Tcell Recognition And Triggering81011

Synopsis: Engagement of the TCR and CD4 or CD8 activates protein tyrosine kinases (TKs) associated with the intracytoplasmic portions of the receptor. TKs trigger second messengers and initiate several signalling pathways which eventually alter proteins which regulate the transcription of genes for cytokines and cytokine receptors. This locks the T cell into activation. Signals provided by additional membrane receptors such as CD28 also play a key role ("second signals").

The Nature of TCR Triggering

The binding of sufficient TCRs to MHC molecules is a necessary condition for T cell activation by antigen. The signal requires the CD3 complex, which includes Y, 6, e, and the long Z chains.89 How does TCR binding to MHC alter CD3? This problem is generally explained by one of two mechanisms:

1.Conformational change: engagement of the V regions alters remote parts of the TCR, which in turn alters the CD3 complex; or

2.Crosslinking: the TCR complexes are brought together by engaging antigen and activate one another. Dimerization of class II molecules may serve to bring TCRs together to aid triggering. This would imply that class II recognition may proceed through a complex of two TCRs and two CD4s.

Crosslinking is a common mechanism of triggering of receptors in general. Class I may be able to form multimers,90 and the dimeric nature of CD8 and of class II suggest that crosslinking could occur.79 Nevertheless, TCR-mediated T cell activation in vivo may reflect molecular changes triggered by the assembly of the TCR-CD3-CD4 or CD8 complex, in which the CD4 or CD8 molecules play key roles, particularly if the affinity of the TCR for the MHC is low.91

The CD3 complex is the transducer which tells the interior of the cell that the TCR has engaged MHC. The Z chains interact directly with the tyrosine kinases. Meanwhile CD4 (or CD8) engage the MHC, and assembly of the complex brings a series of tyrosine kinases together.

The Key Role of Tyrosine Kinases

The CD3-TCR complex is associated with at least three TKs: ZAP, p59fyn, and p50csk. CD4 and CD8 are associated with another protein tyrosine kinase, p56lck. TKs phosphorylate the tyrosine residues in the CD3 molecule Z chain, in key transduction molecules, and in one another. The functions of p56lck and p59fyn have been shown in knockout mice to be nonoverlapping: p59fyn knockouts have defective TCR signalling and p56lck have defective thymocyte development.92,93 The tyrosine kinase p50csk 94 may be particularly involved in negative signalling (tolerizing) the T cell when the TCR is signalled. TKs activate many signalling pathways, including:

1. Ras: the tyrosine kinases can activate the Ras pathway through recently discovered intermediate proteins such as Shc95 and GRB2;96,97 activation of Ras then triggers a cascade which can activate enzymes such as mitogen activated protein kinase (MAP kinase) and eventually impact on cell division.

2. PLC-y1, which lyses the membrane phospholipid phosphatidyl inositol bisphosphate to yield IP3 and diacylglycerol (DAG). DAG activates protein kinase C (PKC) and inositol trisphosphate (IP3) binds to receptors on the ER to release stored calcium and raise intracytosolic Ca2+ levels. The high Ca2+ is then sustained by increased calcium entry through channels in the plasma membrane to maintain high cytosolic Ca2+ con-centrations.98

3. Phosphatidyl inositol-3-kinase and several others.

Each of these pathways has multiple consequences leading to expression of many genes, blast transformation, mitosis, and expression of effector functions. The calcium-dependent pathway is critical for T-cell activation and important in transplantation. High intracellular calcium activates calcium-regulated enzymes, particularly the enzyme calcineurin (CN). This is a calcium- and calmodulin-dependent serine phosphatase. It activates transcription factors for some key cytokines, particular members of the nuclear "factor of activated T cells" or NF^AT family. CN is the target for some of the most important immunosuppressive agents, cyclosporine and tacrolimus (FK506).

Within minutes, mRNA is transcribed from the "immediate" genes, which do not require new protein synthesis. Some of these are transcription factors. The newly synthesized transcription factors, plus the newly activated factors, now activate a second set of genes. The mRNAs and products for IL-2, IFN-y, and other cytokines and certain cytokine receptors then appear.

Costimulation ("Signal 2")

When the naive T cell encounters alloantigen, it requires other signals before proceeding with activation,99 in keeping with the classic two-signal model of lymphocyte activation.100 Signal 1 is the allogeneic MHC antigen, which must be at a high density to trigger a primary T-cell response. High antigen expression may be one reason why antigen presenting cells (dendritic cells and macrophages) are required. "Signal 2" is the nonantigen signal provided by antigen presenting cells.

(A classic belief in immunology is that when T cells engage antigen without appropriate second signals, anergy results. This renders the identity of the second signals crucial for transplantation and immunosuppression. If we could block them, we might induce anergy.)

