Cytokines

Cytokines

Antigenspecific

B cell

B

A

Extracellular

antigen

Fig. 3.17 Role of MHC-associated antigen presentation in recognition of microbial antigens by CD8+ and

CD4+ effector T cells. A, Protein antigens of microbes that live in the cytoplasm of infected cells enter the class I

MHC pathway of antigen processing. As a result, these proteins are recognized by CD8+ cytotoxic T lymphocytes, whose function is to kill infected cells. B, Protein antigens of microbes that are endocytosed from the

extracellular environment by macrophages and B lymphocytes enter the class II MHC pathway of antigen

processing. As a result, these proteins are recognized by CD4+ helper T lymphocytes, whose functions are to

activate macrophages to destroy phagocytosed microbes and activate B cells to produce antibodies against

extracellular microbes and toxins.

CHAPTER 3 Antigen Capture and Presentation to Lymphocytes 71

Finally, it should be mentioned that T cells also

recognize and react against small molecules and even

metal ions in an MHC-restricted manner. In fact,

exposure to some small molecules that are used as

therapeutic drugs and to metals such as nickel and

beryllium often leads to pathologic T cell reactions

(so-called hypersensitivity reactions; see Chapter 11).

There are several ways in which these nonpeptide antigens may be recognized by MHC-restricted CD4+ and

CD8+ T cells. Some of the chemicals are thought to

covalently modify self peptides or the MHC molecules

themselves, creating altered molecules that are recognized as foreign. Other chemicals may bind noncovalently to MHC molecules and alter the structure of the

peptide-binding cleft such that the MHC molecule can

display peptides that are not normally presented and

these peptide-MHC complexes are seen as being foreign.

This chapter began with two questions: how do rare

antigen-specific lymphocytes find antigens, and how are

the appropriate immune responses generated against

extracellular and intracellular microbes? Understanding

the biology of APCs and the role of MHC molecules in

displaying the peptides of protein antigens has provided

satisfying answers to both questions, specifically for T

cell–mediated immune responses.

FUNCTIONS OF ANTIGEN-PRESENTING

CELLS IN ADDITION TO ANTIGEN

DISPLAY

Antigen-presenting cells not only display peptides for

recognition by T cells but, in response to microbes,

also express additional signals for T cell activation.

The two-signal hypothesis of lymphocyte activation was

introduced in Chapters 1 and 2 (see Fig. 2.19), and we will

return to this concept when we discuss the responses of T

and B cells in Chapters 5 and 7. Recall that antigen is the

necessary signal 1, and for T cells, signal 2 is provided by

APCs reacting to microbes. The expression of molecules

in APCs that serve as second signals for lymphocyte activation is part of the innate immune response to different

microbial products. For example, many bacteria produce

a substance called lipopolysaccharide (LPS, endotoxin).

When the bacteria are captured by APCs for presentation

of their protein antigens, LPS acts on the same APCs,

through a TLR, and stimulates the expression of costimulators and the secretion of cytokines. The costimulators

and cytokines act in concert with antigen recognition by

the T cell to stimulate the proliferation of the T cells and

their differentiation into effector and memory cells.

ANTIGEN RECOGNITION BY B CELLS AND

OTHER LYMPHOCYTES

B lymphocytes use membrane-bound antibodies to recognize a wide variety of antigens, including proteins,

polysaccharides, lipids, and small chemicals. These antigens may be expressed on microbial surfaces (e.g., capsular or envelope antigens) or may be in soluble form (e.g.,

secreted toxins). B cells differentiate in response to antigen and other signals into cells that secrete antibodies (see

Chapter 7). The secreted antibodies enter the circulation

and mucosal fluids and bind to the antigens, leading to

their neutralization and elimination. The antigen receptors of B cells and the antibodies that are secreted usually

recognize antigens in the native conformation, with no

requirement for antigen processing or display by a specialized system. Macrophages in lymphatic sinuses and

dendritic cells adjacent to follicles may capture antigens

that enter lymph nodes and present the antigens, in intact

(unprocessed) form, to B lymphocytes in the follicles.

