bind to the same MHC molecules that the TCR binds

and are required for initiation of signaling from the TCR

complex. At the time when the TCR is recognizing the

peptide-MHC complex, CD4 or CD8 binds the class II or

class I MHC molecule, respectively, at a site separate from

the peptide-binding cleft. As discussed in Chapter 3, when

Fig. 5.4 Receptors and ligands involved in T cell activation and inhibition. A, Major surface molecules

of CD4+ T cells involved in the activation of these cells and their corresponding ligands on antigen-presenting

cells. CD8+ T cells use most of the same molecules, except that the TCR recognizes peptide-class I

MHC complexes, and the coreceptor is CD8, which recognizes class I MHC. CD3 is composed of three

polypeptide chains, d, e, and ?, arranged in two pairs (de and ?e); we show CD3 as three chains. Immunoreceptor tyrosine-based activation motifs (ITAMs) are the regions of cytosolic tails of signaling proteins that

are phosphorylated on tyrosine residues and become docking sites for other tyrosine kinases (see Fig. 5.10).

Immunoreceptor tyrosine-based inhibitory motifs are the regions of signaling proteins that are sites for tyrosine phosphatases that counteract actions of ITAMs. B, Important properties of major surface molecules of

T cells involved in functional responses. Cytokines and cytokine receptors are not listed here. The functions

of most of these molecules are described in this chapter; the role of CTLA-4 and PD-1 in shutting off T

cell responses is described in Chapter 9. LFA-1 is an integrin involved in leukocyte binding to endothelium

and other cells. APC, Antigen-presenting cell; ICAM-1, intercellular adhesion molecule 1; LFA-1, leukocyte

function–associated antigen 1; MHC, major histocompatibility complex; PD-1, programmed death-1; TCR,

T cell receptor.

CHAPTER 5 T Cell–Mediated Immunity 101

LFA-1

CD4

CD3

?

TCR

CD28

ICAM-1

Class II

MHC

B7-1/B7-2

CTLA-4 B7-1/B7-2

PD-1 PD-L1/PD-L2

Peptide

Signal

transduction

Receptors and signaling

molecules of CD4+ lymphocyte

Ligands of class II

MHC expressing APC

Antigen

recognition

Signal

transduction

Adhesion

ITAM

Inhibitory motif

A

B

Surface molecules

of T lymphocytes

Function Ligand

None

Signal transduction

(costimulation)

Negative

regulation

Signal transduction

by TCR complex

Antigen recognition

Antigen-presenting

cells

Antigen-presenting

cells

None

B7-1/

B7-2

B7-1/

B7-2

CD3

TCR

?

CD28

All nucleated

cells

All T cells

Signal

transduction

Class I

MHC

PeptideMHC

CD8

CTLA-4

Antigen-presenting

cells

Class II

MHC

Signal

transduction CD4

Name Expressed on

Negative

regulation

Antigen-presenting cells,

tissue cells, tumor cells

PD-L1/

PD-L2 PD-1

Antigen-presenting cells, Adhesion ICAM-1 endothelium LFA-1

102 CHAPTER 5 T Cell–Mediated Immunity

protein antigens are ingested by APCs from the extracellular milieu into vesicles, these antigens are processed into

peptides that are displayed by class II MHC molecules. In

contrast, protein antigens present in the cytosol are processed by proteasomes into peptides displayed by class I

MHC molecules. Thus, because of the specificity of the

coreceptors for different classes of MHC molecules, CD4+

and CD8+ T cells recognize peptides generated through

different protein processing pathways. The TCR and its

coreceptor need to be engaged simultaneously to initiate

the T cell response, and multiple TCRs likely need to be

triggered for T cell activation to occur. Once these conditions are achieved, the T cell begins its activation program.

The biochemical signals that lead to T cell activation are triggered by a set of proteins linked to the

TCR that are part of the TCR complex and by the

CD4 or CD8 coreceptor (see Fig. 5.5). In lymphocytes, antigen recognition and subsequent signaling are

performed by different sets of molecules. The TCR aß

heterodimer recognizes antigens, but it is not able to

transmit biochemical signals to the interior of the cell.

The TCR is noncovalently associated with a complex of

transmembrane signaling proteins including three CD3

proteins and a protein called the ? chain. The TCR, CD3,

and ? chain make up the TCR complex. Although the

a and ß TCRs must vary among T cell clones in order

to recognize diverse antigens, the signaling functions of

TCRs are the same in all clones, and therefore the CD3

and ? proteins are invariant among different T cells. The

mechanisms of signal transduction by these proteins of

the TCR complex are discussed later in the chapter.

T cells can also be activated by molecules that bind to

the TCRs of many or all clones of T cells, regardless of

the peptide-MHC specificity of the TCR. For instance,

some microbial toxins may bind to the TCRs of many

T cell clones and also bind to MHC class II molecules on

APCs without occupying the peptide-binding cleft. By

activating a large number of T cells, these toxins induce

excessive cytokine release and cause systemic inflammatory disease. They are called superantigens because, like

conventional antigens, they bind to MHC molecules and

to TCRs, but to many more than typical antigens do.

Role of Adhesion Molecules in T Cell

Responses

Adhesion molecules on T cells recognize their ligands

on APCs and stabilize the binding of the T cells to the

APCs. Most TCRs bind the peptide-MHC complexes

for which they are specific with low affinity. To induce a

response, the binding of T cells to APCs must be stabilized

for a sufficiently long period to achieve the necessary signaling threshold. This stabilization function is performed

by adhesion molecules on the T cells that bind to ligands

expressed on APCs. The most important of these adhesion molecules belong to the family of heterodimeric

(two-chain) proteins called integrins. The major T cell

integrin involved in binding to APCs is leukocyte function–associated antigen 1 (LFA-1), whose ligand on APCs

is called intercellular adhesion molecule 1 (ICAM-1).

