Unit 6 - Apoptosis, advanced therapies and concepts of immuno-oncology Flashcards Preview

Molecular Medicine > Unit 6 - Apoptosis, advanced therapies and concepts of immuno-oncology > Flashcards

Flashcards in Unit 6 - Apoptosis, advanced therapies and concepts of immuno-oncology Deck (107)
Loading flashcards...
1

4 reasons why cells die

  1. when they get old e.g. lifespan of RBC = 120 days
  2. irreversible damage - exposed to extensive damage e.g. ischaemia, stress e.g. pathophysiological conditions, fever
  3. when they become superfluous - tissue/organ development e.g. loss of interdigital web cells, loss of tadpole tail; elimination of superfluous immune cells after recovery from infectious disease
  4. when they become dangerous to the body/organism - virus infected cells, malignant/cancer cells

2

how do cells die

apoptosis - highly regulated, organised form of cell death

necrosis - dysregulated form of cell death

3

characteristics of apoptotic cells

programmed form of cell death

cell separates from neighbouring cells

cell shrinkage

membrane blebbing

chromatin condenses and the nucleus fragments

cell breaks up into apoptotic bodies which are phagocytosed, such that cellular components or waste products do not produce an inflammatory response

predictable, reproducible sequence of events

4

5

key differences between apoptosis and necrosis

APOPTOSIS

  • single cells die
  • cell shrinks
  • remnants are phagocytosed
  • neat, controlled
  • no trace left
  • no inflammation follows

NECROSIS

  • chunks of tissue die
  • cells swell and burst
  • remnants are not cleared
  • cell content released into EC space
  • inflammation follows

6

too little apoptosis

malignant and pre-malignant conditions

lymphoproliferative disorders

leukemias

lymphomas

solid tumours

7

too much apoptosis

alzheimer's

parkinson's

stroke

atherosclerosis (CV)

ischaemia/reperfusion injury (CV)

dysentery (intestinal)

diarrhea (intestinal)

8

phases of apoptosis

INITIATION

the cell makes the decision to kill itself

EXECUTION

cell commits itself to die and activates the machinery for cellular disassembly

CLEARANCE

the apoptotic cell/bodies are removed from the system

9

what initiates apoptosis

appearance of death signals

withdrawal of survival factors

10

categories of death signals that initiate apoptosis

EXTRINSIC

death signal can derive from environment of cell

hormones and cytokines e.g. death ligands secreted by immune cells can kill infected/cancerous cells

INTRINSIC

can derive from inside of cell

overwhelming stress/irreparable damage (DNA damage, hyperthermia, exposure to toxic compounds)

11

withdrawal of survival factors - initiate apoptosis

most cells in the body depend on the presence of growth factors

in their absence, apoptosis is initiated and cells die

e.g. immune cells depend on presence of IL e.g. T cells on IL-2 and IL-15

12

what are caspases

effectors of apoptosis

cysteine dependent aspartic acid specific proteases

co-ordinate destruction of cellular structures

hallmark of apoptosis - required

13

proteases

enzymes that catalyse the breakdown of proteins into smaller polypeptides or AAs

14

cysteine dependent

active site of the caspase contains cysteine residue that is required for its catalytic activity

15

aspartic acid specific

cleave substrate proteins at aspartic acid residues

16

how are caspases synthesised

as inactive precursors (pro-caspases)

17

2 groups of caspases

initiator - 8, 9, 10

effector - 3, 6, 7

initiator activate effector, which then mediate apoptosis through the proteolytic cleavage of 1000s of proteins

18

initiator caspases

8

9

10

19

effector caspases

3

6

7

20

extrinsic activation pathway is also known as

death receptor mediated pathway

21

intrinsic activation pathway is also known as

mitochondrial mediated pathway

22

death receptors involved in extrinsic pathway

when are they expressed

family involved

subset of TNFR superfamily - TNFR1, Fas, TRAIL-R1, TRAIL-R2

some receptors are constantly present on cell surface, while other are expressed only upon damage

external cysteine rich domain - involved in ligand binding

transmembrane domain

internal death domain (DD) - needed for binding of adapter proteins like FADD

23

MOA of extrinsic pathway

death receptors e.g. TNFR, FasR are transmembrane receptors present on cell surface

binding by death ligands e.g. Fas causes death receptor to oligomerise

death receptors change shape and recruit adaptor molecules e.g. FADD or TRADD

several pro-caspase-8 molecules recruited and transactivate each other

active caspase-8 (initiator caspase) cleaves other caspases promoting irreversible cascade and cell death

