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 of the cells and prepares them to interact with helper T

lymphocytes if the antigen is a protein (Fig. 7.6). The activated B lymphocytes may begin to synthesize more IgM

and to produce some of this IgM in a secreted form. Thus,

antigen stimulation induces the early phase of the humoral

immune response. This response is greatest when the antigen is multivalent, cross-links many antigen receptors,

and activates complement and innate immune receptors

strongly; all these features are typically seen with polysaccharides and other T-independent microbial antigens, as

discussed later, but not most soluble proteins. Therefore, by

themselves, protein antigens typically do not stimulate high

levels of B cell proliferation and differentiation. However,

protein antigens induce changes in B cells that enhance

their ability to interact with helper T lymphocytes.

B cell activation

Iga Igß

Microbial

antigen

BCR

CR2

CD19

CD81

Bound

C3d

Microbial

antigen

A B

PAMP

from

microbe

TLR

B cell activation

Fig. 7.5 Role of innate immune signals in B cell activation. Signals generated during innate immune

responses to microbes and some antigens cooperate with recognition of antigen by antigen receptors to

initiate B cell responses. A, Activation of complement by microbes leads to the binding of a complement

breakdown product, C3d, to the microbes. The B cell simultaneously recognizes a microbial antigen (by the

immunoglobulin receptor) and bound C3d by CR2 (type 2 complement receptor). CR2 is attached to a complex of proteins (CD19, CD81) that are involved in delivering activating signals to the B cell. B, Molecules

derived from microbes (so-called pathogen-associated molecular patterns [PAMPs]; see Chapter 2) may

activate Toll-like receptors (TLRs) of B cells at the same time as microbial antigens are being recognized by

the antigen receptor. BCR, B cell receptor.

144 CHAPTER 7 Humoral Immune Responses

Activated B cells endocytose protein antigen that

binds specifically to the BCR, resulting in degradation

of the antigen and display of peptides bound to class II

MHC molecules, which can be recognized by helper T

cells. Activated B cells migrate out of the follicles and

toward the anatomic compartment where helper T cells

are concentrated. Thus, the B cells are poised to interact

with and respond to helper T cells, which were derived

from naive T cells previously activated by the same antigen presented by dendritic cells.

Antigen binding to

and cross-linking

of membrane Ig

Changes

in activated

B cells

Functional

consequences

Increased survival,

proliferation

Interaction with

helper T cells

Responsiveness

to cytokines

Migration from follicle

to T cell zone

Antibody secretion

Naive B

lymphocyte

Expression of

proteins that promote

survival and cell cycling

Antigen presentation

Increased expression

of cytokine receptors

Generation of

plasma cells

Cytokines

Follicle Chemokines

Increased

expression

of CCR7

T cell

zone

Antigen

IgM

Fig. 7.6 Functional consequences of antigen receptor-mediated B cell activation. The activation of B

cells by antigen in lymphoid organs initiates the process of B cell proliferation and immunoglobulin M (IgM)

secretion and prepares the B cell for interaction with helper T cells.

CHAPTER 7 Humoral Immune Responses 145

The next section describes the interactions of helper

T cells with B lymphocytes in antibody responses to Tdependent protein antigens. Responses to T-independent

antigens are discussed at the end of the chapter.

FUNCTIONS OF HELPER T LYMPHOCYTES

IN HUMORAL IMMUNE RESPONSES

For a protein antigen to stimulate an antibody response,

B lymphocytes and helper T lymphocytes specific for

that antigen must come together in lymphoid organs

and interact in a way that stimulates B cell proliferation and differentiation. We know this process works

efficiently because protein antigens elicit antibody

responses within 3 to 7 days after antigen exposure. The

efficiency of antigen-induced T-B cell interaction raises

many questions. How do B cells and T cells specific for

epitopes of the same antigen find one another, considering that naive B and T lymphocytes specific for any

one antigen are rare, probably less than 1 in 100,000 of

all the lymphocytes in the body? How do helper T cells

specific for an antigen interact with B cells specific for

an epitope of the same antigen and not with irrelevant

B cells? What signals are delivered by helper T cells that

stimulate not only the secretion of antibody but also

the special features of the antibody response to proteins—namely, heavy-chain isotype switching and affinity maturation? As discussed next, the answers to these

questions are now well understood.

