T-Cells NK  Feb 13, 2006
T-cells <ul><li>Antigens that are transported by dendritic cells to lymph nodes are recognized by naive T lymphocytes that...
 
Step 1 <ul><li>The dendritic cell encounters the antigen. </li></ul><ul><li>The antigen interacts with  Toll Like Receptor...
Dendritic TLR
Dendritic TLR  There are 11 TLRs, these are best characterized
Dendritic TLR  TH2 responses  mixed    Strong TH1/TH2     TH1
Distribution of TLR on innate and adaptive cells
Fig. on p. 284
Fig. on p. 284
Therapeutics:  TLR9 <ul><li>Activation of TLR9 has progressed significantly in the development of therapeutics to treat: a...
Therapeutics:  TLR9 <ul><li>CpG ODN mimic bacterial DNA. </li></ul><ul><li>Engagement of the CpG ODN with the intracellula...
TLR2 <ul><li>Pam3Cys-OH a lipoprotein that stimulates the TLR2 leading to a strong TH2 response with secretion of IL-4. </...
Next Step  <ul><li>Now that the dendritic cells have secreted their cytokines to select the T-helper subset, the antigen i...
T-Cells
 
In the T-Helper Lymphocyte <ul><li>Lck:   A Src family non-receptor tyrosine kinase that non-covalently associates with th...
 
In the T-Helper Lymphocyte <ul><li>ZAP-70  (Zeta-associated protein of 70 kD): binds to phosphorylated tyrosines in the cy...
In the T-Helper Lymphocyte <ul><li>Adapter Proteins are involved in serving as scaffolds for the recruitment of other sign...
 
In the T-Helper Lymphocyte <ul><li>Phospholipase C   (PLC  ):  an enzyme that catalyzes hydrolysis of plasma membrane ph...
 
Consequences of Increases in [Ca2+]i and Activation of PKC  <ul><li>The increase in [Ca 2+ ]i influences calmodulin-depend...
Ca 2+ /calmodulin-dependent serine–threonine phosphatase calcineurin <ul><li>Calcineurin is the molecular target for the i...
Ca 2+ /calmodulin-dependent serine–threonine phosphatase calcineurin <ul><li>It is these molecular complexes, not the isol...
CSA & FK506 <ul><li>CSA binds to cyclophilin & FK506 binds to FK binding protein (FKBP). </li></ul><ul><li>This complex in...
Cyclophilin <ul><li>Cyclophilin is a prokaryotic peptidyl-prolyl  cis-trans-isomerase  (also called PPLases), or a rotamas...
Cyclophilin <ul><li>The activity of cyclophilin and FKBP are to serve as cis-trans isomerases.  </li></ul><ul><li>These is...
CSA & FK506 <ul><li>NFAT  is activated by calcium/calmodulin-dependent, calcineurin-mediated dephophorylation that permits...
TCR CSA FK506
 