DISC - Death Inducing Silencing Complex

24

intrinsic cell death pathway

when is it activated

key event

family involved

activated in response to a variety of internal stresses including DNA damage, ER stress, growth factor deprivation

release of mitochondrial intermembrane space proteins is the key event in intrinsic cell death

mitochondrial mediated release of intermembrane space proteins is controlled by the BCL-2 family

25

BCL-2 family

function

what do they have

central controllers of intrinsic cell death

BCL-2 family members have at least 1 of 4 conserved motifs

bcl-2 homology 1 - 4' BH1, BH2, BH3, BH4

important in regulating interaction between family members

26

3 broad classes of BCL2 family members

pro-apoptotic effector proteins - BAX and BAK

anti-apoptotic protein - BCL2, BCL-XL, MCL1

pro-apoptotic BH3-only proteins - BID, BAM, BIM, NOXA

balance between pro and anti apoptotic BCL-2 family members determines cell fate

27

pro-apoptotic effector proteins

BAX

BAK

28

anti-apoptotic proteins

BCL2

BCL-XL

MCL1

29

pro-apoptotic BH3 only proteins

BID

BAD

BIM

NOXA

30

overview of intrinsic pathway

31

apoptosis in cancer

tumour cells under great stress

DNA damage, lack of nutrients, lack of O2

cancer therapies normally induce apoptosis

32

imbalance of apoptosis

autoimmune diseases - Lupus, rheumatoid arthritis, type 1 diabetes

AIDS, CD4+ lymphocyte depletion

33

modified expresson of apoptotic pathway proteins - cancer

increased expression of anti-apoptotic BCL2 family members

elevated XIAP expression (intrinsic)

Bax, TSG mutated in some colon tumours

p53 is a TSG down regulated in many cancers

destruction of pro-apoptotic proteins

proteosome degrades proteins and can be over-active in certain cancers

34

selective BCL-2 inhibitors could treat

chronic lymphocytic leukemia

acute myeloid leukemia...

35

what induces apoptosis in cancer cells through the extrinsic pathway

cell-mediated immunotherapy

immune-checkpoint inhibition

36

cells of the immune system

37

most abundant leukocyte

what do they do

neutrophils are by far the most abundant leukocyte circulating - comprise > 50% of leukocytes

adept at phagocytosing and killing microbes

38

myeloid cells

neutrophil

eosinophil

basophil

immature DC

mast cell precursor

monocyte → macrophage

platelet

erythrocyte

39

macrophages and dendritic cells - function

detecting and instigating immune responses

presenting the components of phagocytosed microbes to cells of the lymphoid system

escalating immune responses through the secretion of multiple cytokines and chemokines

 

40

macrophage

41

dendritic cell

42

lymphoid cells

function

T and B lymphocytes

adaptive immune system

can generate highly specific cell surface receptors through genetic recombination of a relatively limited number of precursors for these receptors

T cell receptors = TCRs

B cell receptors = antibodies

both can be highly specific for particular molecular structures (ANTIGENS)

43

lymphoid cells

B-cell

T-cell

NK

(mature DC)

44

NK cells function

innate immune system

police presence of special antigen-presenting molecules (MHC I)

use germline-encoded receptors (NK receptors) that are distinct from the receptors of T and B cells and are endowed with the ability to kill cells that express abnormal MHC receptor profiles, as well as stress induced molecules

T cells recognise infected or tumour cells in an antigen dependent manner

NK cells recognise infected or tumour cells in an antigen independent manner (no need for antigen exposure or immunological memory)