The B cell–rich lymphoid follicles of the lymph nodes

and spleen contain a population of cells called follicular dendritic cells (FDCs), whose function is to display

antigens to activated B cells. FDCs are not bone-marrow

derived or related to the dendritic cells that process and

present antigens to T cells. FDCs express receptors that

bind antigens coated with antibodies or complement

by-products such as C3b and C3d, with no role for MHC

molecules. The antigens displayed by FDCs are seen by specific B lymphocytes during humoral immune responses,

and they function to select B cells that bind the antigens

with high affinity. This process is discussed in Chapter 7.

Although this chapter has focused on peptide recognition by MHC-restricted CD4+ and CD8+ T cells, there

are other, smaller populations of T cells that recognize

different types of antigens. Natural killer T cells (called

NK-T cells), which are distinct from the natural killer

(NK) cells described in Chapter 2, are specific for lipids

displayed by class I–like CD1 molecules. Mucosal associated innate T cells (MAIT cells) are specific for bacterialderived vitamin B metabolites displayed by class I–like

MR1 molecules. ?d T cells recognize a wide variety of

molecules, some displayed by class I–like molecules and

others apparently requiring no specific processing or display. The functions of these cells and the significance of

their unusual specificities are poorly understood.

72 CHAPTER 3 Antigen Capture and Presentation to Lymphocytes

SUMMARY

• The induction of immune responses to the protein

antigens of microbes depends on a specialized system for capturing and displaying these antigens for

recognition by the rare naive T cells specific for any

antigen. Microbes and microbial antigens that enter

the body through epithelia are captured by dendritic cells located in the epithelia and transported to

regional lymph nodes or captured by dendritic cells

in lymph nodes and spleen. The protein antigens of

the microbes are displayed by the antigen-presenting

cells (APCs) to naive T lymphocytes that recirculate

through the lymphoid organs.

• Molecules encoded in the major histocompatibility

complex (MHC) perform the function of displaying

peptides derived from protein antigens.

• MHC genes are highly polymorphic. Their major

products are class I and class II MHC molecules,

which contain peptide-binding clefts, where the

polymorphic residues are concentrated, and invariant regions, which bind the coreceptors CD8 and

CD4, respectively.

• Proteins that are produced in the cytosol of infected

and tumor cells, or that enter the cytosol from phagosomes, are degraded by proteasomes, transported

into the endoplasmic reticulum by TAP and bind to

the clefts of newly synthesized class I MHC molecules. CD8 binds the invariant part of class I MHC

molecules, so CD8+ cytotoxic T lymphocytes can be

activated only by class I MHC–associated peptides

derived from proteosomal degradation of cytosolic

proteins.

• Proteins that are ingested by APCs from the extracellular environment are proteolytically degraded

within the vesicles of the APCs, and the peptides generated bind to the clefts of newly synthesized class II

MHC molecules. CD4 binds to class II MHC, because

of which CD4+ helper T cells can only be activated

by class II MHC–associated peptides derived mainly

from proteins degraded in vesicles, which are typically ingested extracellular proteins.

• The role of MHC molecules in antigen display

ensures that T cells only recognize cell-associated

protein antigens and that the correct type of T cell

(helper or cytotoxic) responds to the type of microbe

the T cell is best able to combat.

• Microbes activate APCs to express membrane proteins

(costimulators) and to secrete cytokines that provide

signals that function in concert with antigens to stimulate specific T cells. The requirement for these second

signals ensures that T cells respond to microbial antigens and not to harmless, nonmicrobial substances.

• B lymphocytes recognize proteins as well as nonprotein antigens, even in their native conformations.

Follicular dendritic cells display antigens to germinal

center B cells and select high-affinity B cells during

humoral immune responses.

REVIEW QUESTIONS

1. When antigens enter through the skin, in what

organs are they concentrated? What cell type(s) plays

an important role in this process of antigen capture?

2. What are MHC molecules? What are human MHC

molecules called? How were MHC molecules discovered, and what is their function?

3. What are the differences between the antigens that

are displayed by class I and class II MHC molecules?

4. Describe the sequence of events by which class I and

class II MHC molecules acquire antigens for display.

5. Which subsets of T cells recognize antigens presented by class I and class II MHC molecules? What

molecules on T cells contribute to their specificity

for either class I or class II MHC–associated peptide

antigens?

Answers to and discussion of the Review Questions are

available at Student Consult.

