L10 Cytotoxicity Flashcards
what are cytotoxic lymphocytes?
NK cells and CD8+ T cells
NK: recognise and kill through the KIR receptor and and have antibody dependent cellular cytotoxicity (ADCC) through fc gamma receptors/ (FcgammaRIII aka CD16)
CD8 USE TCR/ CD3
CD8+?
CD8+ T cells kill virus-infected and cancer cells with remarkable specificity.
Originate from naive CD8+ T cells in a process named - effector T cell differentiation
Naive CD8+ T cells circulate and upon encountering antigen by a naive t cell presented by professional APCs, these naive T cells slow down—engages the dendritic cell via integrin signaling— and T cell receptor (TCR) activation triggers the formation of the initial signaling “immune synapse” always use the word initial.
This initial ‘signaling’ immune synapse induces remarkable clonal expansion (this effector differentiation occurs over the next 4–5 days)
-> naive CD8+ T cells differentiate into heavily ‘armed’ effector CD8+ T cells or also
known as CTLs that are loaded with specialized cytolytic granules that contain
perforin and granzymes
Once effector CD8+ CTLs recognise their target cells in the periphery, a
“lytic immune synapse” is formed, and the cytolytic granules are secreted.
what do naive CD8+ differentiate into upon effector t cell differentiation?
when the initial immune synapse formation occurs in the lymph node, naive cd8 t cell receive helps from cd4 cells by co stimulation, helping to activate the dendritic cell. so there is a complex of all 3 cells. through release of cytokines and? through expression of CD40L (signalling molecule), cd8 b ecomes differentiated CTLs and memory CD8 cells.
the Naive CD8+ T cells have to differentiate into CTLs
The differentiation
of naive CD8 + T
cells into
functional CTLs
and memory cells
requires not only
antigen
recognition but
also costimulation
and, in some
situations, help
from CD4+ T cells
(co-stimulatory
receptor
signalling and
cytokines)
lytic immune synapse?
after t cell proliferation and differentiated CD8 CTL cells formed in lymph node, they enter circulation and migrate to site of antigen where they form a lytic immune synapse.
Different from an initial immune synapse. to form the lytic synapse only need signal 1.
* TCR-
dependent
(signal 1)
BUT…
* costimulation
(signal 2) or
cytokines
(signal 3) NOT
REQUIRED
- cytotoxic lymphocytes that have been generated are very mobile, leave lymph nodes and survey the body to identify the target cell. The leading edge of a CTL is characterized by actin-rich
lamellipodia and the centrosome (microtubule-organizing center,
MTOC) is localized at the back of the CTL in the uropod. this allows them to rapidly migrate.
Stage 1. Initial interaction of CD8+ CTLs with target cells
* Initial interaction of CTL with a target cell is mediated by non-specific adhesion molecules – integrins: LFA-1
* In the absence of a specific TCR-recognised antigen the cells will
separate
LFA-1 (CD11a/CD18 ICAM-1 (CD54)
LFA-2 (CD2) LFA-3 (CD58)
Stage 2. Early events in CD8+ CTL lytic synapse formation
* Initial contact between the CTL and the target cell occurs through
actin-rich filopodia of the CTL that form an interdigitated contact sites (finger-like contacts) with the target cell.
* TCR–peptide–MHC interactions at the tips of the actin-rich interdigitations.
Step 3. Mature CD8+ CTL lytic immune synapse formation
* In the presence of a specific TCR-recognised antigen bound to class I
MHC cells and CD8 coreceptor engagement, CD8+ CTLs adhere strongly
and the formation of the lytic immune synapse is initiated, leading to
clustering of cytolytic granules towards the target cell.
T cell becomes polarised
- Centrosome/MTOC formation
- clustering of granules at point
of contact. centrosome contains the cytosolic molecules needed to kill the target cell.
Upon strong TCR engagement, the actin-rich lamellipodia move to the
periphery/edges of the lytic synapse leaving an actin-depleted area
(at 6 mins) the centrosome moves and docks at the plasma membrane, and cytotoxic
granules move along microtubules towards the lytic synapse.
➢ The docked centrosome marks the point at which lytic granules that contain cytotoxic
perforin and granzymes are secreted and consequently kill the infected or tumour target
cell by apoptosis
initial synapse signals?
T cell activation requires 2 signals:
Signal 1 – T cell receptor (TCR) signaling. MHC class 1 molecules expressed on APCs bind
to the TCR on T cells. TCR signalling occurs when the TCR is engaged by cognate peptide (Ag)-major histocompatibility complex (MHC).
and Signal 2: co-stimulatory signalling (e.g. CD28) amplifies TCR signalling and
the recruitment of signaling molecules at the initial immune synapse to drive T
cell activation. CD80 and CD86 ligands expressed
on APCs bind to CD28 on T cells CD8
what are the cytolytic? molecules within the centrosome?
perforin- aids in delivering content of granules into the cytoplasm of target cells.
granzymes- serine proteases, which activate apoptosis once in the cytoplasm of target cells.
granulysin- has antimicrobial actions and can induce apoptosis.
Mechanisms of action of perforin and granzymes
Perforin forms large
transmembrane pores that
enable the diffusion of
granzymes into the target
cell cytosol.
Granzymes then initiate
apoptosis of the target cell,
and the cytotoxic lymphocyte
detaches from the dying cell
(indicated by the arrow;
part e) and can interact with
another target cell to carry
out serial killing (not shown).