Third Step <ul><li>IL-2 receptor (CD25) activation  </li></ul><ul><li>Modulation of IL-2r. </li></ul>
Kinetics of gene expression in antigen-stimulated  T lymphocytes.
The low-affinity IL-2 receptor (CD25) <ul><li>Expressed in low levels by about 30% of circulating (resting) lymphocytes </...
The low-affinity IL-2 receptor (CD25) <ul><li>   chain:  TAC, CD 25 (k d  1.4x10 -8  M) </li></ul><ul><li>   chain:  CD ...
The low-affinity IL-2 receptor (CD25) <ul><li> </li></ul>
The low-affinity  IL-2 receptor  (CD25) <ul><li> </li></ul>
IL-2 r <ul><li>The binding of IL-2 to the high- or intermediate-affinity forms of the receptor initiate transmembrane sign...
IL-2 r <ul><li>The IL-2 R   chain has a relatively short cytoplasmic domain, and it appears that its main function is to...
Rapamycin <ul><li>Rapamycin is an immunosuppressive agent that has been used to probe the IL-2/IL-2R pathway.  </li></ul><...
Rapamycin <ul><li>Instead, rapamycin inhibits IL-2–driven T-cell proliferative responses by blocking the function of anoth...
IL-2 <ul><li>Although IL-2–driven T-cell proliferation has been widely considered to be the major mechanism responsible fo...
IL-2 <ul><li>IL-4 and IL-15 are the most likely to function as T-cell growth factors in the absence of IL-2.  </li></ul><u...
IL-2 <ul><li>It is likely that more sustained T cell proliferative responses and recruitment of T cells will occur in inst...
Formation of the  immunological  synapse.
Artificial means <ul><li>Mitogens </li></ul>
Mitogens <ul><li>A number of different reagents have been used to substitute for the stimulating antigen–MHC molecule.  </...
Mitogens <ul><li>Among these reagents are several lectins, plant-derived proteins that bind various carbohydrate groups.  ...
Mitogens <ul><li>Con A  and  PHA  are selective  T-cell  mitogens when compared with their effects on B cells, whereas  PW...
Alternate mitogens <ul><li>In our lab, we use anti-CD-3 and  anti-CD-28 antibody coated plates. </li></ul><ul><li>This is ...
Natural Killer Cells
Natural Killer Cells <ul><li>This small lymphocyte population has remained elusive in many respects. </li></ul><ul><li>Mor...
Natural Killer Cells <ul><li>A major difference is that NK cells can recognize virus-infected cells and many different typ...
Natural Killer Cells <ul><li>At this time, it is believed that two broadly reactive receptors are involved, one that deliv...
Natural Killer Cells
Natural  Killer  Cells
Natural Killer Cells
K Cell (ADCC)
Lymphocyte Apoptosis
Conclusion <ul><li>We have discussed the activation and deactivation of T-lymphocytes </li></ul><ul><li>We have introduced...
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T-Cell Natural Killer Cells PP