On resting naive T cells, which are cells that have not

previously recognized and been activated by antigen,

the LFA-1 integrin is in a low-affinity state. Antigen recognition by a T cell increases the affinity of that cell’s

LFA-1. Therefore, once a T cell sees antigen, it increases

the strength of its binding to the APC presenting that

antigen, providing a positive feedback loop. Integrin-mediated adhesion is critical for the ability of T cells

APC

T cell

Class II MHC

CD3

CD4

?

TCR

ITAM

Signal

transduction

Antigen

recognition

Lck P

P

P

Fig. 5.5 Antigen recognition and signal transduction during

T cell activation. Different T cell molecules recognize antigen

and deliver biochemical signals to the interior of the cell as a

result of antigen recognition. The CD3 and ? proteins are noncovalently attached to the T cell receptor (TCR) a and ß chains

by interactions between charged amino acids in the transmembrane domains of these proteins (not shown). The figure illustrates a CD4+ T cell; the same interactions are involved in the

activation of CD8+ T cells, except that the coreceptor is CD8

and the TCR recognizes a peptide–class I MHC complex. APC,

Antigen-presenting cell; ITAM, immunoreceptor tyrosine-based

activation motifs; MHC, major histocompatibility complex.

CHAPTER 5 T Cell–Mediated Immunity 103

to bind to APCs displaying microbial antigens. Integrins

also play an important role in directing the migration

of effector T cells and other leukocytes from the circulation to sites of infection. This process is described in

Chapter 2 and later in this chapter.

Role of Costimulation in T Cell Activation

The full activation of T cells depends on the recognition of costimulators on APCs in addition to antigen

(Fig. 5.6). We have previously referred to costimulators

as second signals for T cell activation. The name costimulator derives from the fact that these molecules provide

stimuli to T cells that function together with stimulation

by antigen.

The best-defined costimulators for T cells are two

homologous proteins called B7-1 (CD80) and B7-2

(CD86), both of which are expressed on APCs and

whose expression is increased when the APCs encounter

microbes. These B7 proteins are recognized by a receptor called CD28, which is expressed on most T cells.

Different members of the B7 and CD28 families serve

to stimulate or inhibit immune responses (Fig. 5.7). The

binding of CD28 on T cells to B7 on the APCs generates

signals in the T cells that work together with signals generated by TCR recognition of antigen presented by MHC

proteins on the same APCs. CD28-mediated signaling is

essential for the responses of naive T cells; in the absence

of CD28:B7 interactions, antigen recognition by the

TCR is insufficient for initiating T cell responses. The

requirement for costimulation ensures that naive T lymphocytes are activated maximally by microbial antigens

and not by harmless foreign substances or by self antigens, because, as stated previously, microbes stimulate

the expression of B7 costimulators on APCs.

A protein called ICOS (inducible costimulator),

which is homologous to CD28 and also expressed on

Antigen recognition T cell response

"Resting"

(costimulatordeficient) APC Naive

T cell No response

or tolerance

Activated APC:

increased

expression of

costimulators,

secretion of

cytokines

Effector and

memory T cells

T cell proliferation

and differentiation

B7 CD28

CD28

IL-2

Activation of APCs by microbes,

innate immune response

Fig. 5.6 Role of costimulation in T cell activation. Resting antigen-presenting cells (APCs), which have not

been exposed to microbes or adjuvants, may present peptide antigens, but they do not express costimulators

and are unable to activate naive T cells. T cells that recognize antigen without costimulation may die or become

unresponsive (tolerant) to subsequent exposure to antigen. Microbes, as well as cytokines produced during

innate immune responses to microbes, induce the expression of costimulators, such as B7 molecules, on the

APCs. The B7 costimulators are recognized by the CD28 receptor on naive T cells, providing signal 2. In conjunction with antigen recognition (signal 1), this recognition initiates T cell responses. Activated APCs also produce cytokines that stimulate the differentiation of naive T cells into effector cells (not shown). IL, Interleukin.

104 CHAPTER 5 T Cell–Mediated Immunity

T cells, plays an important role in the development and

function of follicular helper T cells during germinal center responses (see Chapter 7).

Another set of molecules that participate in T cell

responses are CD40 ligand (CD40L, or CD154) on activated T cells and CD40 on APCs. These molecules do

not directly enhance T cell activation. Instead, CD40L

expressed on an antigen-stimulated T cell binds to

CD40 on APCs and activates the APCs to express

more B7 costimulators and to secrete cytokines (e.g.,

IL-12) that enhance T cell differentiation. Thus, the

CD40L-CD40 interaction promotes T cell activation

by making APCs better at stimulating T cells. CD40L

on effector CD4+ T cells also enhances activation of B

cells and macrophages, as discussed later.

The role of costimulation in T cell activation explains an

observation mentioned in earlier chapters. Protein antigens,

such as those used in vaccines, fail to elicit T cell–dependent

immune responses unless these antigens are administered

with substances that activate APCs, especially dendritic

cells. Such substances are called adjuvants, and they function mainly by inducing the expression of costimulators

on APCs and by stimulating the APCs to secrete cytokines

that activate T cells. Most adjuvants used in experimental

immunology are products of microbes (e.g., killed mycobacteria, which is often used in experimental studies) or

substances that mimic microbes, and they bind to pattern

recognition receptors of the innate immune system, such as

Toll-like receptors and NOD-like receptors (see Chapter 2).

Adjuvants used in human vaccines are mainly aluminum

Name CD28 CTLA-4 ICOS PD-1

Major

function

Name B7-1

(CD80)

B7-2

(CD86)

PD-L1

(B7-H1,

CD274)

PD-L2

(B7-DC,

CD273)

ICOS-L

(CD275)

Activation

(naive

T cells)

Activation

(follicular

helper T cells

in antibody

responses)

Inhibition

(mediates

suppressive

function of

regulatory

T cells)

Inhibition

Ligands on

APCs and

other cells

Receptors

on T cells

Fig. 5.7 Proteins of the B7 and CD28 families. Ligands on APCs that are homologous to B7 bind to receptors on T cells that are homologous to CD28. Different ligand-receptor pairs serve distinct roles in immune

responses. CD28 and ICOS are stimulatory receptors on T cells, and CTLA-4 and PD-1 are inhibitory receptors. Their functions are discussed in the text.

