Molina-Par?s C., Lythe G. (editors) Mathematical Models and Immune Cell Biology

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3 Tuning of TCR Speciﬁcity by Co-Receptors 73

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Chapter 4

T Cell Activation and Function:

Role of Signal Strength

Asma Ahmed and Dipankar Nandi

Abstract Optimal T cell function lies at the heart of an efﬁcient adaptive response.

T cell activation is a highly regulated process and it is important to ensure that acti-

vation occurs in the proper context to prevent the development of harmful conditions

such as autoimmunity and excessive inﬂammatory responses. One of the important

factors in this process is the strength of the primary activating signal which is de-

livered upon ligation of the T cell receptor (TCR) with the major histocompatibility

complex (MHC) encoded class I or class II molecules bearing the antigenic peptide.

The strength of signal (SOS), in turn, depends on several factors: the afﬁnity/avidity

of the TCR for the MHC–peptide complex, the time of engagement, antigen con-

centrations, costimulatory interactions, etc. This chapter reviews the effects of SOS

on thymocyte selection and education, T cell costimulation, proliferation, survival,

formation of T helper T

1andT

2 subsets, responses to infectious agents etc. The

role of the SOS in modulating diverse T cell responses is well appreciated. However,

further studies are required to understand the mechanisms by which SOS signals

are relayed from the TCR to downstream effectors to modulate T cell activation and

responses.

Introduction

Higher vertebrateshave complex immune systems which allow them to survive in an

environment teeming with infectious agents and also to combat internal abnormali-

ties such as tumors. Immune cells, irrespective of whether they belong to the innate

or adaptive arms, remain quiescent in a healthy individual and swing into action

only when the body is under threat, for example during infections. Usually, the cells

belonging to the innate arm, e.g. neutrophils and macrophages, are the ﬁrst to come

in contact with an invading microbe. These cells produce non-speciﬁc effectors,

D. Nandi (



)

Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

e-mail: nandi@biochem.iisc.ernet.in

C. Molina-Par´ıs and G. Lythe (eds.), Mathematical Models and Immune Cell Biology,

DOI 10.1007/978-1-4419-7725-0

 Springer Science+Business Media, LLC 2011

76 A. Ahmed and D. Nandi

for example free radicals, to contain the infection. In addition, antigen processing

cells (APC) process and present peptides derived from microbial proteins to initiate

the T cell response. Unlike the initial non-speciﬁc innate immune response, the

adaptive immune response is relatively delayed but highly antigen-speciﬁc due to

the presence of clone speciﬁc B cell receptors or T cell receptors (TCR) on B cells

and T cells respectively. The subsequent activation and expansion of speciﬁc T and

B cell clones followed by the secretion of effector molecules, for example antibod-

ies secreted by plasma cells, cytokines by CD4

T cells, granzymes and perforins

by CD8

T cells, play important roles in protecting the host. Subsequently, im-

mune responses are down modulated (involving death of activated cells) although

some memory B cells and T cells survive. Clearly, the ability to induce a speciﬁc

immune response against antigens is an important hallmark of the immune system

in higher organisms.

T cells are one of the most versatile immune cells and perform several functions

ranging from providing B cell help, killing infected/tumor cells and regulating im-

mune responses. The study of T cells becomes important because any perturbation in

their function leads to severe disorders. Hyperactive T cells are observed in several

autoimmune diseases such as multiple sclerosis, insulin dependent diabetes melli-

tus etc. On the other hand, reduced T cell function makes individuals susceptible to

pathogens and tumors as observed in patients with the acquired immunodeﬁciency

syndrome. The importance of the T cell response is highlighted by the fact that im-

munotherapy is very often targeted towards modulating T cell function, for example

suppressing the response during autoimmunity, hypersensitivity and transplantation

or boosting it for successful vaccination purposes.

The adaptive immune response mediated by T cells begins in the secondary lym-

phoid organs with the interaction of dendritic cells (DCs) bearing surface antigen

loaded MHC molecules with speciﬁc TCR bearing T cells. A productive interaction

leads to clonal expansion and differentiation of na¨ıve T cells into effector T cells.

