in figure 6a

A
  • binding of HMBG1 to mutant TLR4 (Asp299Gly) was reduced as compared to its binding in normal (AspGly) TLR4
64
Q

MD-DC

A
  • monocyte-derived dendritic cells
65
Q

what was observed in figure 6b (2)

A
  • MD-DCs from normal (Asp299) individuals cross-presented dying tumour cell antigens to CTL clones in an HMGB1-dependent manner, while MC-DCS with Asp299Gly TLR4 allele did not cross-present
  • defect in Asp299 TLR4 allele MC-DCs was restored with addition of chloroquine
66
Q

what was observed in figure 6c

A
  • women with non-metastatic breast cancer with a wt TLR4 allele had a significantly higher percentage of metastasis-free-survival compared to women in the mutated TLR4 allele group
67
Q

what were the findings from figure 6

A
  • specific mutation in TLR4 with functional relevance may influence immunological component of anthracycline-based chemotherapy in human cancer
68
Q

what are the conclusions of this paper? (6)

A
  • treatment regimes that kill tumour cells induce activation of DCs and the presentation of tumour antigens to T cells
  • DCs are essential for inducing T cell response against tumours
  • TLR4 and MyD88 are critical for DC T cell activation while other TLRs are not important for mouse anti-tumour response
  • alarmin protein HMGB1 is critical for TLR4-dependent tumour immunity
  • HMBG1/TLR4 pathway if important for efficient presentation of tumour antigens to T cells(not for DC maturation/activation)
  • TLR4/HMBG1 pathway may be important for success of tumour treatment protocols in patients

MICB 402 (11 decks)

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