CTL target cell recognition?
CTL target cell recognition is highly specific (TCR-Ag) –
controlled by the lytic immune synapse that prevents
nearby healthy cells from being damaged by cytotoxic proteins.
The CTL may detach and kill other
target cells – serial killers!
CTL recognizes the antigen-
expressing target cell and is
activated.
Activation results in the release
of granule contents from the
CTL into the target cell through
the area of contact (the lytic
immune synapse).
Granule contents deliver a
lethal hit to the target.
The CTL may detach and kill
other target cells.
additional killing mechanisms?
CTLs also use a granule-independent mechanism of killing that is
mediated by interactions of membrane molecules on the CTLs and target
cells. On activation, CTLs express a membrane protein called Fas ligand (FasL) that binds to the death receptor Fas. This interaction also results in activation of caspases and apoptosis
of Fas-expressing target cells.
Indirect killing mechanisms
Adaptive immune cells like CD8+ CTLs can regulate other
immune cells - innate immune cells such as
macrophages and NK BY SECRETING IFN-Y
NK?
regulated by KIR. do not undergo the differentiation process, immediately kill.
Normally they are tolerant but are readily activated by:
* Type I Interferons (IFN-alpha/beta) cytokines
* IL-12, IL-15 cytokines
Killing triggered by:
* Activating receptor on NK cells: induced to kill by FcalphaRIII (CD16) —> Antibody-Dependent Cell-mediated Cytotoxicity (ADCC).
detect abnormal cells with:
* Lack of class I MHC (“missing self ”)
* when cells are damaged/infected express abnormal proteins: Altered self (“stress-induced self”)
NK functions
* kill virus-infected target cells and tumour cells
* produce and release immunoregulatory cytokines (IFN-gamma, TNF-alpha,
GM-CSF and chemokines) that activate macrophages to destroy
phagocytosed microbes (innate NK cells can influence other innate
immune cells – macrophages)
How NK cells regulate other immune cells
NK cell–derived IFN-γ
increases the capacity of
macrophages to kill
phagocytosed bacteria, similar
to IFN-γ produced by T cells.
This allows time for T cell–
mediated immunity to develop
and eradicate the infection. so takes time as immediate response by nk and macrophages but adaptive response which is antigen specific takes several days to develop.
NK cells – how are they regulated
a | NK cells are tolerant to
healthy cells, as the strength of
the activating signals they receive
on encountering these cells is
dampened by the engagement of
inhibitory receptors (tolerance). nk cells express inhibitory receptors designed to keep nk cell toleraised. don’t become activated to mhc class 1 (healthy cells).
b | Tumour cells may lose
expression of MHC class I
molecules. NK cells become
activated in response to these
cells, as they are no longer held
in check by the inhibitory signal
delivered by MHC class I
molecule engagement. This is
known as ‘missing-self’ triggering
of NK cell activation. inhibitory recptor no longer engaged and activating receptor dominates.
c | In addition, NK cells are
selectively activated by
‘stressed’ cells’, which
upregulate activating ligands for
NK cells and thereby overcome
the inhibitory signaling delivered
by MHC class I molecules. This
is known as ‘stress-induced
self’ triggering of NK cell
activation.
NK cell activation leads to
tumour elimination directly
(through NK cell-mediated
cytotoxicity) or indirectly (through
the production of pro-
inflammatory cytokines, such as
interferon-γ).
more info on nk?
NK cells are innate lymphocytes that elicit effector functions following the
ligation of germline-encoded receptors
In humans, NK cells are able to deliver a response immediately
after recognising specific signals, including stress signals, ‘danger
signals’ or signals from molecules of foreign origin (in contrast to naïve
CD8+ T cells)
NK cells therefore participate in the defense against infections, the
regulation of immune responses and the surveillance of stressed or
cancerous cells
NK cells use specific receptor systems
(effector cell differentiation is not required unlike naïve CD8+ T
cells->CTLs) – this is a key distinguishing feature between NK cells and
CD8+ T cells->CTLs
Activating and Inhibitory Receptors of NK cells
NK cells distinguish infected and stressed cells from healthy cells by
balancing the signals from activating receptors and inhibitory
receptors
* These receptors recognize molecules on the surface of other cells and
generate activating or inhibitory signals that promote or inhibit NK
responses
* The activating receptors stimulate protein kinases, while inhibitory
receptors stimulate phosphatases that counteract the kinases, inhibit signalling. suppress activation of nk
* In general, the activating receptors recognize ligands on infected and
injured cells, and the inhibitory receptors recognize ligands on
healthy normal cells
* Engagement of activating receptors stimulates the formation of the NK
cell lytic immune synapse in common with cd8 and killing activity of the NK cells, resulting in
destruction of stressed or infected cells
Activating Receptors on NK cells?
Activating Receptors on NK cells recognize a
diverse group of ligands.
* Ligands are expressed on cells that have undergone stress, infected
with microbes, or neoplastic
* Many of the NK cell–activating receptors are called killer cell
immunoglobulin (Ig)-like receptors (KIRs) (can be activating or inhibiting so regulate nk)
* A second group of activating NK receptors belongs to the family of C-
type lectins, which are proteins with carbohydrate-binding properties
* One well-studied NK cell–activating receptor in the C-type lectin family is
NKG2D, which binds class I MHC–like proteins, including MIC-A and
MIC-B, found on virally infected cells and tumour cells but not normal cells.