45

distinguish between T cells and NK cells

T cells recognise infected cells in antigen dependent manner

NK cells recognise infected cells in antigen independent manner

46

how do the innate and adaptive immune systems work in tandem

infection occurs → innate serves as a rapid rxn force that deploys a range of relatively non-specific weapons to eradicate the infectious agent or at the very least to keep the infection contained

gives time for initially sluggish adaptive immune system to select and clonally expand lymphocytes with receptors (TCR and antibodies) that are capable of making a much more specific response that is uniquely tailored to the infectious agent - adds new weapons to the attack

47

role of macrophage

detect infectious agents - an array of pathogen recognition receptors (PRRs) borne on their plasma membranes e.g. Toll-like receptors as well as other cellular components such as endosomes

DON'T RECOGNISE ANTIGENS, instead:

Pathogen Associated Molecular Patterns (PAMPs)

Danger Associated Molecular Patterns (DAMPs)

examples include certain sugars not seen in humans e.g. lipopolysaccharide (LPS/endotoxin - cell wall of bacteria), nucleic acids

dying cells also release factors capable of activating PRRs

48

how does the macrophage work

  1. macrophage is put on a state of high alert (activated) and is now better at engulfing (phagocytosis) and killing any microorganisms it encounters
  2. macrophage begins to secrete cytokines and chemokines, which enhance vascular permeability and attract other immune cells to cause inflammatory response

49

macrophage activation

50

tissue macrophages initiating immune response to local bacterial skin infection

51

neutrophil extravasion into tissues

cytokines produced by macrophages induce expression of receptors on endothelial cells (selectins) that bind to ligands on neutrophils causing them to decelerate on the BV wall

chemical attractants (chemokines) attract into tissues - WBCs slow down when interacting with these - sense chemokines - chemicals released under tissue - activation of molecules on neutrophil - attaches to endothelial lining and then migrates out - into deeper tissues where inflammatory stimulus is coming from

migration is facilitated by strong adhesion molecules on the neutrophils - integrins

52

what do NK have the ability to do

role of MHC

inspect host cells for signs of abnormal patterns of protein expression that may indicate that such cells might be harboring a virus - immunosurveillance

capable of killing cells that have suffered mutations and are on the way to malignant transformation into tumours

health cells express MHC molecules - NK cells have inhibitory receptors which recognise MHC - if cells lose MHC, they are vulnerable to killing by NK cells (missing self)

predominant mode of killing = release of cytotoxic granules containing lytic proteases Perforin and Granzyme

NK cells also release cytokines to activate immune response

genetic instability (as a result of cancer) and/or viral infection can lead to expression of protein ligands that are recognised by activating receptors on NK cells

53

apart from MHCs, what else do NK cells recognise

antibody coated cells via their Fc receptors, targeting them for destruction (antigen dependent cytotoxicity)

B cells are the source of antibodies

once activated, NK cells can also kill target cells via expression of death inducing ligands, which induce apoptosis (FAS or TRAIL)

54

how do NK cells co-operate with macrophages

cytokines produced by macrophages can activate NK cells

NK cells can recognise PAMPs and DAMPs e.g. nucleic acids from dying cells

cytokines released by activated NK cells important for maturation of dendritic cells and in turn enhance macrophage function

55

how do NK cells form a bridge between innate and adaptive immunity

when NK cells kill cancer cells, the antigens released are taken up by antigen presenting cells - dendritic cells

cytokines produced by NK cells stimulate maturation of dendritic cells

mature dendritic cells present tumour antigens in association with their MHC molecules to helper (CD4) and cytotoxic (CD8) T cells

CD8 cytotoxic cells can then recognise and kill antigen expressing tumour cells

CD4 T cells help B cells produce antibodies against these antigens

56

cells that present antigens

dendritic cells

57

where are DCs produced

how did they get their name

DCs are produced primarily in the bone marrow and derive their name from the multiple long membrane projections or dendrites that these cells possess

they share a common progenitor with macrophages

both macrophages and DCs have somewhat overlapping functions

58

cells responsible for adaptive/acquired immune response

mediated by lymphocytes

→ T lymphocytes (T cells)

→ B lymphocytes (B cells)

59

what characteristic do T and B cells possess

defining characteristics of the acquired immune response

both highly antigen specific

both exhibit immunological memory whereby they respond more vigorously upon re-encounter with specific antigen