CHAPTER 5 T Cell–Mediated Immunity 105

salts that induce local inflammation, which secondarily

leads to dendritic cell costimulator expression. Thus, adjuvants trick the immune system into responding to purified

protein antigens in a vaccine as if these proteins were parts

of infectious microbes.

The increasing understanding of costimulators has led

to new strategies for inhibiting harmful immune responses.

Agents that block B7:CD28 interactions are used in the

treatment of disorders in which T cell activation causes

organ dysfunction, such as certain autoimmune diseases

and graft rejection, and antibodies that block CD40:CD40L

interactions are being tested in these diseases.

Inhibitory Receptors of T Cells

Inhibitory receptors are critical for limiting and terminating immune responses. These inhibitory receptors have been called coinhibitors to contrast them

with the costimulators discussed earlier. Two important

inhibitory receptors—CTLA-4 and PD-1—are structurally related to CD28 (see Fig. 5.7). CTLA-4, like CD28,

recognizes B7-1 and B7-2 on APCs, and PD-1 recognizes

two different but structurally related ligands, PD-L1 and

PD-L2, on many cell types. Both CTLA-4 and PD-1 are

induced in activated T cells, and function to terminate

responses of these cells. CTLA-4 also plays an important role in the suppressive function of regulatory T cells

(see Chapter 9). CTLA-4 and PD-1 prevent responses

to self antigens and are also involved in inhibiting T cell

responses to some tumors and chronic viral infections.

These discoveries are the basis for the use of antibodies that block CTLA-4 or PD-1 to enhance immune

responses to tumors in cancer patients (see Chapter 10).

Because the normal function of these inhibitory receptors is to prevent immune responses against self antigens, genetic deletion or blockade of these molecules in

mice and humans results in autoimmune disease. The

function of these inhibitory receptors is discussed in

more detail in Chapter 9, in the context of maintaining

unresponsiveness to self antigens.

Stimuli for Activation of CD8+ T Cells

The activation of CD8+ T cells is stimulated by recognition of class I MHC–associated peptides and

requires costimulation and helper T cells. CD8+ T cells

function in much the same manner to kill infected cells

and tumor cells, and their responses to microbial antigens and tumor antigens are essentially similar. However, the responses of CD8+ T cells differ in several ways

from responses of CD4+ T lymphocytes:

• The initiation of CD8+ T cell activation often requires

cytosolic antigen from one cell (e.g., virus-infected or

tumor cells) to be cross-presented by dendritic cells

(see Fig. 3.16).

• The differentiation of naive CD8+ T cells into fully

active cytotoxic T lymphocytes (CTLs) and memory cells may require the concomitant activation of

CD4+ helper T cells (Fig. 5.8). When virus-infected

or tumor cells are ingested by dendritic cells, the

APCs may present viral or tumor antigens from the

cytosol in complex with class I MHC molecules and

from vesicles in complex with class II MHC molecules. Thus, both CD8+ T cells and CD4+ T cells

APC

CD8+

T cell

Costimulator

CD4+

helper

T cell

CD4+ helper T cells

produce molecules

that stimulate

CTL differentiation Differentiated

CTLs, memory

CD8+ T cells

Fig. 5.8 Activation of CD8+ T cells. Antigen-presenting cells (APCs), principally dendritic cells, may ingest

and present microbial antigens to CD8+ T cells (cross-presentation) and to CD4+ helper T cells. Sometimes,

the APC may be infected and can directly present antigens (not shown). The helper T cells then produce

cytokines that stimulate the expansion and differentiation of the CD8+ T cells. Helper cells also may activate

APCs to make them potent stimulators of CD8+ T cells. CTLs, Cytotoxic T lymphocytes.

106 CHAPTER 5 T Cell–Mediated Immunity

specific for viral or tumor antigens are activated near

one another. The CD4+ T cells may produce cytokines or membrane molecules that help to activate

the CD8+ T cells. This requirement for helper T cells

in CD8+ T cell responses is the likely explanation for

the increased susceptibility to viral infections and

cancers in patients infected with the human immunodeficiency virus (HIV), which kills CD4+ but not

CD8+ T cells.

Now that we have described the stimuli required to

activate naive T lymphocytes, we next consider the biochemical pathways triggered by antigen recognition and

other stimuli.

BIOCHEMICAL PATHWAYS OF T CELL

ACTIVATION

Following the recognition of antigens and costimulators, T cells express proteins that are involved in

their proliferation, differentiation, and effector functions (Fig. 5.9). Naive T cells that have not encountered

antigen have a low level of protein synthesis. Within

minutes of antigen recognition, new gene transcription and protein synthesis are seen in the activated

T cells. These newly expressed proteins mediate many of

the subsequent responses of the T cells. The expression

of these proteins is a consequence of signal transduction pathways emanating from the TCR complex and

costimulatory receptors.

Antigen recognition activates several biochemical

mechanisms that lead to T cell responses, including

the activation of enzymes such as kinases, recruitment

of adaptor proteins, and production or activation of

functional transcription factors (Fig. 5.10). These biochemical pathways are initiated when TCR complexes

and the appropriate coreceptor are brought together

by binding to MHC-peptide complexes on the surface

of APCs. In addition, there is an orderly movement of

proteins in both the APC and T cell membranes at the

region of cell-to-cell contact, such that the TCR complex, CD4/CD8 coreceptors, and CD28 coalesce to the

center and the integrins move to form a peripheral ring.

