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T-Cells NK . Feb 13, 2006. T-cells. Antigens that are transported by dendritic cells to lymph nodes are recognized by naive T lymphocytes that recirculate through these lymph nodes.

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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-guanine 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’-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:


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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 [Ca2+]i and Activation of PKC

  • The increase in [Ca2+]i 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 serine–threonine phosphatase calcineurin is now well established.


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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.


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Ca2+/calmodulin-dependent serine–threonine 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 cyclophilin’s 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 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.


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Rapamycin

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).


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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 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


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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.


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