TCells NK

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T-Cells NK
Feb 13, 2006
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T-cells

• Antigens that are transported by dendritic cells
  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.

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Step 1

• The dendritic cell encounters the 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.

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Dendritic TLR
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Dendritic TLR
There are 11 TLRs, these are best characterized
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Dendritic TLR
TH2 responses mixed Strong TH1/TH2
TH1
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Distribution of TLR on innate and adaptive cells
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Fig. on p. 284
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Fig. on p. 284
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Therapeutics TLR9

• Activation of TLR9 has progressed significantly
  in the development of therapeutics to treat
  asthma, cancer, and sepsis.
• CpG ODN(unmethylated cytosine-phosphorothioate-gua
  nine oligodeoxynucleotide) sequences are being
  used to stimulate TH1 responses.

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Therapeutics TLR9

• CpG ODN mimic bacterial DNA.
• Engagement of the CpG ODN with the intracellular
  TLR9 induces IL-12, IL-18, and IFN-g.
• CpG-A ODN 2216
• 5-GGG_G_G_A_C_G_A_T_C_G_T_C_GGGGGG-3
• ( ) are phosphothiorate linkages, and
• ( _ ) are phosphodiester linkages

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TLR2

• Pam3Cys-OH a lipoprotein that stimulates the TLR2
  leading to a strong TH2 response with secretion
  of IL-4.
• This is a synthetic lipoamino acid
  N-palmitoyl-S-2,3-bis(palmitoyloxy)-(2RS)-propyl
  -(R)-cysteine (Pam3Cys), which is derived from
  the N-terminus of bacterial lipoprotein.

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Next Step

• Now that the dendritic cells have 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.

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T-Cells
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In the T-Helper Lymphocyte

• Lck A Src family non-receptor tyrosine 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.

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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.

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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.

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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.

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Consequences of Increases in Ca2i and
Activation of PKC

• The increase in Ca2i influences
  calmodulin-dependent events, including the
  activation of calcineurin (PP2B) and
  Ca2/calmodulin-dependent kinase (CAM-kinase).
• A critical role for the Ca2/calmodulin-dependent
  serinethreonine phosphatase calcineurin is now
  well established.

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Ca2/calmodulin-dependent serinethreonine
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.

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Ca2/calmodulin-dependent serinethreonine
phosphatase calcineurin

• 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.

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CSA FK506

• CSA binds to cyclophilin FK506 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)

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Cyclophilin

• Cyclophilin is a prokaryotic peptidyl-prolyl
  cis-trans-isomerase (also called PPLases), or a
  rotamase.

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Cyclophilin

• The activity of cyclophilin and FKBP are to serve
  as cis-trans isomerases.
• These isomerases act on calcineurin.
• Calcineurin thereby dephosphorylates NFAT.
• Tacrolimus and cyclosporine inhibit cyclophilins
  isomerase activity.

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CSA FK506

• NFAT is activated by calcium/calmodulin-dependent,
  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.

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TCR
CSA FK506
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Third Step

• IL-2 receptor (CD25) activation
• Modulation of IL-2r.

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Kinetics of gene expression in antigen-stimulated
T lymphocytes.
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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

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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)

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The low-affinity IL-2 receptor (CD25)

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IL-2 r

• The binding of IL-2 to the high- or
  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.

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IL-2 r

• The IL-2 R a chain has a relatively short
  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.

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Rapamycin

• Rapamycin is an immunosuppressive 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 drugreceptor 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.

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Rapamycin

• Instead, rapamycin inhibits IL-2driven T-cell
  proliferative responses by blocking the function
  of another enzyme, called mTOR (for mammalian
  target of rapamycin also called FRAP, for
  FKBP12-rapamycinassociated protein).
• mTOR is a member of a larger family of proteins
  with PI-kinase domains.

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IL-2

• Although IL-2driven 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).

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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.

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Formation of the immunological synapse.
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Artificial means

• Mitogens

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Mitogens

• A number of different reagents have been used to
  substitute for the stimulating antigenMHC
  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
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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.

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Natural Killer Cells
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Natural Killer Cells

• This small lymphocyte population has remained
  elusive in many respects.
• Morphologically, most NK cells fit into the
  population of large granular lymphocytes.
• Functionally, they are able to kill
  virus-infected 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.

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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.

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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)

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Natural Killer Cells
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Natural Killer Cells
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Natural Killer Cells
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K Cell (ADCC)
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Lymphocyte Apoptosis
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Conclusion

• We have discussed the activation and 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.