Depending on their surface homing receptors, these effectors migrate to speciﬁc tis-

sues, under the inﬂuence of chemokine gradients, to perform their functions. There

are several outcomes downstream of T cell activation and multiple factors deter-

mine the differentiation program of na¨ıve T cells, whether they proliferate or die

and, ﬁnally, whether a productive T cell response clears the infection/tumor. One of

these factors is the strength of the activating signal delivered to the cell upon interac-

tion of the TCR with the MHC–peptide complex on APCs. The strength or potency

(weak or strong) of the signal is, primarily, determined by the afﬁnity/avidity of the

TCR–MHC interaction, the duration of the interaction, the dose of antigen available

and presence of costimulatory signals. This chapter will give a brief introduction

to T cell activation and will delve deeper into the mechanisms by which the SOS

impacts cell fate decisions in the thymus and inﬂuences T cell function and death in

the periphery.

4 T Cell Activation and Function: Role of Signal Strength 77

The T Cell Receptor

A T cell recognizes processed antigenic peptides presented on MHC class I or class

II molecules with the help of speciﬁc cell surface receptors known as the TCR. It is

important to point out that the TCR is different from the B cell receptor in that it does

not bind antigen alone but only does so in the context of self MHC molecules. This

attribute is known as self-MHC restriction, which was ﬁrst described by Doherty and

Zinkernagel [1]. The TCR is a heterodimer of two disulphide linked chains, either

˛ˇ or ı. About 95% of TCRs are a heterodimer of ˛ and ˇ chains and a small frac-

tion (2–5%) expresses the ı heterodimer. On account of having an immunoglobulin

like domain structure, TCRs are considered members of the immunoglobulin super-

family. Each ˛ and ˇ chain has an amino terminal variable (V) and a constant (C)

region much like an antibody. In addition, each chain has a transmembrane region

of 21 or 22 amino acids and a very short 5–12 amino acid long cytoplasmic tail. In

humans the ˛ and ı gene segments are located on chromosome 14 and the ˇ and 

segments on chromosome 7. The generation of functional TCR molecules is due to

rearrangement of V and J segments of the ˛ and  chains and V, D and J segments

of the ˇ and ı chains. This mechanism is similar to antibody gene rearrangements

and occurs in the thymus.

As TCRs contain a very short cytoplasmic tail, signal transduction after MHC–

TCR binding occurs via the CD3 molecule. In fact, TCRs are associated with the

CD3 complex (Fig. 4.1) consisting of ﬁve invariant chains which form two het-

erodimers (" and "ı) and one homodimer (). The cytoplasmic tails of the CD3

chains contain special immunoreceptor tyrosine-based activation motifs (ITAM)

which interact with tyrosine kinases and play important roles in signal transduc-

tion. The sequence of a typical ITAM is YXXL/IX

68

YXXL. , ı and " have single

ITAMs while  has three in a row, taking the tally up to ten ITAMs per TCR–CD3

complex.

Apart from the TCR–CD3 complex, T cells express several other important sur-

face molecules, the most vital among them being the CD4 and CD8 co-receptors,

which are also members of the immunoglobulinsuperfamily. T cells are, broadly, di-

vided into two subsets: CD4

T cells recognize antigen in context of MHC class II,

and CD8

in context of MHC class I (Fig. 4.2). CD4 is a monomer whereas CD8

is a heterodimer of ˛ and ˇ chains. These co-receptors perform two functions: ﬁrst,

their extracellular domains bind regions on the MHC molecule thus strengthening

interaction with the TCR. Second, their intracellular domains associate with the

Src family tyrosine kinase, Lck, and this association helps in signal transduction.

Mice lacking CD4 generate reduced MHC class II restricted T cells, produce less

Interleukin (IL)-2 upon activation and are unable to mount an efﬁcient antibody

response [2,3].

78 A. Ahmed and D. Nandi

Fig. 4.1 Structure of the T cell receptor–CD3 complex. Most T cells express a cell surface TCR

consisting of a heterodimer of ˛ and ˇ chains bearing variable antigen binding (blue) and constant

(pink) regions. The TCR does not transduce a signal upon antigen binding and this function is

performed by the associated CD3 complex comprising of , , ı and  chains. CD3 subunits have

long cytoplasmic tails bearing ITAMs (green) which assist in signal transduction

Signalling Pathways Leading to T Cell Activation

T cell activation is initiated when a na¨ıve circulating T cell comes in contact with

an APC expressing on its surface the cognate MHC–peptide complex. The TCR

binds to the MHC–peptide complex and a cascade of signalling events is triggered,

the end result of which is the secretion of IL-2 and clonal expansion (Fig. 4.3).