The process of T-B cell interaction and T cell–

dependent antibody responses is initiated by recognition of different epitopes of the same protein antigen by

the two cell types and occurs in a series of sequential

steps (Fig. 7.7):

• Naive CD4+ T cells are activated in the T cell zone

of a secondary lymphoid organ by antigen (in the

form of processed peptides bound to class II MHC

molecules) presented by dendritic cells, and differentiate into functional (cytokine-producing) helper

T cells.

• Naive B cells are activated in the follicles of the same

lymphoid organ by an exposed epitope on the same

protein (in its native conformation) that is transported to the follicle.

• The antigen-activated helper T cells and B cells

migrate toward one another and interact at the edges

of the follicles, where the initial antibody response

develops.

• Some of the cells migrate back into follicles to form

germinal centers, where the more specialized antibody responses are induced.

Next we describe each of these steps in detail.

Activation and Migration of Helper T Cells

and B cells

Helper T cells that have been activated by dendritic

cells migrate toward the B cell zone and interact

with antigen-stimulated B lymphocytes in parafollicular areas of the peripheral lymphoid organs (see

Fig. 7.7A).

• The initial activation of T cells requires antigen recognition and costimulation, as described in Chapter

5. The antigens that stimulate CD4+ helper T cells are

proteins derived from microbes that are internalized,

processed in late endosomes and lysosomes, and displayed as peptides bound to class II MHC molecules

of antigen-presenting cells (APCs) in the T cell–rich

zones of peripheral lymphoid tissues. T  cell activation is induced best by microbial protein antigens

and, in the case of vaccines, by protein antigens that

are administered with adjuvants, which stimulate

the expression of costimulators on APCs. The CD4+

T cells differentiate into effector cells capable of producing various cytokines and CD40 ligand, and some

of these T lymphocytes migrate toward the edges of

lymphoid follicles.

• B lymphocytes are activated by antigen in the follicles, as described above, and the activated B cells

begin to move out of the follicles toward the T cells.

The directed migration of activated B and T cells

toward one another depends on changes in the expression of certain chemokine receptors on the activated

lymphocytes. Activated T cells reduce expression of the

chemokine receptor CCR7, which recognizes chemokines

produced in T cell zones, and increase expression of the

chemokine receptor CXCR5, which binds a chemokine

produced in B cell follicles. Activated B cells undergo

precisely the opposite changes, decreasing CXCR5

and increasing CCR7 expression. As a result, antigenstimulated B and T cells migrate toward one another

and meet at the edges of lymphoid follicles or in interfollicular areas. The next step in their interaction occurs

here. Because antigen recognition is required for these

changes, the cells that move towards one another are the

ones that have been stimulated by antigen. This regulated migration is one mechanism for ensuring that rare

146 CHAPTER 7 Humoral Immune Responses

antigen-specific lymphocytes can locate one another

and interact productively during immune responses to

the antigen.

Presentation of Antigens by B Lymphocytes to

Helper T Cells

The B lymphocytes that bind protein antigens by their

membrane Ig antigen receptors endocytose these

antigens, process them in endosomal vesicles, and

display class II MHC–associated peptides for recognition by CD4+ helper T cells (Fig. 7.8). The membrane Ig

of B cells is a high-affinity receptor that enables a B cell

to specifically bind a particular antigen, even when the

extracellular concentration of the antigen is very low. In

addition, antigen bound by membrane Ig is endocytosed efficiently and is delivered to late endosomal vesicles

and lysosomes, where proteins are processed into peptides that bind to class II MHC molecules (see Chapter

3). Therefore, B lymphocytes are efficient APCs for the

antigens they specifically recognize.