73

Structure of Lymphocyte Antigen

Receptors and Development

of Immune Repertoires

4

Antigen receptors serve critical roles in the maturation

of lymphocytes from progenitors and in all adaptive

immune responses. In adaptive immunity, naive lymphocytes recognize antigens to initiate responses, and

effector T cells and antibodies recognize antigens to

perform their functions.

B and T lymphocytes express different receptors that recognize antigens: membrane-bound

antibodies on B cells and T cell receptors (TCRs)

on T lymphocytes. The principal function of cellular receptors in the immune system, as in other biologic systems, is to detect external stimuli and trigger

responses of the cells on which the receptors are

expressed. To recognize a large variety of different

antigens, the antigen receptors of lymphocytes must

be able to bind to and distinguish between many,

often closely related, chemical structures. Antigen

receptors are clonally distributed, meaning that each

lymphocyte clone is specific for a distinct antigen and

has a unique receptor, different from the receptors of

all other clones. (Recall that a clone consists of a parent cell and its progeny.) The total number of distinct

lymphocyte clones is very large, and this entire collection makes up the immune repertoire. Although each

clone of B lymphocytes or T lymphocytes recognizes

a different antigen, the antigen receptors transmit

biochemical signals that are fundamentally the same

in all lymphocytes and are unrelated to specificity.

Antigen Recognition in the

Adaptive Immune System

CHAPTER OUTLINE

Antigen Receptors of Lymphocytes, 74

Antibodies, 76

Binding of Antigens to Antibodies, 78

Monoclonal Antibodies, 78

T Cell Receptors for Antigens, 80

Antigen Recognition by the T Cell Receptor, 80

Development of B and T Lymphocytes, 83

Lymphocyte Development, 84

Production of Diverse Antigen Receptors, 86

Inherited Antigen Receptor Genes, 86

Somatic Recombination and Expression of Antigen

Receptor Genes, 86

Mechanisms of V(D)J Recombination, 88

Generation of Ig and TCR Diversity, 89

Maturation and Selection of B Lymphocytes, 89

Early Steps in B Cell Maturation, 89

Role of the Pre-BCR Complex in B Cell Maturation, 91

Completion of B Cell Maturation, 92

Selection of Mature B Cells, 92

Subsets of Mature B Cells, 92

Maturation and Selection of T Lymphocytes, 92

Early Steps in T Cell Maturation, 93

Selection of Mature T Cells, 94

 Summary, 94

74 CHAPTER 4 Antigen Recognition in the Adaptive Immune System

These features of lymphocyte recognition and antigen

receptors raise the following questions:

• How do the antigen receptors of lymphocytes recognize extremely diverse antigens and transmit activating signals to the cells?

• What are the differences in the recognition properties of antigen receptors on B cells and T cells?

• How is the vast diversity of receptor structures in

the lymphocyte repertoire generated? The diversity

of antigen recognition implies the existence of many

structurally different antigen receptor proteins, more

than can be encoded in the inherited genome (germline). Therefore, special mechanisms must exist for

generating this diversity.

In this chapter, we describe the structures of the antigen receptors of B and T lymphocytes and how these

receptors recognize antigens. We also discuss how the

diversity of antigen receptors is generated during the

process of lymphocyte development, thus giving rise to

the repertoire of mature lymphocytes. The process of

antigen-induced lymphocyte activation is described in

later chapters.

ANTIGEN RECEPTORS OF LYMPHOCYTES

The antigen receptors of B and T lymphocytes have several features that are important for their functions in

adaptive immunity (Fig. 4.1). Although these receptors

have many similarities in terms of structure and mechanisms of signaling, there are fundamental differences

related to the types of antigenic structures that B cells

and T cells recognize.

• Membrane-bound antibodies, which serve as the

antigen receptors of B lymphocytes, can recognize

many types of chemical structures, while T cell antigen receptors recognize only peptides bound to major

histocompatibility complex (MHC) molecules. B

lymphocyte antigen receptors and the antibodies that

B cells secrete can recognize the shapes, or conformations, of macromolecules, including proteins, lipids,

carbohydrates, and nucleic acids, as well as simpler,

smaller chemical moieties. This broad specificity of

B cells for structurally different types of molecules in

their native form enables the humoral immune system to recognize, respond to, and eliminate diverse

microbes and toxins. In striking contrast, T cells see

only peptides displayed on antigen-presenting cells

(APCs) bound to MHC molecules. This specificity

ensures that T cells never interact with free or soluble

antigens and that they only interact with microbial or

tumor antigens present inside other cells in the body.