This redistribution of signaling and adhesion molecules is required for optimal induction of activating

signals in the T cell. The region of contact between the

APC and T cell, including the redistributed membrane

proteins, is called the immune synapse. Although

the synapse was first described as the site of delivery

of activating signals from membrane receptors to the

cell’s interior, it may serve other functions. Some effector molecules and cytokines may be secreted through

this region, ensuring that they do not diffuse away

but are targeted to the cell in contact with the T cell.

Enzymes that degrade or inhibit signaling molecules

are also recruited to the synapse, so it may be involved

in terminating lymphocyte activation as well.

The cytoplasmic tails of the CD4 and CD8 coreceptors have a constitutively attached protein tyrosine

kinase called Lck. As discussed in Chapter 4, several

transmembrane signaling proteins are associated with

the TCR, including the CD3 and ? chains. CD3 and ?

contain motifs, each with two tyrosine residues, called

immunoreceptor tyrosine-based activation motifs

(ITAMs), which are critical for signaling. Lck, which

is brought near the TCR complex by the CD4 or CD8

molecules, phosphorylates tyrosine residues contained within the ITAMs of the CD3 and ? proteins,

and this is the event that launches signal transduction in the T cells. The importance of the coreceptors is that by binding to MHC molecules, they bring

the kinase close to its critical substrates in the TCR

complex. The phosphorylated ITAMs of the ? chain

become docking sites for a tyrosine kinase called

ZAP-70 (zeta-associated protein of 70 kD), which

also is phosphorylated by Lck and thereby made enzymatically active. The active ZAP-70 then phosphorylates various adaptor proteins and enzymes, which

assemble near the TCR complex and mediate additional signaling events.

The major signaling pathways linked to TCR complex activation are the calcium-NFAT pathway, the

Ras– and Rac–MAP kinase pathways, the PKC?–NF-?B

pathway, and the PI-3 kinase pathway:

• Nuclear factor of activated T cells (NFAT) is a

transcription factor present in an inactive phosphorylated form in the cytosol of resting T cells.

NFAT activation and its nuclear translocation

depend on the concentration of calcium (Ca2+) ions

in the cytosol. This signaling pathway is initiated by

phosphorylation and activation of an enzyme called

phospholipase C? (PLC?) by a kinase, Itk, that

becomes attached to one of the adaptor proteins

in the signaling complex. Activated PLC? catalyzes

the hydrolysis of a plasma membrane phospholipid called phosphatidylinositol 4,5-bisphosphate

CHAPTER 5 T Cell–Mediated Immunity 107

Maximum level (percent)

Hours Days

Time after activation

B

A

Naive

T cell

TCR CD69

Retention in

lymph node Proliferation Effector

functions

Control of

response

IL-2R CD40L CTLA-4

100

75

50

25

0

1 2 3 4 5

c-Fos CD40

ligand

IL-2

CD69

IL-2Ra

(CD25) Cell

division

1 2 3 4 5 6 12

Fig. 5.9 Proteins produced by antigen-stimulated T cells. Antigen recognition by T cells results in the

synthesis and expression of a variety of proteins, examples of which are shown. The kinetics of production of these proteins (A) are approximations and may vary in different T cells and with different types of

stimuli. The possible effects of costimulation on the patterns or kinetics of gene expression are not shown.

The functions of some of the surface proteins expressed on activated T cells are shown in (B). CD69 is a

marker of T cell activation involved in cell migration; the interleukin-2 receptor (IL-2R) receives signals from

the cytokine IL-2 that promotes T cell survival and proliferation; CD40 ligand is an effector molecule of T

cells; CTLA-4 is an inhibitor of immune responses. c-Fos (shown in A) is a transcription factor. TCR, T cell

receptor.

(PIP2). One by-product of PLC?-mediated PIP2

breakdown, called inositol 1,4,5-triphosphate (IP3),

binds to IP3 receptors on the endoplasmic reticulum

(ER) membrane and the mitochondria and initiates

release of Ca2+ into the cytosol. In response to the

loss of calcium from intracellular stores, a plasma

membrane calcium channel is opened, leading to

the influx of extracellular Ca2+ into the cell, which

further increases the cytosolic Ca2+ concentration

and sustains this for hours. The elevated cytosolic

Ca2+ leads to activation of a phosphatase called calcineurin. This enzyme removes phosphates from

cytoplasmic NFAT, enabling the transcription factor

to migrate into the nucleus, where it binds to and

activates the promoters of several genes, including

the genes encoding the T cell growth factor IL-2

108 CHAPTER 5 T Cell–Mediated Immunity

and components of the IL-2 receptor. Calcineurin

inhibitors (cyclosporine and tacrolimus) are drugs

that block the phosphatase activity of calcineurin,

and thus suppress the NFAT-dependent production

of cytokines by T cells. These drugs are widely used

as immunosuppressants to prevent graft rejection

and other T cell–mediated inflammatory conditions

(see Chapter 10).

• The Ras/Rac–MAP kinase pathways include the

guanosine triphosphate (GTP)-binding Ras and Rac

proteins, several adaptor proteins, and a cascade of

enzymes that eventually activate one of a family of

CD4/

CD8

T cell

Adapter

proteins

Initiation of

TCR-mediated

signals

Active

enzymes

Transcription

factors

Biochemical

intermediates

ZAP-70

PLC?1

activation

GTP/GDP

exchange on

Ras, Rac

Diacylglycerol

(DAG)

Increased

cytosolic Ca2+

Calcineurin PKC ERK, JNK

ITAM

PLC?1

PI3-Kinase

ITK

GDP/GTP

exchange

factor

CD3

?