Any one B cell may bind a conformational epitope of

a native protein antigen, internalize and process the protein, and display multiple peptides from that protein for T

cell recognition. Therefore, B cells recognize one epitope

of a protein antigen first, and helper T cells recognize different epitopes of the same protein later. Because B cells

efficiently internalize and process the antigen for which

they have specific receptors, and helper T cells recognize

peptides derived from the same antigen, the ensuing

interaction remains antigen specific. B cells are capable

of activating previously differentiated effector T cells but

are inefficient at initiating the responses of naive T cells.

B

Dendritic cell Antigen

Helper

T cell

Initial T-B interaction

T cell zone B cell zone

(primary follicle)

B cell

Antigen

Extrafollicular

helper T cell

Short-lived

plasma cells

Long-lived plasma cells

Follicular helper

T (Tfh) cell

Germinal center B cells

Memory B cells

Follicular

dendritic cell

Extrafollicular

focus

A

Germinal

center

reaction

Fig. 7.7 Sequence of events in helper T cell–dependent antibody responses. A, T and B lymphocytes

independently recognize the antigen in different regions of peripheral lymphoid organs and are activated.

The activated cells migrate toward one another and interact at the edges of lymphoid follicles. B, Antibodysecreting plasma cells are initially produced in the extrafollicular focus where the antigen-activated T and B

cells interact. Some of the activated B and T cells migrate back into the follicle to form the germinal center,

where the antibody response develops fully.

CHAPTER 7 Humoral Immune Responses 147

The idea that a B cell recognizes one epitope of an

intact antigen and displays different epitopes (peptides)

for recognition by helper T cells was first demonstrated

by studies using hapten-carrier conjugates. A hapten is

a small chemical that is recognized by B cells but stimulates strong antibody responses only if it is attached to a

carrier protein. In this situation, the B cell binds the hapten portion, ingests the conjugate, and displays peptides

derived from the carrier to helper T cells. The antibody

response is, of course, specific for the epitope that the

B cell recognized (the hapten in this example), and the

peptides derived from the carrier protein simply bring

helper T cells into the reaction. This concept has been

exploited to develop effective vaccines against microbial

polysaccharides (Fig. 7.9). Some bacteria have polysaccharide-rich capsules, and the polysaccharides themselves stimulate T-independent antibody responses,

which are weak in infants and young children. If the

polysaccharide is coupled to a carrier protein, however,

effective T-dependent responses are induced against the

polysaccharide because helper T cells specific for the

carrier are engaged in the response. In this situation,

the B cell recognizes the polysaccharide (equivalent to

the hapten) and the T cell recognizes peptides from the

attached protein (the carrier); the antibody response is

specific for the polysaccharide, but it is much stronger

than conventional T-independent responses because

helper T cells are “forced” to participate. Such conjugate

vaccines have been very useful for inducing protective

immunity against bacteria such as Haemophilus influenzae, especially in infants, and current vaccines against

pneumococcus are also conjugate vaccines.

Mechanisms of Helper T Cell–Mediated

Activation of B Lymphocytes

Activated helper T lymphocytes that recognize antigen presented by B cells use CD40 ligand (CD40L)

and secreted cytokines to activate the antigen-specific

B cells (Fig. 7.10). The process of helper T cell–mediated

B lymphocyte activation is analogous to the process of T

cell–mediated macrophage activation in cell-mediated

immunity (see Chapter 6, Fig. 6.6). CD40L expressed on

activated helper T cells binds to CD40 on B lymphocytes.