 

60

where do T cells develop

from bone marrow precursors in the thymus

61

3 major functions carried out by T lymphocytes

  1. providing assistance to other cells in the immune response - helper T cells
  2. Limiting excessive or undesired immune responses – regulatory T cells
  3. killing cells infecting with pathogens - cytotoxic T cells

62

function of B cells

develop fully within bone marrow

produce antibodies - humoral immunity

63

response that recognises antigens

specific acquired immune response

64

shape of antigen

3-D shape that is complementary to antibody molecules that act as the antigen receptor on B lymphocytes

antigen-specific antibody molecules are subsequently released in a soluble (secreted) version by plasma cells derived from B cells following their activation

antigens can be proteins, CHOs, lipids, nucleic acids, small molecules

 

65

apart from B cells, what other cells recognise antigens

T cells

usually in form of proteins that are digested from the original polypeptide into short peptides

peptides are then shown to the antigen receptor on the surface of T cells using a MHC which is specialised to show peptides to the T cell receptor (TCR)

T cell therefore recognises a shape that is a combination of antigen-derived peptide and MHC

66

structure of antibody

variable region - devoted to binding to the individual antigen (antigen recognition function)

constant region - concerned with linking to the complement, phagocytes (e.g. macrophages) and NK cells (effector function)

body must make millions of antibody molecules with different antigen-recognition sites but that all share the property of recruiting other elements of the immune response

67

Antibodies and Effector Cells (7 mins)

ADCC = Antigen Dependent Cellular Cytotoxicity

68

B cell activation and antibody production

when the antigen binds to a complementary B cell receptor - a complex containing an antibody on the surface of the B cells → activating signal leading to increased proliferation and survival of B cell

69

normal antibody production

where does it take place

MOA

takes place in germline centre of lymph node where Follicular Dendritic Cells present antigen to the B cells

only those cells that receive the strongest survival signal due to the best antigen-antibody fit are selected and survive - others undergo apoptosis

further help from helper T cells enable the selected B cells to mature into either anitbody producing plasma cells or memory B cells, which are primed waiting to mount a rapid antibody response in case the antigen is encountered in the future

70

what is the MHC

how are MHCs recognised

structure

genetic locus that control histocompatability

in early transplantation experiments, the donor and recipient were found to need the same MHC locus in order to avoid graft rejection

MHC molecules are recognised by T cells via TCRs

MHC = polygenic and polymorphic

3 regions = class I (contains genes encoding 6 MHC proteins) and class II (variable number of genes that ultimately encode 3 heterodimeric MHC proteins)

MHC genes are highly polymorphic with 1000s of different alleles within population

MHC antigens in humans = HLA antigens (human leukocyte antigens) as they were first identified on WBCs

71

MHC antigens = 

HLA antigens

72

how does the TCR work

T cells express TCRs - recognise antigens being presented by MHC molecules

CD8 + T cells recognised internally derived antigen being presented on MHC class I molecules allowing CD8 + T cells to scan cells for internal threats

CD4 + T cells recognise endocytosed antigen presented on MHC class II molecules of professional anitgen-presenting cells, thereby allowing recognition of EC antigens

73

structure of TCR

highly variable

heterodimer consisting of either α and β OR γ and δ chains

these chains undergo random rearrangement at the genetic level to generate a wide variety of TCRs

74

what do most T cells express

αβ TCRs with which they recognise their antigen being presented by MHC

75

how does the TCR signal

forms a complex with CD3 molecules

TCR itself has a short cytoplasmic tail with no signalling capability

it forms a complex with CD3γ, δ, ε and ζ chains

the cytoplasmic tail of CD3ζ contains an immunoreceptor tyrosine-based activation motif (ITAM), which is phosphorylated, allowing it to signal

76

what does the cytoplasmic tail of CD3ζ contain

immunoreceptor tyrosine-based activation motif (ITAM), which is phosphorylated, allowing it to signal

77

what is the TCR similar to

how do they differ

similar to Fab fragments of B-cell receptors/antibodies

both composed of 2 different peptide chains and have variable regions for binding antigen, constant regions and hinge regions

principal differences - TCRs remain membrane bound and contain only a single antigen-binding site