• Antigen receptor molecules consist of regions

(domains) involved in antigen recognition—

therefore varying between clones of lymphocytes—

and other regions required for structural integrity

and effector functions—thus relatively conserved

among all clones. The antigen-recognizing domains

of the receptors are called variable (V) regions, and

the conserved portions are the constant (C) regions.

Even within each V region, most of the sequence variation is concentrated within short stretches, which

are called hypervariable regions, or complementarity-determining regions (CDRs), because they form

the parts of the receptor that bind antigens (i.e., they

are complementary to the shapes of antigens). By

concentrating sequence variation in small regions of

the receptor, it is possible to maximize the variability

of the antigen-binding part while retaining the basic

structure of the receptors. As discussed later, special

mechanisms exist in developing lymphocytes to create genes that encode different variable regions of

antigen receptor proteins in individual clones.

• Antigen receptor chains are associated with invariant membrane proteins whose function is to deliver

intracellular signals following antigen recognition

(see Fig. 4.1). These signals, which are transmitted to

the cytosol and the nucleus, may cause a lymphocyte

to divide, to differentiate, to perform effector functions, or in certain circumstances to die. Thus, the

two functions of lymphocyte receptors for antigen—

specific antigen recognition and signal transduction—

are mediated by different polypeptides. This again

allows variability to be segregated in one set of molecules—the antigen receptors themselves—while

leaving the conserved function of signal transduction to the other invariant proteins. The set of plasma

membrane antigen receptor and signaling molecules

in B lymphocytes is called the B cell receptor (BCR)

complex, and in T lymphocytes it is called the T cell

receptor (TCR) complex. When antigens bind to

the extracellular portions of the antigen receptors of

lymphocytes, intracellular portions of the associated

signaling proteins are phosphorylated on conserved

tyrosine residues by enzymes called protein tyrosine

kinases. Phosphorylation triggers complex signaling cascades that culminate in the transcriptional

Antigen-presenting cell Membrane Ig

Igß CD3

?

TCR

MHC

Secreted

antibody

Forms of

antigens

recognized

Diversity

Antigen

recognition is

mediated by:

Signaling

functions are

mediated by:

Effector

functions are

mediated by:

Mainly peptides displayed

by MHC molecules

on APCs

Linear epitopes

Each clone has a unique

specificity; potential for

~1016 distinct specificities

TCR does not perform

effector functions

Macromolecules (proteins,

polysaccharides, lipids,

nucleic acids),

small chemicals

Conformational and

linear epitopes

Each clone has a unique

specificity; potential* for ~1011

distinct specificities

Variable (V) regions of

heavy and light chains

of membrane Ig

Proteins (Iga and Igß)

associated with

membrane Ig

Proteins (CD3 and ?)

associated with the TCR

Constant (C) regions of

secreted Ig

Variable (V) regions of

a and ß chains of the TCR

Feature

or function

Antibody

(immunoglobulin)

Iga

Antigen

Signal

transduction

Effector

functions:

complement

fixation,

phagocyte

binding

Signal

transduction

T cell

receptor (TCR)

Antigen

Fig. 4.1 Properties of antibodies and T cell antigen receptors (TCRs). Antibodies (also called immunoglobulins) may be expressed as membrane receptors or secreted proteins; TCRs only function as membrane

receptors. When immunoglobulin (Ig) or TCR molecules recognize antigens, signals are delivered to the lymphocytes by proteins associated with the antigen receptors. The antigen receptors and attached signaling

proteins form the B cell receptor (BCR) and TCR complexes. Note that single antigen receptors are shown

recognizing antigens, but signaling typically requires the binding of two or more receptors to adjacent antigen

molecules. The important characteristics of these antigen-recognizing molecules are summarized. *The total

number of possible receptors with unique binding sites is very large, but only ~107–109

 clones with distinct

specificities are present in adults. APCs, Antigen-presenting cells; Ig, immunoglobulin; MHC, major histocompatibility complex.