"Signal 2" may involve certain adhesion molecules of the Ig superfamily, notably B7-1 and B7-2 (also called B70) on the APC, engaging CD28 on the T cells.101-105 CD28 activates systems in the T cell which synergize with the signals from the T-cell receptor. CD28 amplifies and prolongs signal 1, increasing IL-2

transcription and prolonging the half life of IL-2 mRNA.106 In CD28 knockout mice, T-cell triggering can still occur, indicating that other systems can compensate.107 Other signals from the antigen presenting cell, which could contribute to signalling, include other adhesion molecule ligand receptor pairs on the APC and T cell respectively (ICAM-1-LFA-1 and LFA-3-CD2), and cytokines such as IL-1 and IL-6 produced by the antigen presenting cell.

Stimulation of the primary T-cell response may require all of these, in a "conversation" between T cells and APCs initiated by high density of the allogeneic class II molecules on the APCs in the context of cytokines and adhesion molecules. The signals from the triggered CD4 T cells then activate the APCs to increase the signals to the T cell in a cascade of reciprocal activation.

One of the key sites for regulating signal 2 may be a expression of CD40 ligand. CD45 is a tyrosine phosphatase on the surface of all marrow-derived cells whose function may be to keep the key tyrosine in tyrosine kinases (Ick and fyn) dephosphorylated and ready to participate in triggering.108

Details of Signal Transduction and T-Cell Activation: Control of Cytokine Expression

PLC-y1, activated by tyrosine phosphorylation, lyses membrane phosphatidyl inositol bisphosphate (PIP2), releasing DAG and IP3. DAG activates PKC which is also activated through other pathways, including calcium flux. PKC activation leads to the transcription of several genes which encode transcription factors such as fos and jun which form the complex called AP-1, composed of the Jun and Fos proteins.109

IP3 binds to receptors on the endoplasmic reticulum which release calcium into the cytosol. The high cytosolic calcium is then sustained by changes in membrane transport.110 The high calcium activates calcium-dependent enzymes, one of which is CN. CN activates cytosolic factors called NF-AT, which is free to translocate from the cytosol to the nucleus.111,112 When cytoplasmic and nuclear factors assemble to form the full NF-AT complex, transcription of IL-2 mRNA begins. While the NF-AT sites account for the majority of inducible IL-2 expression, it is likely that the NF-kB site113 and the octamer site are also critical. The characteristic behavior of the IL-2 gene requires the interaction of multiple transcription factors binding to these sites.

Similar events occur with other cytokine genes, although less is known about them. The result is a wave of transcription of cytokine mRNAs. Note that this is the "second wave" of protein synthesis, the first being the nuclear factors which control the cytokine promoters. In this sense the cytokines are "early", not "immediate" genes.113

Naive CD4 T cells make predominantly IL-2 in their first encounter with antigen, whereas previously stimulated or memory T cells make other cytokines. IL-2 engages its receptor, and other cytokines engage through their receptors, giving waves of receptor triggering and signal transduction. The cell becomes committed to activation, differentiation, mitosis, and clonal expansion. Effector functions emerge such as cytotoxicity in CD8 cells. Eventually the molecules associated with memory and recirculation, such as the "very late antigens" or VLA molecules,114 appear.

Cytokines and Their Receptors9

The term "cytokine" includes the interleukins, interferons, and colony stimulating factors of the hematopoietic and host defense system. They are protein mediators which signal cells through specific membrane receptors. Cytokines and their receptors are related in structure and function to protein hormones and their receptors. Cytokines have certain characteristics:

1. Short half-life: cytokine mRNAs and cytokines themselves have short half-lives to permit fine regulation.

2. Relatively small size: the typical cytokine gene is about 4-5 kb in length, with about four exons. Numerous AT sequences at its 3' end confer a short half life on the mRNA.115 The protein is typically a polypeptide chain of about 10-20 kD, often glycosylated and/or multimerized to a higher molecular weight.

3. a-helical structure: many cytokines are folded into a bundle of four to six a-helices, sometimes with very short p strands. Exceptions include TNF-a, a sandwich or "jelly roll" of antiparallel p strands, and TGF-p, which has both a-helices and p-pleated sheets.116

4. Multimer formation is common: IFN-y and TGF-p are dimers, and TNF-a is a trimer.117

5. Cytokines are generally not stored but are synthesized and secreted when needed. They are not usually expressed as membrane proteins, but some have membrane-bound variants, e.g., TNF-a118 and IL-1.

6. The main control of cytokine production is transcriptional, although post-transcriptional control is known, e.g., for TNF-a.119

Cytokines often act in concert with other cytokines: interactions (synergy, competition, and antagonism) are common. Cytokines are pleiotropic (i.e., have many effects) and redundant (i.e., have overlapping effects). Cytokines commonly induce other cytokines in a cascade. Self-amplifying circuits are common to facilitate rapid potent responses. The potency of the cytokine response is impressive as is well known to the clinician who observes the cytokine release syndrome after OKT3 treatment (see below).