Activation of

PI3-Kinase

PIP3

Akt,

mTOR

P

P

P

P

P

P

P

P

P

P

P

P

Ras•GTP,

Rac•GTP

NFAT NF-?B AP-1

Lck

 Protein

synthesis

APC

Fig. 5.10 Signal transduction pathways in T lymphocytes. Antigen recognition by T cells induces early

signaling events, which include tyrosine phosphorylation of molecules of the T cell receptor (TCR) complex

and the recruitment of adaptor proteins to the site of T cell antigen recognition. These early events lead to

the activation of several biochemical intermediates, which in turn activate transcription factors that stimulate

transcription of genes whose products mediate the responses of the T cells. The possible effects of costimulation on these signaling pathways are not shown. These signaling pathways are illustrated as independent of

one another, for simplicity, but may be interconnected in more complex networks. AP-1, Activating protein 1;

APC, antigen-presenting cell; GTP/GDP, guanosine triphosphate/diphosphate; ITAM, immunoreceptor tyrosine-based activation motif; mTOR, mammalian target of rapamycin; NFAT, nuclear factor of activated T cells;

PKC, protein kinase C; PLC?1, ?1 isoform of phosphatidylinositol-specific phospholipase C; PI-3, phosphatidylinositol-3; ZAP-70, zeta-associated protein of 70 kD.

CHAPTER 5 T Cell–Mediated Immunity 109

mitogen-activated protein (MAP) kinases. These

pathways are initiated by ZAP-70–dependent phosphorylation and accumulation of adaptor proteins at

the plasma membrane, leading to the recruitment of

Ras or Rac, and their activation by exchange of bound

guanosine diphosphate (GDP) with GTP. Ras•GTP

and Rac•GTP, the active forms of these proteins, initiate different enzyme cascades, leading to the activation of distinct MAP kinases. The terminal MAP

kinases in these pathways, called extracellular signal–

regulated kinase (ERK) and c-Jun amino-terminal

(N-terminal) kinase (JNK), respectively, induce the

expression of a protein called c-Fos and the phosphorylation of another protein called c-Jun. c-Fos

and phosphorylated c-Jun combine to form the transcription factor activating protein 1 (AP-1), which

enhances the transcription of several T cell genes.

• Another major pathway involved in TCR signaling consists of activation of the ? isoform of the serine-threonine kinase called protein kinase C (PKC?),

which leads to activation of the transcription factor

NF-?B. PKC is activated by diacylglycerol, which, like

IP3, is generated by PLC-mediated hydrolysis of membrane inositol lipids. PKC? acts through adaptor proteins recruited to the TCR complex to activate NF-?B.

• TCR signal transduction also involves a lipid kinase

called PI-3 kinase, which phosphorylates the membrane phospholipid PIP2 to generate phosphatidyl

inositol (3,4,5)-trisphosphate (PIP3). PIP3 is required

for the activation of a number of targets, including a

serine-threonine kinase called Akt, or protein kinase B,

which has many roles, including stimulating expression

of antiapoptotic proteins and thus promoting survival of

antigen-stimulated T cells. The PI-3 kinase/Akt pathway

is triggered not only by the TCR but also by CD28 and

IL-2 receptors. Akt activates mTOR (mammalian target

of rapamycin), a serine-threonine kinase that is involved

in stimulating protein translation and promoting cell

survival and growth. Rapamycin, a drug that binds to

and inactivates mTOR, is used to treat graft rejection.

The various transcription factors that are induced or

activated in T cells, including NFAT, AP-1, and NF-?B,

stimulate transcription and subsequent production of

cytokines, cytokine receptors, cell cycle inducers, and

effector molecules such as CD40L (see Fig. 5.9). All

of these signals are initiated by antigen recognition,

because binding of the TCR and coreceptors to peptide-MHC complexes is necessary to bring together critical enzymes and substrates in T cells.

As stated earlier, recognition of costimulators, such as

B7 molecules, by their receptor CD28 is essential for full

T cell responses. The biochemical signals transduced by

CD28 on binding to B7 costimulators are less well defined

than are TCR-triggered signals. CD28 engagement likely

amplifies some TCR signaling pathways that are triggered

by antigen recognition (signal 1) and may induce other

signals that complement TCR signals.

Lymphocyte activation is associated with a profound change in cellular metabolism. In naive (resting)

T cells, low levels of glucose are taken up and used to generate energy in the form of adenosine triphosphate (ATP)

by mitochondrial oxidative phosphorylation. Upon activation, glucose uptake increases markedly, and the cells

switch to aerobic glycolysis. This process generates less

ATP but facilitates the synthesis of more amino acids,

lipids, and other molecules that provide building blocks

for organelles and for producing new cells. As a result, it

is possible for activated T cells to more efficiently manufacture the cellular constituents that are needed for their

rapid increase in size and for producing daughter cells.

Having described the stimuli and biochemical pathways in T cell activation, we now discuss how T cells

respond to antigens and differentiate into effector cells

capable of combating microbes.

FUNCTIONAL RESPONSES OF

T LYMPHOCYTES TO ANTIGEN

AND COSTIMULATION

The recognition of antigen and costimulators by T cells initiates an orchestrated set of responses that culminate in the

expansion of the antigen-specific clones of lymphocytes

and the differentiation of the naive T cells into effector cells

and memory cells (see Fig. 5.3). Many of the responses of

T cells are mediated by cytokines that are secreted by the T

cells and act on the T cells themselves and on many other

cells involved in immune defenses. Each component of the

biologic responses of T cells is discussed next.

Secretion of Cytokines and Expression of

Cytokine Receptors

In response to antigen and costimulators, T lymphocytes, especially CD4+ T cells, rapidly secrete the

cytokine IL-2. We have already discussed cytokines in

innate immune responses, which are produced mainly

by dendritic cells and macrophages (see Chapter 2). In

adaptive immunity, cytokines are secreted by T cells,

mainly CD4+ cells. Because most of these cytokines are

110 CHAPTER 5 T Cell–Mediated Immunity

produced by effector T cells and serve diverse roles in

host defense, we describe them in Chapter 6 when we

discuss the effector mechanisms of cell-mediated immunity.