76 CHAPTER 4 Antigen Recognition in the Adaptive Immune System

activation of many genes and the production of

numerous proteins that mediate the responses of the

lymphocytes. We return to the processes of T and B

lymphocyte activation in Chapters 5 and 7, respectively.

• Antibodies exist in two forms—as membrane-bound

antigen receptors on B cells and as secreted proteins—but TCRs exist only as membrane receptors on

T cells. Secreted antibodies are present in the blood

and mucosal secretions, where they provide protection against microbes (i.e., they are the effector molecules of humoral immunity). Antibodies

are also called immunoglobulins (Igs), referring to

immunity-conferring proteins with the physical

characteristics of globulins. Secreted antibodies recognize microbial antigens and toxins by their variable

domains, the same as the membrane-bound antigen

receptors of B lymphocytes. The constant regions of

some secreted antibodies have the ability to bind to

other molecules that participate in the elimination of

antigens: these molecules include receptors on phagocytes and proteins of the complement system. Thus,

antibodies serve different functions at different stages

of humoral immune responses: membrane-bound

antibodies on B cells recognize antigens to initiate B

cell activation, and secreted antibodies neutralize and

eliminate microbes and their toxins in the effector

phase of humoral immunity. In cell-mediated immunity, the effector function of microbe elimination is

performed by T lymphocytes themselves and by other

leukocytes responding to the T cells. The antigen

receptors of T cells are involved only in antigen recognition and T cell activation, and these proteins are

not secreted and do not mediate effector functions.

With this introduction, we describe next the antigen receptors of lymphocytes, first antibodies and then

TCRs.

Antibodies

An antibody molecule is composed of four polypeptide chains—two identical heavy (H) chains and two

identical light (L) chains—with each chain containing

a variable region and a constant region (Fig. 4.2). The

four chains are assembled to form a Y-shaped molecule.

Each light chain is attached to one heavy chain, and the

two heavy chains are attached to each other, all by disulfide bonds. A light chain is made up of one V and one

C domain, and a heavy chain has one V and three or

four C domains. Each domain folds into a characteristic

three-dimensional shape, called the immunoglobulin

(Ig) domain (see Fig. 4.2D). An Ig domain consists of

two layers of a ß-pleated sheet held together by a disulfide bridge. The adjacent strands of each ß-sheet are

connected by short, protruding a-helical loops; in the

V regions of Ig molecules, three of these loops make

up the three CDRs responsible for antigen recognition.

Ig domains without hypervariable loops are present in

many other proteins in the immune system, as well as

outside the immune system, and most of these proteins

are involved in responding to stimuli from the environment and from other cells. All of these proteins are said

to be members of the immunoglobulin superfamily.

The antigen-binding site of an antibody is composed of the V regions of both the heavy chain and

the light chain, and the core antibody structure contains two identical antigen binding sites (see Fig. 4.2).

Each variable region of the heavy chain (called VH) or of

the light chain (called VL) contains three hypervariable

regions, or CDRs. Of these three, the greatest variability

is in CDR3, which is located at the junction of the V

and C regions. As may be predicted from this variability,

CDR3 is also the portion of the Ig molecule that contributes most to antigen binding.

Functionally distinct portions of antibody molecules

were first identified based on proteolysis, which generated fragments that were composed of different parts of

antibody proteins. The fragment of an antibody that contains a whole light chain (with its single V and C domains)

attached to the V and first C domains of a heavy chain is

required for antigen recognition and is therefore called

the Fab (fragment, antigen-binding) region. The remaining heavy-chain C domains make up the Fc (fragment,

crystalline) region; because this fragment is identical in

all antibody molecules of a particular type, it tends to

crystallize in solution. In each Ig molecule, there are two

identical Fab regions that bind antigen attached to one Fc

region that is responsible for most of the biologic activity

and effector functions of the antibodies. (As discussed

later, some types of antibodies exist as multimers of two

or five Ig molecules attached to one another.) Linking

the Fab and Fc regions of most antibody molecules is a

flexible portion called the hinge region. The hinge allows

the two antigen-binding Fab regions of each antibody

molecule to move independent of each other, enabling

them to simultaneously bind antigen epitopes that are

separated from one another by varying distances.

CHAPTER 4 Antigen Recognition in the Adaptive Immune System 77