T Cell Signaling and Activation: No Simple Matter

By Noemí Bronstein-Sitton, Ph.D.

T cells are central players of the adaptive immune response, which help protect the host against different pathogens ranging from bacteria to fungi and viruses. In order to perform their function, T cells need to be activated, a process that could lead to a variety of responses including proliferation, migration, cytokine production and even apoptosis. The “decision” by T cells to become activated or not is crucial: an inappropriate or exaggerated response could lead to autoimmune diseases while a failure to respond could lead to infection and death. To perform such a complex and sensitive task, T cells must respond to environmental cues that stimulate a complex signaling cascade. In the last few years, the signaling mechanisms that govern T cell activation and their subsequent cellular responses have been closely studied. In the present review we will examine the main signaling cascades involved and discuss several molecules that are being used to specifically block some of these pathways.

Introduction

T cell activation that leads to a productive response (i.e. cytotoxicity of target cells or stimulation of antibody production by B cells) needs two signals. The primary signal (or signal one) is provided by the binding of foreign antigens (usually small peptides from the respective pathogen) to the T cell antigen receptor (TCR) in the context of the class II major histocompatibility complex (MHC). The TCR is a complex of six different polypeptides. The clonotypic α and β chains provide the specificity of the ligand binding by a process of genetic rearrangement that provides millions of receptor variants. While the α and β heterodimer binds directly to the peptide/MHC complex, the engagement of the intracellular signaling machinery is through the invariant components of the TCR: the γ, δ and ε chains (collectively known as the CD3 complex) and the ζ chains^1,2.

Signal two or the costimulatory signal is provided by interaction of coreceptors such as CD28 or CD4 with their counterparts in the antigen presenting cell (APC)^1,2.

The main signaling pathways elicited by binding of the TCR and some of the coreceptors are depicted in Figure 1.

**Figure 1. Major Signaling Pathways in T Cell Activation**
Please note that not all the molecules involved in the signaling cascades are illustrated here for simplification purposes.

The Tyrosine Kinases (and their kinases) Signaling Pathway

The principal signaling pathway following TCR engagement is protein tyrosine phosphorylation. The tyrosine phosphorylation cascade will ultimately activate several transcription factors such as NF-AT and AP-1 which in turn direct the transcription of the new genes needed for T cell response.

The earliest detectable event following ligation of the TCR by the peptide-MHC complex is the activation of the Src family protein tyrosine kinases Lck and Fyn³. They phosphorylate specific motifs (called immunoreceptor tyrosinebased activation motifs or ITAMs) present on the ζ and CD3 subunits of the TCR complex. Phosphorylation of these motifs promotes recruitment and activation of ZAP-70, another tyrosine kinase which in turn activates several target proteins including the adaptor/linker proteins LAT and SLP-76^4,5,6. Phosphorylation of LAT and SLP-76 recruits into their vicinity and facilitates the activation by tyrosine phosphorylation of the phospholipase Cγ1 (PLCγ1). PLCγ1 catalyzes the formation of the second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), which, respectively, trigger Ca²⁺ flux and contribute to protein kinase C (PKC) and Ras activation (see also below)⁷.

Since protein tyrosine phosphorylation is such a crucial force in T cell signaling it must be carefully controlled. This is achieved mainly by the action of the tyrosine kinase Csk which phosphorylates a critical Tyr at the C-terminal domain of Lck rendering it inactive³.

The preponderancy of the tyrosine kinase cascade in T cell signaling is demonstrated by the use of tyrosine kinase inhibitors such as Genistein (#G-300) which are able to block T cell function both in vitro and in vivo^8,9. Other non-specific kinase inhibitors such as Staurosporine (#S-350) can also be used to block T cell function.

PKC and Ras activation results in the activation of several members of the mitogen-activated protein kinase (MAPK) superfamily. The MAPKs are serine/threonine kinases that activate kinases (and other) signaling cascades that result in the activation of the transcription factors NF-κB and AP-1, both of which are necessary for the transcription of several key genes involved in the T cell immune response. Three MAPK subfamilies have been described: the extracellular signal regulated kinase (ERK), the Jun-NH2-terminal kinase (JNK) and the p38, all of which play an important role in T cell activation.

Use of specific inhibitors for the different MAPK such as the p38 inhibitor SB203580 (#S-370) or the ERK inhibitor U0126 (#U-400) have helped unravel the complex signaling cascades activated by TCR-dependent T cell activation^27,28.

**Genistein: A Tyrosine Kinase and Topoisomerase Inhibitor**
Genistein (#G-300) inhibits the Tyrosine phosphorylation of 28 kDa protein. Hela cells were grown to 70% confluency, and the cells serum starved for 18 h. The cells were preincubated for 30 min in presence or absence of 500 nM Genistein and simulated for 2 h with or without 20% fetal calf serum. The cell extracts were analyzed by Western blotting by probing with phospho-Tyr specific antibodies.

**SB 203580: MAPK Homolog – p38 Inhibitor**
SB203580 (#S-370) inhibits the phosphorylation of Elk1 and Atf2 caused by p38 MAPK. 3T3-L1 cells were grown to 70% confluence and then serum starved for 3 h. The cells were incubated for 2 h with various concentrations of SB203580 and stimulated with 5μM Anisomycin (#A-520). The cell proteins were resolved by SDS PAGE, probed with antiphospho-p38 MAPK, anti-p38 MAPK, anti-phospho-Elk1 and anti-phospho-Atf2. The phophorylation of Elk1 and Atf2 was reduced as a function of SB203580 concentration.