IL-2 is produced within 1 to 2 hours after activation

of CD4+ T cells. Activation also transiently increases the

expression of the high-affinity IL-2 receptor, thus rapidly

enhancing the ability of the T cells to bind and respond

to IL-2 (Fig. 5.11). The receptor for IL-2 is a three-chain

molecule. Naive T cells express two signaling chains,

ß and ?, which constitute the low-affinity receptor for

IL-2, but these cells do not express the a chain (CD25)

that enables the receptor to bind IL-2 with high affinity.

Within hours after activation by antigens and costimulators, the T cells produce the a chain of the receptor,

and now the complete IL-2 receptor is able to bind IL-2

strongly. Thus, IL-2 produced by antigen-stimulated T

cells preferentially binds to and acts on the same T cells,

an example of autocrine cytokine action.

The principal functions of IL-2 are to stimulate the

survival and proliferation of T cells, resulting in an

increase in the number of the antigen-specific T cells;

because of these actions, IL-2 was originally called T cell

growth factor. The high-affinity IL-2 receptor is constitutively expressed in regulatory T cells, so these cells are very

sensitive to IL-2. In fact, IL-2 is essential for the maintenance of regulatory T cells and thus for controlling immune

responses, as we discuss in Chapter 9. Activated CD8+ T

cells and natural killer (NK) cells express the low-affinity

ß? receptor and respond to higher concentrations of IL-2.

Clonal Expansion

T lymphocytes activated by antigen and costimulation begin to proliferate within 1 or 2 days, resulting

T cell activation

by antigen

+ costimulator

Secretion of IL-2

Expression of

IL-2Ra chain;

formation of

high-affinity

IL-2Raß? complex

IL-2–induced

T cell

proliferation

IL-2Rß?c

complex IL-2Rß?c

IL-2Raß?c

complex IL-2Raß?c

Low-affinity

IL-2R

(Kd ~ 10-9 M)

High-affinity

IL-2R

(Kd ~ 10-11 M)

APC

Costimulator

(B7) CD28

Resting

(naive)

T cell

IL-2

Fig. 5.11 Role of interleukin-2 and IL-2 receptors in T cell proliferation. Naive T cells express the lowaffinity IL-2 receptor (IL-2R) complex, made up of the ß and ?c chains (?c designates common ? chain, so

called because it is a component of receptors for several cytokines). On activation by antigen recognition and

costimulation, the cells produce IL-2 and express the a chain of the IL-2R (CD25), which associates with the

ß and ?c chains to form the high-affinity IL-2 receptor. Binding of IL-2 to its receptor initiates proliferation of

the T cells that recognized the antigen. APC, Antigen-presenting cell.

CHAPTER 5 T Cell–Mediated Immunity 111

in expansion of antigen-specific clones (Fig. 5.12).

This expansion quickly provides a large pool of antigenspecific lymphocytes from which effector cells can be

generated to combat infection.

The magnitude of clonal expansion is remarkable,

especially for CD8+ T cells. Before infection, the frequency of CD8+ T cells specific for any one microbial

protein antigen is approximately 1 in 105 or 1 in 106

lymphocytes in the body. At the peak of some viral

infections, possibly within a week after the infection, as

many as 10% to 20% of all the lymphocytes in the lymphoid organs may be specific for that virus. This means

that the numbers of cells in antigen-specific clones have

increased by more than 10,000-fold, with an estimated

doubling time of approximately 6 hours. This enormous

expansion of T cells specific for a microbe is not accompanied by a detectable increase in bystander cells that do

not recognize that microbe.

The expansion of CD4+ T cells appears to be 100-

fold to 1000-fold less than that of CD8+ cells. This difference may reflect differences in the functions of the

two types of T cells. CD8+ CTLs are effector cells that

kill infected and tumor cells by direct contact, and many

CTLs may be needed to kill large numbers of infected or

tumor cells. By contrast, each CD4+ effector cell secretes

cytokines that activate many other effector cells, so a

relatively small number of cytokine producers may be

sufficient.

Differentiation of Naive T Cells into Effector

Cells

Some of the progeny of antigen-stimulated, proliferating T cells differentiate into effector cells

whose function is to eradicate infections and some

cancers. This process of differentiation is the result

of changes in gene expression, such as the activation

of genes encoding cytokines (in CD4+ T cells) or

cytotoxic proteins (in CD8+ CTLs). It begins in concert with clonal expansion, and differentiated effector cells appear within 3 or 4 days after exposure to

microbes. Effector cells of the CD4+ lineage acquire

the capacity to produce different sets of cytokines.

The subsets of T cells that are distinguished by their

cytokine profiles are named Th1, Th2, and Th17 (Fig.

5.13). Many of these cells leave the peripheral lymphoid organs and migrate to sites of infection, where

their cytokines recruit other leukocytes that destroy

the infectious agents. The development and functions

Contraction

(homeostasis)

CD8+

T cells

CD4+

T cells

Infection Memory

106

104

102

7 14 200

Days after infection

Number of microbe-specific T cells

Clonal

expansion

Fig. 5.12 Expansion and decline of T cell responses. The numbers of CD4+ and CD8+ T cells specific

for various antigens in inbred mice and the clonal expansion and contraction during immune responses are

illustrated. The numbers are approximations based on studies of model microbial and other antigens in inbred

mice; in humans, the numbers of lymphocytes are approximately 1000-fold greater.

112 CHAPTER 5 T Cell–Mediated Immunity

of these effector cells are described in Chapter 6, when

we discuss cell-mediated immunity. Other differentiated CD4+ T cells remain in the lymphoid organs and

migrate into lymphoid follicles, where they further

differentiate into T follicular helper cells and help B

lymphocytes to produce antibodies (see Chapter 7).

