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T-Cells NK
Feb 13, 2006
T-cells
• Antigens that are transported by dendritic cells
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to lymph nodes are recognized by naive T
lymphocytes that recirculate through these lymph
nodes.
The T cells are activated to differentiate into
effector and memory cells, which may remain in
the lymphoid organs or migrate to nonlymphoid
tissues.
At sites of infection, the effector cells are again
activated by antigens and perform their various
functions, such as macrophage activation.
Step 1
• The dendritic cell encounters the
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antigen.
The antigen interacts with Toll Like
Receptors (TLR) on the dendritic cell.
Depending on which set of the TLR
receptors that are activated,
determines the type of immune
response.
Dendritic TLR
Dendritic TLR
There are 11 TLRs, these are best characterized
Dendritic TLR
TH2 responses
mixed
TH1/TH2
Strong
TH1
Distribution of TLR on innate and adaptive cells
Fig. on p. 284
Fig. on p. 284
Therapeutics: TLR9
• Activation of TLR9 has progressed
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significantly in the development of
therapeutics to treat: asthma, cancer,
and sepsis.
CpG ODN(unmethylated cytosinephosphorothioate-guanine
oligodeoxynucleotide) sequences are
being used to stimulate TH1 responses.
Therapeutics: TLR9
• CpG ODN mimic bacterial DNA.
• Engagement of the CpG ODN with the
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intracellular TLR9 induces IL-12, IL-18, and
IFN-g.
CpG-A ODN 2216
5’-G*G*G_G_G_A_C_G_A_T_C_G_T_C_G*G*G*G*G*G-3’
( * ) are phosphothiorate linkages, and
( _ ) are phosphodiester linkages:
TLR2
• Pam3Cys-OH a lipoprotein that
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stimulates the TLR2 leading to a
strong TH2 response with secretion
of IL-4.
This is a synthetic lipoamino acid Npalmitoyl-S-[2,3-bis(palmitoyloxy)(2RS)-propyl]-(R)-cysteine (Pam3Cys),
which is derived from the N-terminus
of bacterial lipoprotein.
Next Step
• Now that the dendritic cells have
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secreted their cytokines to select
the T-helper subset, the antigen is
presented to the T-helper
lymphocyte.
This step determines the fate of the
immune response: humoral or cellular.
T-Cells
In the T-Helper Lymphocyte
• Lck: A Src family non-receptor tyrosine
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kinase that non-covalently associates
with the cytoplasmic tails of CD4 and
CD8 molecules of the T-cells and is
involved in the early signaling events of
antigen-induced T cell activation.
Lck mediates tyrosine phosphorylation
of the cytoplasmic tail of the CD3 and z
(zeta) proteins of the TCR complex.
In the T-Helper Lymphocyte
• ZAP-70 (Zeta-associated protein of 70
kD): binds to phosphorylated tyrosines
in the cytoplasmic tail of the CD3 and z
(zeta) proteins and phosphorylates
adapter proteins.
In the T-Helper Lymphocyte
• Adapter Proteins are involved in serving
as scaffolds for the recruitment of
other signaling molecules.
• During lymphocyte activation, adapter
proteins may be phosphorylated on
tyrosine residues to enable them to bind
other proteins containing Scr homology
2 (SH2) domains.
In the T-Helper Lymphocyte
• Phospholipase Cg (PLCg): an enzyme
that catalyzes hydrolysis of plasma
membrane phospholipid PIP2 to
generate IP3 and DAG.
• This leads to increased intra-cellular
calcium and activation of PKC.
Consequences of Increases in
[Ca2+]i and Activation of PKC
• The increase in [Ca2+]i influences
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calmodulin-dependent events, including
the activation of calcineurin (PP2B) and
Ca2+/calmodulin-dependent kinase
(CAM-kinase).
A critical role for the Ca2+/calmodulindependent serine–threonine
phosphatase calcineurin is now well
established.
Ca2+/calmodulin-dependent serine–
threonine phosphatase calcineurin
• Calcineurin is the molecular target for
the immunosuppressives CsA and FK506,
drugs that have revolutionized clinical
organ transplantation.
• CsA and FK506 form molecular
complexes with their cellular receptors,
cyclophilin and FKBP, respectively.
Ca2+/calmodulin-dependent serine–
threonine phosphatase calcineurin
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It is these molecular complexes, not the
isolated drugs, that inhibit the phosphatase
function of calcineurin.
Calcineurin is expressed ubiquitously but is
expressed at only low levels in T-lymphocytes.
This probably accounts for the relative
specificity of the immunosuppressive drugs in
targeting T-cell function.
CSA & FK506
• CSA binds to cyclophilin & FK506
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binds to FK binding protein (FKBP).
This complex inhibits the cis-trans
isomerase activity of cyclophilin and
that is required for calcineurin
activation of nuclear factor of
activated T-cells (NFAT)
Cyclophilin
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Cyclophilin is a
prokaryotic
peptidyl-prolyl cistrans-isomerase
(also called
PPLases), or a
rotamase.
Cyclophilin
• The activity of cyclophilin and FKBP
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are to serve as cis-trans isomerases.
These isomerases act on calcineurin.
Calcineurin thereby dephosphorylates
NFAT.
Tacrolimus and cyclosporine inhibit
cyclophilin’s isomerase activity.
CSA & FK506
• NFAT is activated by calcium/calmodulindependent, calcineurin-mediated
dephophorylation that permits NFAT to
translocate into the nucleus and bind to
consensus binding sequences in the
regulatory regions of IL-2, IL-4, and
others in association with AP-1.