**Staurosporine: A Protein Kinase Inhibitor and Apoptosis Inducer**
Staurosporine (#S-350) induces apoptosis and inhibits phosphorylation. Jurkat cells were grown to 70% confluency, then 2 μM Staurosporine or vehicle was added for 3 or 6 hours. At the end of the incubation period, the cells extracts were probed for Caspase 3 and cleaved Caspase 3 with specific antibodies.

**U0126: A Specific MAPK Pathway Inhibitor**
U0126 (#U-400) inhibits P42/44 MAPK phosphorylation. C6 glioma cells were grown to 70% confl uence and then serum starved for 1.5 h. The cells were then incubated for 2 h with various concentrations of U0126 as indicated in the picture and stimulated with or without 7ng/ml PDGFAA. The cell proteins were resolved by SDS PAGE, probed with anti phospho-P42/44 MAPK (upper panel) or with anti P42/44 MAPK (lower panel). The inhibition of P42/44 MAPK phosphorylation by U0126 increased respectively with the increase in U0126 concentration.

**Cyclosporin A: A Protein Phosphatase 2B Inhibitor and Immunosuppressant**
Cyclosporin A (#C-900) inhibits the antiadipogenic effect of Ca²⁺ dependent protein phosphatase (calcineurin). 3T3-L1 preadipocytes were induced to undergo adipocyte differentiation with 1μg/ml insulin for 4 days (A, B and C ). Cells were additionally treated for the first 4 days of differentiation with 1 μM Ionomycin (#I-700) (B-D) in the presence of 10 ng/ml (C) or 50ng/ml (D) Cyclosporin A. After 10 days the cells were stained with Oil Red O and visualized with light microscopy.

Ca²⁺-dependent Signaling and the Elusive CRAC Channel

A sustained elevation of intracellular Ca²⁺ is an indispensable step for a productive T cell activation. Elevated intracellular Ca²⁺ levels need to be maintained for several hours in order to permit downstream signaling events such as NF-AT translocation to the nucleus¹⁰. This sustained Ca²⁺ elevation is accomplished by the concerted action of several receptors, kinases and ion channels.

As mentioned above, PLCγ1 catalyzes the formation of IP₃ which binds to the IP₃ receptor (IP₃R) located in the endoplasmic reticulum (ER) membrane. Activation of the IP₃R induces a brief spike in intracellular Ca²⁺concentration due to the release of Ca²⁺ from the ER stores. The continuous activation of the IP₃R depletes the ER Ca²⁺ stores, an event that triggers the opening of the Ca²⁺ release-activated-Ca²⁺ channel (CRAC) in the plasma membrane by an as yet unknown mechanism. Only the opening of the plasma membrane CRAC channel permits entry of sufficient Ca²⁺ in order to maintain the required high intracellular levels. Because Ca²⁺ entry depolarizes the cell and thus reduces the driving force for further Ca²⁺ entry, a sustained Ca²⁺ entry can only be achieved by the participation of a voltage-gated K⁺ channel. In the case of T cells, this channel is the voltage-dependent K⁺ channel K_V1.3. The K_V1.3 channel is located in the plasma membrane and opens in response to the membrane depolarization caused by Ca²⁺ entry. Opening of the channel produces an efflux of K⁺ thus restoring the negative membrane potential that will permit further Ca²⁺ entry¹¹. Another channel involved in the regulation of Ca²⁺ entry is the intermediate conductance Ca²⁺-activated K⁺ channel, K_Ca3.1 (also known as KCNN4, SK4 or IK_Ca1). This channel is structurally similar to the K_V1.3 channel but it opens in response to a rise in intracellular Ca²⁺ instead of membrane depolarization.

The pivotal role of the K⁺ channels in controlling T cell activation and proliferation was demonstrated by the use of several channel-blocking compounds such as Charybdotoxin (#STC-325), Margatoxin (#STM-325) and Stichodactyla toxin (ShK) (#STS-400) that were able to block T cell function both in vitro and in vivo^22-24. As K_V1.3 channel levels were shown to be upregulated in T cells infiltrating the brain of multiple sclerosis patients, treatment of these patients with K⁺-channel blockers could offer a very attractive therapy in the near future²⁴.

Remarkably, about 75% of the T cell activation regulated genes show a dependence on Ca²⁺ influx through the CRAC channels. The importance of Ca²⁺ entry through CRAC channels in T cell activation is highlighted by the fact that patients with a complete absence of CRAC channel activity show a phenotype of severe immunodeficiency^13,14.

Although a great deal is known regarding the biophysical, electrophysiolocical and pharmacological characteristics of the CRAC channel, the molecular identity of the channel remains elusive. Several members of the transient receptor potential (TRP) channel superfamily have been proposed to be good candidates to encode the CRAC channel including TRPC1¹⁵, TRPC3¹⁶, TRPV6¹⁷, and TRPM2¹⁸, as well as members of the voltage-gated Ca²⁺ (Ca_V) channel family^19-21.

The rise in intracellular Ca²⁺ permits the activation of the phosphatase calcineurin, among others. Calcineurin dephosphorylates NF-AT in the cytosol inducing its activation and allowing it to translocate to the nucleus¹². The importance of the calcineurin-NF-AT pathway is underscored by the fact that the widely used immunosuppressants Cyclosporin A (#C-500) and FK-506 (#F-900) target this particular interaction. Their immunosuppressive activity stems from the fact that inhibition of NF-AT activation and translocation to the nucleus results in the inhibition of the transcription of key genes involved in the immune response²⁵.

Concluding Remarks

A great deal of progress has been made in the last few years towards elucidating the molecular mechanisms involved in the control of T cell activation. The major signaling cascades are now fairly well understood and some of the complex interactions between them have been also deciphered.

What is certain is that activating a T cell is not a simple matter, but given the potentially disastrous results of an inappropriate T cell response, this may be a good thing.