As we discuss in Chapters 6 and 7, CD4+ helper T

cells activate phagocytes and B lymphocytes through

the actions of the plasma membrane protein CD40L

and secreted cytokines. In addition, the interaction

of CD40L on T cells with CD40 on dendritic cells

increases the expression of costimulators on these

APCs and the production of T cell–stimulating cytokines, thus providing a positive feedback (amplification) mechanism for APC-induced T cell activation.

Effector cells of the CD8+ lineage acquire the ability

to kill infected and tumor cells; their development

and function are also described in Chapter 6.

Development of Memory T Lymphocytes

A fraction of antigen-activated T lymphocytes differentiates into long-lived memory cells. These cells are a

pool of lymphocytes that are induced by microbes and

are ready to respond rapidly if the microbe returns. We

Effector T cells and

antibodies enter circulation

and go to sites of infection

Tfh cells remain

in lymphoid organ,

migrate into follicles

Naive CD4+

T cell

Lymph node

Help B cells

to produce

high-affinity

antibodies

Elimination

of microbes

Th1

cell

Th2

cell

Th17

cell

B cell

Microbe

Infected tissue

Follicular

helper T cells

(Tfh)

Fig. 5.13 Development of effector CD4+ T cells. When naive CD4+ T cells are activated in secondary lymphoid organs, they proliferate and differentiate into effector cells. Some of the effectors (the Th1, Th2, and

Th17 populations) mostly exit the lymphoid organ and function to eradicate microbes in peripheral tissues.

Other differentiated cells, called follicular helper T (Tfh) cells, remain in the lymphoid organ and help B cells

to produce potent antibodies.

CHAPTER 5 T Cell–Mediated Immunity 113

do not know what factors determine whether the progeny of antigen-stimulated lymphocytes will differentiate

into effector cells or memory cells. Memory cells have

several important characteristics.

• Memory cells survive even after the infection is eradicated and antigen is no longer present. Certain cytokines, including IL-7 and IL-15, which are produced

by stromal cells in tissues, may serve to keep memory

cells alive and cycling slowly.

• Memory T cells may be rapidly induced to produce

cytokines or kill infected cells on encountering

the antigen that they recognize. These cells do not

perform any effector functions until they encounter antigen, but once activated, they respond much

more vigorously and rapidly than do naive lymphocytes.

• Memory T cells can be found in lymphoid organs,

in various peripheral tissues, especially mucosa

and skin, and in the circulation. They can be distinguished from naive and effector cells by several criteria (see Chapter 1). A subset of memory T cells,

called central memory cells, populate lymphoid

organs and are responsible for rapid clonal expansion after reexposure to antigen. Another subset,

called effector memory cells, localize in mucosal and

other peripheral tissues and mediate rapid effector

functions on reintroduction of antigen to these sites.

A third subset, called tissue-resident memory cells,

reside in the skin and mucosal tissues and may be

incapable of entering the circulation. They mediate

rapid secondary responses to antigens encountered

in tissues.

Memory T cells can likely be activated in lymphoid

and nonlymphoid tissues, and their activation, unlike

that of naive T cells, does not require high levels of

costimulation or antigen presentation by dendritic cells.

In fact, various APCs, including B cells, may be capable

of activating memory T cells.

MIGRATION OF T LYMPHOCYTES IN

CELL-MEDIATED IMMUNE REACTIONS

As we discussed at the beginning of this chapter, T cell

responses are initiated primarily in secondary lymphoid

organs, and the effector phase occurs mainly in peripheral tissue sites of infection (see Fig. 5.2). Thus, T cells

at different stages of their lives have to migrate in

different ways:

• Naive T cells must migrate between blood and secondary (peripheral) lymphoid organs throughout the

body, until they encounter dendritic cells within the

lymphoid organ that display the antigens the T cells

recognize (see Chapter 3).

• After the naive T cells are activated and differentiate into effector cells, these cells must migrate back

to the sites of infection, where they function to kill

microbes.

The migration of naive and effector T cells is controlled

by three families of proteins—selectins, integrins, and

chemokines—that regulate the migration of all leukocytes, as described in Chapter 2 (see Fig. 2.16). The routes

of migration of naive and effector T cells differ significantly because of selective expression of different adhesion molecules and chemokine receptors on naive T cells

versus effector T cells, together with the selective expression of endothelial adhesion molecules and chemokines

in lymphoid tissues and sites of inflammation (Fig. 5.14).

Naive T cells express the adhesion molecule

L-selectin (CD62L) and the chemokine receptor

CCR7, which mediate the selective migration of the

naive cells into lymph nodes through specialized

blood vessels called high endothelial venules (HEVs)

(see Fig. 5.14). HEVs are located in the T cell zones of

lymphoid tissues and are lined by specialized endothelial cells, which express carbohydrate ligands that bind

to L-selectin. HEVs also display chemokines that are

made only in lymphoid tissues and are specifically recognized by CCR7. The migration of naive T cells proceeds in a multistep sequence like that of migration of all

leukocytes through blood vessels (see Chapter 2):

• Naive T cells in the blood engage in L-selectin–

mediated rolling interactions with the HEV, allowing

chemokines to bind to CCR7 on the T cells.

• CCR7 transduces intracellular signals that activate

the integrin LFA-1 on the naive T cell, increasing the

binding affinity of the integrin.

• The increased affinity of the integrin for its ligand,

ICAM-1, on the HEV results in firm adhesion and

arrest of the rolling T cells.

• The T cells then exit the vessel through the endothelial junctions and are retained in the T cell zone of the

lymph node because of the chemokines produced there.