Engagement of CD40 generates signals in the B cells that

stimulate proliferation and the synthesis and secretion

of antibodies. At the same time, cytokines produced by

the helper T cells bind to cytokine receptors on B lymphocytes and stimulate more B cell proliferation and Ig

production. The requirement for the CD40L-CD40 interaction ensures that only T and B lymphocytes in physical

contact engage in productive interactions. As described

previously, the antigen-specific lymphocytes are the

cells that physically interact, thus ensuring that the antigen-specific B cells are the cells that receive T cell help and

are activated. The CD40L-CD40 interaction also stimulates heavy-chain isotype switching and affinity maturation, which explains why these changes typically are seen

in antibody responses to T-dependent protein antigens.

Extrafollicular and Germinal Center

Reactions

The initial T-B interaction, which occurs outside the

lymphoid follicles, results in the production of low

levels of antibodies, which may be of switched isotypes (described next) but are generally of low affinity

Microbial

protein

antigen

B cell

Receptormediated

endocytosis

of antigen

B cell recognition

of native protein

antigen

Antigen

processing and

presentation

T cell recognition

of antigen

Class II

MHC-peptide

complex

CD4+

T cell

Fig. 7.8 Antigen presentation by B lymphocytes to helper T

cells. B cells specific for a protein antigen bind and internalize

that antigen, process it, and present peptides attached to class

II major histocompatibility complex (MHC) molecules to helper

T cells. The B cells and helper T cells are specific for the same

antigen, but the B cells recognize native (conformational) epitopes, and the helper T cells recognize peptide fragments of

the antigen bound to class II MHC molecules.

148 CHAPTER 7 Humoral Immune Responses

(see  Fig. 7.7B). The plasma cells that are generated in

these extra-follicular foci are typically short-lived and

produce antibodies for a few weeks, and few memory B

cells are generated.

Many of the events in fully developed antibody

responses occur in germinal centers that are formed

in lymphoid follicles and require the participation

of a specialized type of helper T cell (Fig. 7.11). Some

of the activated helper T cells express high levels of the

chemokine receptor CXCR5, which draws these cells into

the adjacent follicles. The CD4+ T cells that migrate into

B cell–rich follicles are called follicular helper T (Tfh)

cells. The generation and function of Tfh cells are dependent on a receptor of the CD28 family called inducible

Tetanus toxoid

protein (TT) Tetanus toxoidspecific Th cell

Bacterial

capsular

polysaccharide

conjugated to

protein

Polysaccharidespecific B cell Long-lived plasma

cells and memory

B cells

Polysaccharide-specific

high affinity IgG

B cell activation

and differentiation

(germinal center reaction)

Presentation of

TT peptide to

helper T cell

Processing of

TT protein

Fig. 7.9 The principle of conjugate vaccines: the hapten-carrier concept. In order to generate strong

antibody responses against a microbial polysaccharide, the polysaccharide is coupled to a protein (in this case,

tetanus toxoid). B cells that recognize the polysaccharide ingest it and present peptides from the protein to

helper T cells, which stimulate the polysaccharide-specific B cells. Thus isotype switching, affinity maturation, and long-lived plasma cells and memory cells (all features of responses to proteins) are induced in a

response to polysaccharides. (Note that some B cells will also recognize the tetanus toxoid and antibodies

will be produced against the carrier protein, but this has no bearing on the antipolysaccharide response.) Ig,

Immunoglobulin.

CD40

B cell T cell

Cytokine

receptor

Activated

helper T cell

expresses CD40L,

secretes cytokines

B cells are

activated by

CD40 engagement,

cytokines

B cell

proliferation

and

differentiation

Cytokines

CD40

ligand

Fig. 7.10 Mechanisms of helper T cell–mediated activation of B lymphocytes. Helper T cells recognize

peptide antigens presented by B cells on the B cells. The helper T cells are activated to express CD40 ligand

(CD40L) and secrete cytokines, both of which bind to their receptors on the same B cells and activate the B cells.