Events downstream of the binding of the TCR upon activation can be classiﬁed as

early and late. Early responses include formation of the immunological synapse, a

specialized structure at the contact point(s) between the T cell and APC, phosphory-

lation and activation of kinases and other proteins, massive remodelling of the actin

cytoskeleton, changes in cytosolic Ca

concentrations and, ﬁnally, activation of

transcription factors such as NF-AT and their translocation into the nucleus [4].

Subsequent events include transcription and translation of genes, such as IL-2,

4 T Cell Activation and Function: Role of Signal Strength 79

Fig. 4.2 Optimal T cell activation requires a combination of primary and secondary signals.

CD4

and CD8

T cells recognize antigenic peptides in context of MHC class II and MHC class I

molecules, respectively. CD4 and CD8 co-receptors stabilize the TCR–MHC peptide interaction

which sends signal 1 to the T cell. Binding of CD28 on T cells with CD80/CD86 on the APCs

or target cells delivers signal 2 which drives T cell cytokine secretion and clonal expansion. Also

important is the interaction between CD40 on APCs and CD40L on CD4

T cells for T dependent

B cell responses and isotype switching

appearance of activation markers such as the CD25 (the high afﬁnity IL-2 receptors),

DNA replication and cell division. Further down the line are events which lead to

differentiation of cells into effector and memory types and the eventual clearance of

effector cells to maintain homeostasis.

The ﬁrst signalling event downstream of the TCR is the phosphorylation of

the ITAMs by the kinases Lck and Fyn. Multiple pathways might be operating

in coupling the binding of TCR and the cognate-MHC–peptide complex to phos-

phorylation of ITAMs in CD3: First, the proximity of CD4 linked Lck to the

TCR–CD3 complex may be responsible. Second, conformational changes in the

CD3 polypeptides induced by TCR ligation may make the ITAMs more accessible

to phosphorylation. Alternately, the exclusion of Csk and other phosphatases may

80 A. Ahmed and D. Nandi

Fig. 4.3 An overview of

T cell signalling pathways

leading to transcription

of IL-2. Activation via the

TCR–CD3 complex

stimulates multiple pathways

that upregulate three major

transcription factors (shown

in red), leading to enhanced

IL-2 production in T cells

push the momentum towards phosphorylation. Phosphorylated ITAMs now serve as

docking sites for Src homology (SH)2 domain containing protein tyrosine kinases,

e.g. Zeta associated protein 70 kDa (ZAP70), spleen tyrosine kinase (Syk), and a

Tec family member, IL-2 induced tyrosine kinase (Itk) and Receptor like kinase

(Rlk) [4,5].

A dramatic increase in cytosolic Ca

due to release from ER stores is one of

the early events which takes place after ligation of the TCR–CD3 complex with the

cognate MHC–peptide complex. The trigger for release of Ca

from smooth endo-

plasmic reticulum stores is the binding of IP3 (generated by the activation of PLC)

to its receptors (Fig. 4.4). Cytosolic Ca

amounts in a resting T cell are about

0.1 mM but increase to 0.5–2 mM as soon as the smooth endoplasmic reticulum

stores are opened. However, these stores are soon exhausted and this depletion

acts as a trigger for Ca

from the extracellular milieu to ﬂow into the cell through

release activated channels (CRAC). This process known as capacitative Ca

entry ensures that cytosolic Ca

levels are maintained at higher than basal amounts,

e.g. 0.2–0.5 mM, which is required for sustained T cell activation [6].

The Immunological Synapse

The immunological synapse (IS) is a highly specialized structure which is formed

at the interface of a T cell and APC. Molecules present in the IS are the TCR,

CD4 associated with Lck, CD28, PKC and LFA-1 on the T cell side and pMHC,

4 T Cell Activation and Function: Role of Signal Strength 81

Fig. 4.4 A simpliﬁed overview of the Ca

signalling pathway. First, signals downstream of the

TCR–CD3 complex lead to generation of InsP3. Second, the InsP3 binds to its receptors on the

SER leading to release of Ca

in to the cytosol. Soon, the intracellular Ca

stores are exhausted.