Thus, many naive T cells that are carried by the blood

into an HEV migrate to the T cell zone of the lymph node

stroma. This happens constantly in all lymph nodes and

mucosal lymphoid tissues in the body. Effector T cells

114 CHAPTER 5 T Cell–Mediated Immunity

Blood

vessel

L-selectin

L-selectin

ligand

ICAM-1

L-selectin

ligand

CCL19 or CCL21

Integrin (LFA-1

or VLA-4)

Integrin (LFA-1)

LFA-1 (ß2-integrin) or

VLA-4 (ß1 integrin)

E- or Pselectin

ligand

E- and Pselectin ligand

E- or Pselectin

CXCR3

CXCL10,

others

E- or Pselectin

L-selectin

CCR7

ICAM-1

or VCAM-1

ICAM-1 or VCAM-1

T cell

homing receptor

Ligand on

endothelial cell

Function of receptor:

ligand pair

Naive T cells

Activated (effector

and memory) T cells

Adhesion of naive T cells to

high endothelial venule (HEV)

in lymph node

Activation of integrins and

chemotaxis

CXCR3, others CXCL10, others Activation of integrins and

chemotaxis

Initial weak adhesion of effector and

memory T cells to cytokine-activated

endothelium at peripheral site

of infection

Stable arrest on cytokine-activated

endothelium at peripheral

site of infection

LFA-1 (ß2-integrin) ICAM-1 Stable arrest on HEV

Peripheral

venule

A

B

CCL19/

CCL21

CCR7

Artery

Activated

T cell

Naive T cell

S1PR1

S1P

High endothelial

venule in lymph node

Endothelium

at the site of infection

Lymph node Peripheral tissue

Efferent

lymphatic

vessel

CHAPTER 5 T Cell–Mediated Immunity 115

Fig. 5.14 Migration of naive and effector T lymphocytes. A, Naive T lymphocytes home to lymph nodes as a

result of L-selectin, integrin, and chemokine receptor CCR7 binding to their ligands on high endothelial venules

(HEVs). Chemokines expressed in lymph nodes bind to CCR7 on naive T cells, enhancing integrin-dependent

adhesion and migration through the HEV. The phospholipid, sphingosine 1-phosphate (S1P), plays a role in the

exit of T cells from lymph nodes, by binding to its receptor, called S1PR1 (type 1 sphingosine 1-phosphate

receptor). Activated T lymphocytes, including the majority of effector cells, home to sites of infection in peripheral tissues, and this migration is mediated by E-selectin and P-selectin, integrins, and chemokines secreted at

inflammatory sites. Follicular helper T (Tfh) cells (not shown) are effector cells that remain in lymphoid organs,

because they express a chemokine receptor (CXCR5) that draws them into lymphoid follicles, where they can

interact with resident B lymphocytes. B, This table summarizes the functions of the principal T cell homing

receptors and chemokine receptors and their ligands. ICAM-1, Intercellular adhesion molecule 1; LFA-1, leukocyte function–associated antigen 1; VCAM-1, vascular cell adhesion molecule 1; VLA-4, very late antigen 4.

do not express CCR7 or L-selectin, and thus they are not

drawn into lymph nodes.

The phospholipid sphingosine 1-phosphate (S1P)

plays a key role in the egress of T cells from lymph

nodes. The levels of S1P are higher in the blood and lymph

than inside lymph nodes. S1P binds to and induces internalization of its receptor, which keeps the expression of the

receptor on circulating naive T cells low. When a naive T

cell enters the node, it is exposed to lower concentrations

of S1P, and expression of the receptor begins to increase. If

the T cell does not recognize any antigen, the cell leaves the

node through efferent lymphatic vessels, following the gradient of S1P into the lymph. If the T cell does encounter specific antigen and is activated, the surface expression of the

S1P receptor is suppressed for several days by CD69, which

is transiently expressed following T cell activation. As a

result, recently activated T cells stay in the lymph node long

enough to undergo clonal expansion and differentiation.

When this process is completed, S1P receptor is reexpressed

on the cell surface; at the same time, the cells lose expression of L-selectin and CCR7, which previously attracted

the naive T cells to the lymph nodes. Therefore, activated

T cells are drawn out of the nodes into the draining lymph,

which then transports the cells to the circulation. The net

result of these changes is that differentiated effector T

cells leave the lymph nodes and enter the circulation. The

importance of the S1P pathway has been highlighted by the

development of a drug (fingolimod) that binds to the S1P

receptor and blocks the exit of T cells from lymph nodes.

This drug is approved for the treatment of the inflammatory disease multiple sclerosis.

Effector T cells migrate to sites of infection because

they express adhesion molecules and chemokine

receptors that bind to ligands expressed or displayed

on vascular endothelium at sites of infection. The

process of differentiation of naive T lymphocytes into

effector cells is accompanied by changes in the types of

adhesion molecules and chemokine receptors expressed

on these cells (see Fig. 5.14). The migration of activated

T cells into peripheral tissues is controlled by the same

kinds of interactions involved in the migration of other

leukocytes into tissues (see Chapter 2):

• Activated T cells express high levels of the glycoprotein

ligands for E- and P-selectins and the integrins LFA-1

and VLA-4 (very late antigen 4). Innate immune cytokines produced in response to the infection, such as TNF

and IL-1, act on the endothelial cells to increase expression of E- and P-selectins, as well as ligands for integrins,

especially ICAM-1 and vascular cell adhesion molecule

1 (VCAM-1), the ligand for the VLA-4 integrin.

• Effector T cells that are passing through the blood

vessels at the infection site bind first to the endothelial selectins, leading to rolling interactions.

• Effector T cells also express receptors for chemokines

that are produced by macrophages and endothelial

cells at these inflammatory sites and are displayed

on the surface of the endothelium. The rolling T cells

bind these chemokines, leading to increased affinity

of the integrins for their ligands and firm adhesion of

the T cells to the endothelium.

• After the effector T lymphocytes are arrested on the

endothelium, they engage other adhesion molecules

at the junctions between endothelial cells, crawling

through these junctions into the tissue. Chemokines that

were produced by macrophages and other cells in the tissues stimulate the motility of the transmigrating T cells.