  1. 1. T-Cells NK Feb 13, 2006
  2. 2. T-cells <ul><li>Antigens that are transported by dendritic cells to lymph nodes are recognized by naive T lymphocytes that recirculate through these lymph nodes. </li></ul><ul><li>The T cells are activated to differentiate into effector and memory cells, which may remain in the lymphoid organs or migrate to nonlymphoid tissues. </li></ul><ul><li>At sites of infection, the effector cells are again activated by antigens and perform their various functions, such as macrophage activation. </li></ul>
  3. 4. Step 1 <ul><li>The dendritic cell encounters the antigen. </li></ul><ul><li>The antigen interacts with Toll Like Receptors (TLR) on the dendritic cell. </li></ul><ul><li>Depending on which set of the TLR receptors that are activated, determines the type of immune response. </li></ul>
  4. 5. Dendritic TLR
  5. 6. Dendritic TLR There are 11 TLRs, these are best characterized
  6. 7. Dendritic TLR TH2 responses mixed Strong TH1/TH2 TH1
  7. 8. Distribution of TLR on innate and adaptive cells
  8. 9. Fig. on p. 284
  9. 10. Fig. on p. 284
  10. 11. Therapeutics: TLR9 <ul><li>Activation of TLR9 has progressed significantly in the development of therapeutics to treat: asthma, cancer, and sepsis. </li></ul><ul><li>CpG ODN(unmethylated cytosine-phosphorothioate-guanine oligodeoxynucleotide) sequences are being used to stimulate TH1 responses. </li></ul>
  11. 12. Therapeutics: TLR9 <ul><li>CpG ODN mimic bacterial DNA. </li></ul><ul><li>Engagement of the CpG ODN with the intracellular TLR9 induces IL-12, IL-18, and IFN-  . </li></ul><ul><li>CpG-A ODN 2216 </li></ul><ul><li>5’-G*G*G_G_G_A_C_G_A_T_C_G_T_C_G*G*G*G*G*G-3’ </li></ul><ul><li>( * ) are phosphothiorate linkages, and </li></ul><ul><li>( _ ) are phosphodiester linkages: </li></ul>
  12. 13. TLR2 <ul><li>Pam3Cys-OH a lipoprotein that stimulates the TLR2 leading to a strong TH2 response with secretion of IL-4. </li></ul><ul><li>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. </li></ul>
  13. 14. Next Step <ul><li>Now that the dendritic cells have secreted their cytokines to select the T-helper subset, the antigen is presented to the T-helper lymphocyte. </li></ul><ul><li>This step determines the fate of the immune response: humoral or cellular. </li></ul>
  14. 15. T-Cells
  15. 17. In the T-Helper Lymphocyte <ul><li>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. </li></ul><ul><li>Lck mediates tyrosine phosphorylation of the cytoplasmic tail of the CD3 and  zeta  proteins of the TCR complex. </li></ul>
  16. 19. In the T-Helper Lymphocyte <ul><li>ZAP-70 (Zeta-associated protein of 70 kD): binds to phosphorylated tyrosines in the cytoplasmic tail of the CD3 and  zeta  proteins and phosphorylates adapter proteins. </li></ul>
  17. 20. In the T-Helper Lymphocyte <ul><li>Adapter Proteins are involved in serving as scaffolds for the recruitment of other signaling molecules. </li></ul><ul><li>During lymphocyte activation, adapter proteins may be phosphorylated on tyrosine residues to enable them to bind other proteins containing Scr homology 2 (SH2) domains. </li></ul>
  18. 22. In the T-Helper Lymphocyte <ul><li>Phospholipase C  (PLC  ): an enzyme that catalyzes hydrolysis of plasma membrane phospholipid PIP 2 to generate IP 3 and DAG. </li></ul><ul><li>This leads to increased intra-cellular calcium and activation of PKC. </li></ul>
  19. 24. Consequences of Increases in [Ca2+]i and Activation of PKC <ul><li>The increase in [Ca 2+ ]i influences calmodulin-dependent events, including the activation of calcineurin (PP2B) and Ca 2+ /calmodulin-dependent kinase (CAM-kinase). </li></ul><ul><li>A critical role for the Ca 2+ /calmodulin-dependent serine–threonine phosphatase calcineurin is now well established. </li></ul>
  20. 25. Ca 2+ /calmodulin-dependent serine–threonine phosphatase calcineurin <ul><li>Calcineurin is the molecular target for the immunosuppressives CsA and FK506 , drugs that have revolutionized clinical organ transplantation. </li></ul><ul><li>CsA and FK506 form molecular complexes with their cellular receptors, cyclophilin and FKBP , respectively. </li></ul>
  21. 26. Ca 2+ /calmodulin-dependent serine–threonine phosphatase calcineurin <ul><li>It is these molecular complexes, not the isolated drugs, that inhibit the phosphatase function of calcineurin . </li></ul><ul><li>Calcineurin is expressed ubiquitously but is expressed at only low levels in T-lymphocytes. </li></ul><ul><li>This probably accounts for the relative specificity of the immunosuppressive drugs in targeting T-cell function. </li></ul>
  22. 27. CSA & FK506 <ul><li>CSA binds to cyclophilin & FK506 binds to FK binding protein (FKBP). </li></ul><ul><li>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 ) </li></ul>
  23. 28. Cyclophilin <ul><li>Cyclophilin is a prokaryotic peptidyl-prolyl cis-trans-isomerase (also called PPLases), or a rotamase. </li></ul>
  24. 29. Cyclophilin <ul><li>The activity of cyclophilin and FKBP are to serve as cis-trans isomerases. </li></ul><ul><li>These isomerases act on calcineurin. </li></ul><ul><li>Calcineurin thereby dephosphorylates NFAT. </li></ul><ul><li>Tacrolimus and cyclosporine inhibit cyclophilin’s isomerase activity. </li></ul>
