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
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
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
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
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
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
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
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
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
10
Q
what are the 3 possible interactions between our tumours and our immune system (3)
A
- elimination
- equilibrium
- escape
11
Q
tumours and the immune system: elimination
A
- immune system successfully eliminates the tumour
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
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
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
15
Q
what are the main cell types involved in tumour immunity (3)
A
- CD4+ T cells
- CD8+ T cells and CTL
- mature DC
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
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
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
19
Q
what is IL-2 production indicative of
A
- the successful MHC-antigen presentation occurring between DCs and T cells
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
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
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)
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
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
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
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”
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)
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