Third, this depletion is sensed and conveyed by stromal interaction molecule 1 (STIM1) to the

plasma membrane CRAC channels, which now open and allow Ca

inﬂux from extrcellular en-

vironment. Fourth, sufﬁcient Ca

is now present to bind calmodulin and enable activation of

phosphatase calcineurin. Finally, calcineurin dephosphorylates cytosolic NF-AT (cNF-AT), leads

to its nuclear translocation and nNF-AT, along with other transcription factors, enhances transcrip-

tion of IL-2

CD80/CD86 and ICAM-1 on the APC side. Circulating T cells which have not

encountered antigen are symmetrical, round and non-polarized. Also, their adhe-

sion molecules are in a low activity state. However, when they migrate to inﬂamed

tissue or extravasate into secondary lymphoid tissue they become polarized, possi-

bly under the inﬂuence of chemokine gradients. These T cells now have a leading

F-actin rich lamellipodium and a myosin-II rich uropod (lagging end), migrating

with a speed of 5–10m/min. The lamellipodium is ﬁve- to tenfold more sensi-

tive to antigen than the uropod. Enhanced actin polymerization at the leading edge

also leads to the activation of integrins. The chances of adhesion and formation of

a synapse between the T cell and APC bearing the agonist MHC–peptide complex

are now increased manifold. Recognition of agonist MHC–peptide complex by the

TCR leads to elevation of Ca

, which serves as a stop signal for T cell migration.

Formation of the IS begins with a few TCR–MHC–peptide complexes and, subse-

quently, these clusters come together to form a supra-molecular complex [7].

82 A. Ahmed and D. Nandi

Costimulation

Optimal T cell activation, in addition to the signal generated by TCR–MHC–peptide

complex ligation, requires a second costimulatory signal. This interaction involves

costimulatory receptors like CD28 on the T cells and their ligands CD80/CD86 on

APCs. This is known as the two signal hypothesis [8] and is required to drive T cell

proliferation and effector functions. In addition, it prevents anergy, a state of unre-

sponsiveness during a secondary encounter with antigen [9]. In vitro experiments

performed to study anergy primarily characterized it as a condition of reduced pro-

liferation and cytokine production (mainly IL-2) upon restimulation of T cells. The

situation is slightly different in vivo where the phenomenon of un-responsiveness is

termed as adaptive tolerance and is required to prevent autoreactivity.

CD28, a 44 kDa glycoprotein homodimer, is a member of the immunoglobulin

superfamily and is constitutively expressed on T cells [10]. Structurally, it has a

leader sequence, an extracellular domain comprising of three complementarity de-

termining regions (CDRs), a transmembrane region and an intracellular domain. Its

ligands are CD80 and CD86 expressed on APCs. CD86 is constitutively expressed

on APCs but CD80, almost absent on resting cells, is upregulated during inﬂamma-

tion. The ligand binding motif of CD28 consists of MYPPY which is present in the

CDR3 region and shows high conservation in CD28 from different species. CD28

is present at the immunological synapse and functions mainly as an ampliﬁer of

TCR signals [11] and enhances recruitment and redistribution of lipid rafts [12]. It

boosts Ca

signalling and prolongs T cell–APC interactions [13]. The signalling

molecules, Itk, PI3K and PLC, assemble on the CD28 intracellular tail to form a

signalling unit [13]. Consequently, CD28 signalling leads to higher nuclear levels

of transcription factors like NF-AT and AP-1 and enhanced transcription of Il-2.

Also, CD28 signalling stabilizes IL-2 mRNA levels. All this leads to enhanced IL-2

levels and cell cycle progression in cells which receive both the ﬁrst and the second

signal [14]. CD28 prolongs T cell responses as is highlighted by the phenotype of

Cd28

=

mice which can initiate but not sustain T cell activation [15].

CD28 also enhances T cell survival by upregulating anti-apoptotic proteins such

as Bcl-2 and BclX

and inhibiting FasL expression [16]. CD28 apart from amplify-

ing TCR signals can generate its own unique signal. After it has bound to its ligand,

Tyr residues on its cytoplasmic tail get phosphorylated and lead to the recruitment

of PI3K [17]. Among other molecules that get activated downstream of CD28 are

Tec, Itk [18] and the guanine nucleotide exchange factor Vav-1 (involved in actin

polymerization) and PLC1[19]. A comparative study of expression in cells ac-

tivated with TCR alone, CD28 alone or the two together showed both quantitative

and qualitative differences in genes modulated by CD28 [11].

While CD28 is the prototypic positive costimulatory receptor, at the other end

of the spectrum is the cytotoxic T lymphocyte associated molecule 4 (CTLA4 or

CD152). Interestingly, CD28 and CTLA4 bind to the same ligands, i.e. CD80 and

CD86. CTLA4, a 33–45 kDa glycoprotein, binds to CD80 and CD86 but with a man-

ifold higher afﬁnity than CD28. Also, CTLA4 is expressed only on activated T cells,

unlike CD28. CTLA4 is a negative regulator of T cell responses as concluded from