  25. 30. CSA & FK506 <ul><li>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. </li></ul>
  26. 31. TCR CSA FK506
  27. 33. Third Step <ul><li>IL-2 receptor (CD25) activation </li></ul><ul><li>Modulation of IL-2r. </li></ul>
  28. 34. Kinetics of gene expression in antigen-stimulated T lymphocytes.
  29. 35. The low-affinity IL-2 receptor (CD25) <ul><li>Expressed in low levels by about 30% of circulating (resting) lymphocytes </li></ul><ul><li>CD25 has been proposed to be associated with T-lymphocyte memory </li></ul>
  30. 36. The low-affinity IL-2 receptor (CD25) <ul><li> chain: TAC, CD 25 (k d 1.4x10 -8 M) </li></ul><ul><li> chain: CD 122 (k d 1.2 x 10 -7 M) </li></ul><ul><li> chain: functional component of: </li></ul><ul><li>IL-4r, IL-7r, and IL-9r. </li></ul><ul><li> chain: (k d 1.3 x 10 -11 M) </li></ul>
  31. 37. The low-affinity IL-2 receptor (CD25) <ul><li> </li></ul>
  32. 38. The low-affinity IL-2 receptor (CD25) <ul><li> </li></ul>
  33. 39. IL-2 r <ul><li>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. </li></ul><ul><li>Such signal transduction events also account for other effects of IL-2, such as the up-regulation of transcription of the IL-2R  chain. </li></ul>
  34. 40. IL-2 r <ul><li>The IL-2 R  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. </li></ul><ul><li>It is the  and  chains that are responsible for the signal transduction function of the receptor. </li></ul>
  35. 41. Rapamycin <ul><li>Rapamycin is an immunosuppressive agent that has been used to probe the IL-2/IL-2R pathway. </li></ul><ul><li>It binds to the same cellular receptor as FK506, FKBP , and it is the drug–receptor complex that mediates inhibition of T-cell function. </li></ul><ul><li>However, unlike FK506, rapamycin does not inhibit the induction of IL-2 gene, nor is its target calcineurin. </li></ul>
  36. 42. Rapamycin <ul><li>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). </li></ul><ul><li>mTOR is a member of a larger family of proteins with PI-kinase domains. </li></ul>
  37. 43. IL-2 <ul><li>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. </li></ul><ul><li>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. </li></ul>
  38. 44. IL-2 <ul><li>IL-4 and IL-15 are the most likely to function as T-cell growth factors in the absence of IL-2. </li></ul><ul><li>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). </li></ul>
  39. 45. IL-2 <ul><li>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. </li></ul>
  40. 46. Formation of the immunological synapse.
  41. 47. Artificial means <ul><li>Mitogens </li></ul>
  42. 48. Mitogens <ul><li>A number of different reagents have been used to substitute for the stimulating antigen–MHC molecule. </li></ul><ul><li>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. </li></ul>
  43. 49. Mitogens <ul><li>Among these reagents are several lectins, plant-derived proteins that bind various carbohydrate groups. </li></ul><ul><li>These lectins, phytohemagglutinin ( PHA ), concanavalin A ( Con A ), and pokeweed mitogen ( PWM ), were among the first recognized polyclonal activators of T cells. </li></ul><ul><li>Because they can induce the proliferative responses, they are among a class of reagents termed mitogens. </li></ul>
  44. 50. Mitogens <ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>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. </li></ul>KNOW
  45. 51. Alternate mitogens <ul><li>In our lab, we use anti-CD-3 and anti-CD-28 antibody coated plates. </li></ul><ul><li>This is a means to stimulate lymphocytes in a selective manner. </li></ul>
  46. 52. Natural Killer Cells
  47. 53. Natural Killer Cells <ul><li>This small lymphocyte population has remained elusive in many respects. </li></ul><ul><li>Morphologically, most NK cells fit into the population of large granular lymphocytes. </li></ul><ul><li>Functionally, they are able to kill virus-infected or malignant cells with low or absent MHC molecules. </li></ul><ul><li>NK cells are neither T nor B lymphocytes: TcR and immunoglobulin genes are in the unrearranged genomic configuration. </li></ul>
  48. 54. Natural Killer Cells <ul><li>A major difference is that NK cells can recognize virus-infected cells and many different types of malignant cells without clonal restriction. </li></ul><ul><li>In other words, their recognition mechanisms are relatively nonspecific and common to all NK cells. </li></ul>
  49. 55. Natural Killer Cells <ul><li>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. </li></ul><ul><li>The triggering or activating receptor ( NKAR, NKR-Pl ) </li></ul>
  50. 56. Natural Killer Cells
  51. 57. Natural Killer Cells
  52. 58. Natural Killer Cells
  53. 59. K Cell (ADCC)
  54. 60. Lymphocyte Apoptosis
  55. 61. Conclusion <ul><li>We have discussed the activation and deactivation of T-lymphocytes </li></ul><ul><li>We have introduced the mechanism of action of CSA, FK506, and Rapamycin. </li></ul><ul><li>We have introduced the concepts of effort functions of cytotoxic cells. </li></ul>
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