TCR
CSA
FK506
Third Step
• IL-2 receptor (CD25) activation
• Modulation of IL-2r.
Kinetics of gene expression in antigen-stimulated
T lymphocytes.
The low-affinity IL-2
receptor (CD25)
• Expressed in low levels by about 30%
of circulating (resting) lymphocytes
• CD25 has been proposed to be
associated with T-lymphocyte
memory
The low-affinity IL-2 receptor (CD25)
 a chain: TAC, CD 25 (kd 1.4x10-8 M)
 b chain: CD 122 (kd 1.2 x 10-7 M)
 g chain: functional component of:
IL-4r, IL-7r, and IL-9r.
 abg chain: (kd 1.3 x 10-11 M)
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The low-affinity IL-2
receptor (CD25)
The low-affinity
IL-2 receptor
(CD25)
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IL-2 r
• The binding of IL-2 to the high- or
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intermediate-affinity forms of the
receptor initiate transmembrane signals
in order to induce the events that
promote the progression of T cells
through the cell cycle.
Such signal transduction events also
account for other effects of IL-2, such
as the up-regulation of transcription of
the IL-2R a chain.
IL-2 r
• The IL-2 R a chain has a relatively short
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cytoplasmic domain, and it appears that
its main function is to increase the
sensitivity of the receptor by increasing
its binding affinity for IL-2.
It is the b and g chains that are
responsible for the signal transduction
function of the receptor.
Rapamycin
• Rapamycin is an immunosuppressive
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agent that has been used to probe
the IL-2/IL-2R pathway.
It binds to the same cellular receptor
as FK506, FKBP, and it is the drug–
receptor complex that mediates
inhibition of T-cell function.
However, unlike FK506, rapamycin
does not inhibit the induction of IL-2
gene, nor is its target calcineurin.
Rapamycin
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Instead, rapamycin inhibits IL-2–
driven T-cell proliferative responses
by blocking the function of another
enzyme, called mTOR (for mammalian
target of rapamycin; also called FRAP,
for FKBP12-rapamycin–associated
protein).
mTOR is a member of a larger family
of proteins with PI-kinase domains.
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IL-2
Although IL-2–driven T-cell proliferation
has been widely considered to be the major
mechanism responsible for T-cell growth,
under some circumstances, T-cell
proliferation can occur independently of
IL-2.
For instance, murine T-cell cytolytic clones
can proliferate in response to anti-TCR
mAb in the absence of detectable IL-2, as
can resting human T cells.
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IL-2
IL-4 and IL-15 are the most likely to
function as T-cell growth factors in the
absence of IL-2.
Hence, if IL-4 production predominates in
a particular T-cell response, as it does in
response to parasites and allergens, one
may observe T-cell proliferation, but this
proliferation may be restricted only to
certain subsets of T cells (i.e., Th2 T-cell
clones).
IL-2
• It is likely that more sustained T cell
proliferative responses and
recruitment of T cells will occur in
instances in which T-cell growth
factors such as IL-2, IL-4, or IL-15
are produced.
Formation of the
immunological
synapse.
Artificial means
• Mitogens
Mitogens
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A number of different reagents have been
used to substitute for the stimulating
antigen–MHC molecule.
Many of these stimuli represent reagents
that can polyclonally activate T cells,
thereby eliminating the difficulties
encountered in studying small numbers of
antigen-specific responding cells within
complex polyclonal T-cell populations.
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Mitogens
Among these reagents are several lectins,
plant-derived proteins that bind various
carbohydrate groups.
These lectins, phytohemagglutinin (PHA),
concanavalin A (Con A), and pokeweed mitogen
(PWM), were among the first recognized
polyclonal activators of T cells.
Because they can induce the proliferative
responses, they are among a class of
reagents termed mitogens.
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Mitogens
Con A and PHA are selective T-cell mitogens
when compared with their effects on B cells,
whereas PWM is a T- and B-cell mitogen.
Their mitogenic effects for T cells are felt to
depend on their ability to bind and cross-link
relevant receptors involved in physiologic T-cell
activation.
Studies with PHA and Con A suggest that these
lectins can bind to component chains of the TCR
and that their ability to activate T cells is
dependent on the expression and function of
the TCR.
KNOW
Alternate mitogens
• In our lab, we use anti-CD-3 and
anti-CD-28 antibody coated plates.
• This is a means to stimulate
lymphocytes in a selective manner.
Natural Killer Cells
Natural Killer Cells
• This small lymphocyte population has
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remained elusive in many respects.
Morphologically, most NK cells fit into the
population of large granular lymphocytes.
Functionally, they are able to kill virusinfected or malignant cells with low or
absent MHC molecules.
NK cells are neither T nor B lymphocytes:
TcR and immunoglobulin genes are in the
unrearranged genomic configuration.
Natural Killer Cells
• A major difference is that NK cells can
recognize virus-infected cells and many
different types of malignant cells without
clonal restriction.
• In other words, their recognition
mechanisms are relatively nonspecific and
common to all NK cells.
Natural Killer Cells
• At this time, it is believed that two
broadly reactive receptors are involved,
one that delivers activating signals, and
the other that delivers inhibitory signals.
• The triggering or activating receptor
(NKAR, NKR-Pl)
Natural Killer Cells
Natural
Killer
Cells
Natural Killer Cells
K Cell (ADCC)
Lymphocyte Apoptosis
Conclusion
• We have discussed the activation and
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deactivation of T-lymphocytes
We have introduced the mechanism
of action of CSA, FK506, and
Rapamycin.
We have introduced the concepts of
effort functions of cytotoxic cells.