78

T cell receptor activation

TCR is assisted by CD4 or CD8 receptors

recognises peptide antigen in context of MHC molecules

TCR activation signals are propogated via the CD3 co-receptor complex, which is made up of CD3 γ, δ, ε and ζ chains

co-clustering of CD4 or CD8 with the TCR complex facilitates signal propogation through phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) with the CD3 ζ chain

79

T cell adhesion and activation

 

initial stable interaction between APCs or target cells and T cells following antigen recognition is facilitated by adhesion molecules, which causes cells to stick more tightly together

in addition to antigen recognition via TCR along with CD3 signalling, T cells require a 2nd activating signal (signal 2) to fully activate and avoid anergy (unresponsive) or apoptosis

co-stimulatory molecules on APC's bind to CD28 and this provides this 2nd signal

by upregulating an anti-apoptotic protein Bcl-xL, CD28 stimulation blocks TCR-mediated signals that would otherwise result in apoptosis (activation-induced cell death)

 

80

what do cytokines do to signal

provide signal 3 to promote clonal expansion and proliferation

further stimulation and activation (signal 3) can be provided by cytokines e.g. IL 2 (released by dendritic cells or macrophages)

81

T cell adhesion and activation

initial stable interaction between APCs or target cells and T lymphocytes following antigen recognition is facilitated by adhesion molecules, which cause cells to stick more tightly together

82

killing by cytotoxic lymphocytes and NK cells

NB NK cells - innate killers

Antigen independent

Recognise virally infected cell or tumour cell because it has

1. Lost MHC molecule

2. Upregulated stress molecules - danger associated molecular patterns - ABNORMAL

Adaptive response - B cells produce antibodies - NK cells may further contribute to ongoing immune response through ADCC

Virally Infected or cancer cells - sometimes downregulate MHC class I so T cells no longer recognise them but this then makes them vulnerable to killing by NK cells

83

controlling T cell activation

uncontrolled T cell activation could be very harmful and lead to autoimmune disease

dampening down of T cell responses occurs via a number of mechanisms, some of which operate at the level of the actvated T cell itself, while others operate via additional T cell subsets - regulatory T cells

molecules present on activated T cells that serve as "off switches" for such T cells represent important immunological checkpoints, helping to keep T cell responses within certain limits

84

immune checkpoint receptors and their ligands

immune checkpoint receptors - PD-1 and CTLA4

ligands - PD-L1 and CD80/86

T cells expressing these molecules are suppressed - molecules are ligated by PD L1

Propogation of inhibitory signals in T cell that turns off T cell cycle - no further cytotoxic T cell activation

Important in normal homeostasis

 in cancer this is exploited by tumour - T cell exhaustion

85

immune checkpoints and activation induced cell death

signalling via TCR and CD28 leads to T cell activation

inhibitory receptors or "immune checkpoints" e.g. CTLA-4 and PD-1 are upreg. on activated T cells, usually after prolonged activation in the presence of their ligands on APC's or tumour cells can induce T cell inhibition

activated T cells upregulate Fas receptor

in absence of co-stimulation by CD28 Fas ligand expressing cells can induce apoptosis (activation induced cell death)

86

regulatory T cells

how do they work

Tregs are able to suppress T cell activation by expressing ligands for immune checkpoints, releasing suppressive cytokines or eliminating effector CD8/CD4 T cells

Tregs are important in preventing autoreactivity (autoimmune disease)

in cancer, Tregs are often over-active, leading to an impaired immune response

87

what is the immune system designed for

how does cancer pose a challenge

immune system has evolved to deal with infection (non-self antigens) rather than cancer (altered self)

recognising and mounting a robust immune response against mutated cancer cells that may only have subtle differences from normal cells is challenging

mutational processes associated with the development of cancer frequently generates neoantigens that, in principle, can elicit T cell responses, in practice such responses are highly muted because of mechanisms that serve to prevent the emergence of autoimmunity