CHAPTER 7 Humoral Immune Responses 149

costimulator (ICOS), which binds to its ligand expressed

on B cells and other cells. Inherited mutations in the

ICOS gene are the cause of some antibody deficiencies

(see Chapter 12). Tfh cells may secrete cytokines, such as

interferon (IFN)-?, interleukin (IL)-4, or IL-17, which are

characteristic of Th1, Th2, and Th17 subsets; the role of

these cytokines in B cell responses is described below. In

addition, most Tfh cells secrete the cytokine IL-21, which

has an important but incompletely understood role in Tfh

cell function.

A few of the activated B cells from the extrafollicular

focus migrate back into the lymphoid follicle, together

with Tfh cells, and begin to divide rapidly in response to

signals from the Tfh cells. It is estimated that these B cells

have a doubling time of approximately 6 hours, so one cell

may produce several thousand progeny within a week.

The region of the follicle containing these proliferating

B cells is the germinal center, so named because it was

once incorrectly thought that these were the sites where

new lymphocytes are generated (germinated). In the germinal center, B cells undergo extensive isotype switching and somatic mutation of Ig genes; both processes are

described below. The highest-affinity B cells are the ones

that are selected during the germinal center reaction to

differentiate into memory B cells and long-lived plasma

cells. Proliferating B cells reside in the dark zone of the

germinal center (see Fig. 7.11), while selection occurs in

the less dense light zone.

Heavy-Chain Isotype (Class) Switching

Helper T cells stimulate the progeny of IgM– and IgD–

expressing B lymphocytes to change the heavy-chain

Germinal

center

Activation of B cells

and migration

into germinal center

Somatic mutation

and affinity

maturation;

isotype switching

B cell

proliferation

Exit of high-affinity

antibody-secreting cells,

and memory B cells

Memory

B cell

Low affinity

IgG High affinity

IgG

Helper

T cell

Tfh

cell

B cell

Dark

zone

Light

zone

Follicular

dendritic

cell Fc

receptor

Long-lived plasma cell

Fig. 7.11 The germinal center reaction. B cells that have been activated by T helper cells at the edge of a

primary follicle migrate into the follicle and proliferate, forming the dark zone of the germinal center. Germinal

center B cells undergo extensive isotype switching and somatic mutation of Ig genes and migrate into the

light zone, where B cells with the highest-affinity Ig receptors are selected to survive, and they differentiate

into plasma cells or memory cells, which leave the germinal center. The right panel shows the histology of a

secondary follicle with a germinal center in a lymph node. The germinal center includes a basal dark zone and

an adjacent light zone. The mantle zone is the part of the follicle outside the germinal center.

150 CHAPTER 7 Humoral Immune Responses

isotypes (classes) of the antibodies they produce, without changing their antigen specificities (Fig. 7.12). Different antibody isotypes perform different functions, and

therefore the process of isotype switching broadens the

functional capabilities of humoral immune responses. For

example, an important defense mechanism against the

extracellular stages of most bacteria and viruses is to coat

(opsonize) these microbes with antibodies and cause them

to be phagocytosed by neutrophils and macrophages. This

reaction is best mediated by antibody classes, such as IgG1

and IgG3 (in humans), that bind to high-affinity phagocyte Fc receptors specific for the Fc portion of the ? heavy

chain (see Chapter 8). Helminths, in contrast, are too large

to be phagocytosed, and they are best eliminated by eosinophils. Therefore, defense against these parasites involves

coating them with antibodies to which eosinophils bind.

The antibody class that is able to do this is IgE, because

eosinophils have high-affinity receptors for the Fc portion

of the e heavy chain. Thus, effective host defense requires

that the immune system make different antibody isotypes

in response to different types of microbes, even though

all naive B lymphocytes specific for all these microbes

express antigen receptors of the IgM and IgD isotypes.

Another functional consequence of isotype switching is that the IgG antibodies produced are able to bind

to a specialized Fc receptor called the neonatal Fc receptor (FcRn). FcRn expressed in the placenta mediates the

transfer of maternal IgG to the fetus, providing