Some cytokines are produced in normal tissues at low levels and affect growth, development, and homeostasis, e.g., the maturation of T and B lymphocytes. But their most characteristic effects are in inflammation and host response to injury or infection.

"Knockout mice" are providing important insights into the roles of cytokines and their receptors.120-123 In knockout mice, both copies of the target gene have been mutated to prevent expression. Such strategies may underestimate the importance of the deleted structure because the deletion forces the embryo to use other cytokines to develop, thereby maximizing apparent redundancy. Moreover, the laboratory mouse, protected from many of the usual pathogens of its species, tolerates immune defects which would be more serious in the natural environment.

Surprises arise in knockouts: for example the IL-2 and IL-10 knockouts, as well as some TCR knockouts, get inflammatory bowel disease for unknown reasons.124,125

Cytokine receptors are typically multimers of different transmembrane proteins, one or more which have an external ligand-binding domain, and an intracyto-plasmic signalling domain. One or more chains may bind the cytokine with high affinity, but the multimer is required for internalization and/or signalling. Cytokine receptors are classified into families on the basis of their external, ligand binding domain.126-128

1. The hemopoietins, e.g., IL-2R p chain, use a 200 kd external domain with four conserved cysteines and one tryptophan residue at the N terminal, and aromatic residues (Trp-Ser-X-Trp-Ser) at the C terminal. A few receptors in this group have typical Ig domains in their extracellular regions.

2. The interferon and IL-10 receptors, e.g., IFN-yR, have two external domains distantly related to Ig domains, with characteristic conserved cysteines.

3. The TNF receptor and its relatives have an external domain with cysteine-rich repeats.128

4. IL-8 and its relatives have a "seven pass" membrane receptor associated with G proteins, similar to many endocrine receptors.

Unlike the cytokines, which are often predominantly a-helical, the external ligand binding domain of a hematopoietin or interferon receptor is often two p-pleated sheets. A second chain of the receptor may or may not actually engage the cytokine: in the IL-2 receptor it does, but in the IFN-y receptor the binding site is formed by the single receptor protein with the second receptor component presumably playing other roles. Binding of the cytokine to the external domain of the receptor may alter the cytoplasmic domain, triggering second messengers usually through a kinase, usually a protein tyrosine kinase or less commonly a serine/threonine kinase. The signalling systems are similar to those already described: PTKs activate PLC-y, PI-3 kinase and other second messengers with downstream activation of serine-threonine kinases, e.g., PKC and release of intracellular calcium.

The final effect is often on transcription factors, but other events are common, such as direct effects on membrane receptors or cytoplasmic effector mechanisms.

Signal transduction, through the IFN-y receptor,129 is a useful example of a cytokine system which we can watch in operation in transplant rejection. IFN-y engages the IFN-yR and activates two tyrosine kinases, JAK1 and JAK2, which phosphorylate a factor called STAT 91. This induces transcription of selected genes by moving to the nucleus and engaging specific sites in their promoters. We will expand on some features of the IFN-y response later as an example of cytokine signal transduction. The TNF receptor acts through a sphingomyelin pathway to induce NF-kB to be released from its cytoplasmic binding protein (IkB) to enter the nucleus and bind to specific DNA regulatory sites.118

From the above, the passage of signals from hematopoietin and interferon receptors to the interior of the cell involves the regulation of tyrosine phosphorylation.

The cytoplasmic regions of many membrane receptors for protein hormones have intrinsic tyrosine kinase activity, but cytokine receptors are associated with separate tyrosine kinases (like JAKs which associated with the IFNL-yR). Engagement of the receptor by its ligand activates the tyrosine kinase activity, which results in phosphorylation of one or more key tyrosine residues in the cytoplasmic region of the receptor. This phosphorylated tyrosine can then be recognized by other proteins via specific regions in those proteins called "src homology-2" or SH2 domains.130,131 The sequence "ligand-receptor-tyrosine kinase activation-tyrosine phosphorylation—recognition and binding of a second messenger via its SH2 domain activation of second messenger by tyrosine phosphorylation—is probably a common pattern for linking membrane receptors to second messengers like STAT proteins.

Several cytokine receptors, including IL-2R, apparently utilize a signal transduction pathway which involves the activation and phosphorylation of an enzyme called the "Target of Rapamycin" or TOR. TOR in turn activates p70 S6 kinase.132,133 The role of TOR was discovered because the immunosuppressive drug rapamycin acts at this point. The role of TOR kinase is probably crucial in the initiation of cell division by cytokines. TOR acts to increase the translation of existing mRNAs for proteins which control the cell cycle.

0 0

Post a comment