88

how does cancer manipulate the immune system

well-meaning regulatory T cell responses and other mechanisms that serve to limit the development of autoimmunity (such as CTLA-4 and PD-1 mediated downregulation of T cell responses) conspire to suppress the immune response against cancer

tumours also actively manipulate the immune system to minimise immune responses that do emerge

tumours recruit macrophages, neutrophils and other innate immune cells and "re-educate" such cells towards a wound-healing phenotype for the purposes of supporting tumour growth and survival

"cancer = wound that does not heal"

 

89

cancer progression over time

90

immune editing

gradual stepwise development of tumours over long periods of time permits the selection of cells that are effectively invisible to the immune system - must avoid killing by immune system

if they are not, such cells are weeded out by the immune system as the tumour develops

this process selects for the "fittest" tumours that are very difficult for the immune system to deal with

91

how can the immune system become tolerant of tumour antigens

in the absence of sufficient activation

Cytotoxic T cells can't deal with cancer

Antigens taken up by dendritic cells

Co-stim. signals

Proper functioning T cells

If dendritic cells take up antigen - various cytokines

T cells - recognised antigen but cannot mount response

92

ways in which cancer cells can evade or suppress the immune system

loss of MHC expression by cancer cells means T cells unable to recognise tumour antigen

however this can make them vulnerable to NK cells

expression of certain ligands can either suppress (e.g. PD-L1) or kill (Fas Ligand) immune cells

93

T cell checkpoint inhibition

how does it work

what are the molecules involved

T cell checkpoint molecules (CTLA-4, PD-1) are frequently engaged by tumours to suppress anti-tumour T cell responses

cancer cells frequently engage immune checkpoint molecules on T cells, such as PD-1 and CTLA-4, which has the effect of anergizing T cells in the tumour environment

blocking antibodies against CTLA-4, PD-1 and PD-L1 can reactivate tumour-specific T cells and can now play a role in treatment of solid cancers

94

7 approaches to cancer immunotherapy

  1. passive immunotherapy with monoclonal antibodies
  2. unmasking of latent T cell responses by targeting immune checkpoint molecules
  3. antigen-independent cytokine therapy
  4. vaccination approaches to stimulate immune responses against tumour antigens or the tumour vasculature
  5. adoptive transfer of ex vivo expanded T, NK or DCs
  6. adoptive transfer of ex vivo generated chimeric antigen receptor (CAR) T cells
  7. bispecific T cell engagers

 

95

approved monoclonal antibodies

96

approved monoclonal antibodies

97

MOA - monoclonal antibodies

Recognition by NK via Fc receptor

NK recognises Fc - release cytotoxic granules - perforin forms pores in tumour cells to allow other contents in - lysis

98

CD20 monoclonal antibodies

CD20 = antigen present on mature B cells

when CD20 antibodies bind to CD20 antigens on surface of malignant B cells it can induce cell death by:

fixing complement leading to cell lysis

direct apoptotic signals

ADCC (NK cells)

phagocytosis (macrophages bind antibody via Fc receptors)

99

therapeutic use - how to reactivate cytotoxic CD8 lymphocytes

CTLA-4, PD-1 and PD-L1 blocking antibodies

100

retuxemab

CD20 antibody

non-hodgkins lymphoma

chronic lymphocytic leukemia

101

CAR T cell therapy

acute lymphoblastic leukemia

no MHC needed

102

chimeric antigen receptors

103

chimeric antigen receptors - MOA

In normal t cell receptor CD28 would be needed for co stim. But all built in car t cell molecule - recognises tumour cell antigen even in absence of MHC - sufficient to activate killing by T cell

104

CAR-T procedure

HIV - retroviruses - good at stable genetic modification

Leukapheresis - T cells extracted and we put in retrovirus that can genetically express

Need cytokines to proliferate and expand

After chemotherapy (done to get rid of lymphocytes)

105

challenges of CARs

106

what ensures survival of MM cells

BCMA

represents a good target for immunotherapy

  • Can recognise Tumour antigen (BCMA)
  • Other arm - recognises CD3
  • Brings t cell to close proximity to target cell and leads to recognition of tumour cell by t cell - recognition and release

107

options other than monoclonal antibodies for targeting Myeloma antigens

CAR T cells

bispecific T cell engagers

bispecific antibodies

antibody-drug conjugates