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. 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 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

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

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.

Therapeutics: TLR9 CpG ODN mimic bacterial DNA. Engagement of the CpG ODN with the intracellular TLR9 induces IL-12, IL-18, and IFN- . CpG-A ODN ’-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 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.

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.

T-Cells

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   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   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 C  (PLC  ): an enzyme that catalyzes hydrolysis of plasma membrane phospholipid PIP 2 to generate IP 3 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 [Ca 2+ ]i influences calmodulin-dependent events, including the activation of calcineurin (PP2B) and Ca 2+ /calmodulin-dependent kinase (CAM-kinase). A critical role for the Ca 2+ /calmodulin- dependent serine–threonine phosphatase calcineurin is now well established.

Ca 2+ /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.

Ca 2+ /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.

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)

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

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.

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.

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)    chain: TAC, CD 25 (k d 1.4x10 -8 M)   chain: CD 122 (k d 1.2 x M)   chain: functional component of: IL-4r, IL-7r, and IL-9r.   chain: (k d 1.3 x M)

The low-affinity IL-2 receptor (CD25)

The low-affinity IL-2 receptor (CD25)

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  chain.

IL-2 r 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. It is the  and  chains that are responsible for the signal transduction function of the receptor.

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.

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.

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.

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

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.

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.

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

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.

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

K Cell (ADCC)

Lymphocyte Apoptosis

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.