Table 9-1. Features of Primary and Secondary Antibody Responses
Feature
Primary response
Secondary response
Time lag after
immunization
Usually 5-10 days
Usually 1-3 days
Peak response
Smaller
Larger
Antibody isotype
Usually IgM > IgG
Relative increase in IgG and, under
certain situations, in IgA or IgE
Antibody affinity
Lower average affinity, more variable
Higher average affinity (affinity
maturation)
Induced by
All immunogens
Only protein antigens
Required
immunization
Relatively high doses of antigens, optimally with Low doses of antigens; adjuvants may
adjuvants (for protein antigens)
not be necessary
BOX 9-1 Assays for B Lymphocyte Activation
It is technically difficult to study the effects of antigens on normal B cells
because, as the clonal selection hypothesis predicted, very few lymphocytes in
an individual are specific for any one antigen. To examine the effects of antigen
binding to B cells, investigators have attempted to isolate antigen-specific B
cells from complex populations of normal lymphocytes or to produce cloned B
cell lines with defined antigenic specificities. These efforts have met with little
success. However, transgenic mice have been developed in which virtually all
B cells express a transgenic Ig of known specificity, so that most of the B cells
in these mice respond to the same antigen. Another approach to circumventing
this problem is to use anti-Ig antibodies as analogues of antigens, with the
assumption that anti-Ig will bind to constant (C) regions of membrane Ig
molecules on all B cells and will have the same biologic effects as an antigen
that binds to the hypervariable regions of membrane Ig molecules on only the
antigen-specific B cells. To the extent that precise comparisons are feasible,
this assumption appears generally correct, indicating that anti-Ig antibody is a
valid model for antigens. Thus, anti-Ig antibody is frequently used as a
polyclonal activator of B lymphocytes. The same concept underlies the use of
antibodies against framework determinants of TCRs or against receptorassociated CD3 molecules as polyclonal activators of T lymphocytes (see
Chapter 8).
Much of our knowledge of B cell activation is based on in vitro experiments, in
which different stimuli are used to activate B cells and their proliferation and
differentiation can be measured accurately. The same assays may be done
with B cells recovered from mice exposed to different antigens or with
homogeneous B cells expressing transgene-encoded antigen receptors. The
most frequently used assays for B cell responses are the following.
ASSAYS FOR B CELL PROLIFERATION The proliferation of B lymphocytes,
like that of other cells, is measured in vitro by determining the amount of 3Hlabeled thymidine incorporated into the replicating DNA of cultured cells.
Thymidine incorporation provides a quantitative measure of the rate of DNA
synthesis, which is usually directly proportional to the rate of cell division.
Cellular proliferation in vivo can be measured by injecting the thymidine
analogue bromodeoxyuridine (BrdU) into animals and staining cells with antiBrdU antibody to identify and enumerate nuclei that have incorporated BrdU
into their DNA during DNA replication. The same assays may be used to
measure the proliferation of T cells (see Box 8-1).
ASSAYS FOR ANTIBODY PRODUCTION Antibody production is measured in
two different ways: with assays for cumulative Ig secretion, which measure the
amount of Ig that accumulates in the supernatant of cultured lymphocytes or in
the serum of an immunized individual; and with single-cell assays, which
determine the number of cells in an immune population that secrete Ig of a
particular specificity or isotype. The most accurate, quantitative, and widely
used techniques for measuring the total amount of Ig in a culture supernatant
or serum sample are radioimmunoassay (RIA) and enzyme-linked
immunosorbent assay (ELISA), described in Appendix III. By use of antigens
bound to solid supports, it is possible to use RIA or ELISA to quantitate the
amount of a specific antibody in a sample. In addition, the availability of anti-Ig
antibodies that detect Igs of different heavy or light chain classes allows
measurement of the quantities of different isotypes in a sample. Other
techniques for measuring antibody levels include hemagglutination for
antierythrocyte antibodies and complement-dependent lysis for antibodies
specific for known cell types. Both assays are based on the demonstration that
if the amount of antigen (i.e., cells) is constant, the concentration of antibody
determines the amount of antibody bound to cells, and this is reflected in the
degree of cell agglutination or subsequent binding of complement and cell lysis.
Results from these assays are usually expressed as antibody titers, which are
the dilution of the sample giving half-maximal effects or the dilution at which the
end point of the assay is reached.
A single-cell assay for antibody secretion that has been used in the past is the
hemolytic plaque assay, in which the antigen is either an erythrocyte protein or
a molecule covalently coupled to an erythrocyte surface. Such erythrocytes
serve as "indicator cells." They are mixed with lymphocytes, among which are
the specific antibody-producing cells, and are incubated in a semisolid
supporting medium to allow secreted antibody to bind to the erythrocyte
surface. If the antibody binds complement avidly, the subsequent addition of
complement leads to lysis of the indicator cells that are coated with specific
antibody. As a result, clear zones of lysis, called plaques, are formed around
individual B lymphocytes or plasma cells that secrete the specific antibody.
These antibody-secreting cells are also called plaque-forming cells. This assay
can also be used to detect antibodies that do not fix complement by
incorporating into the medium a complement-binding anti-Ig antibody that will
coat indicator cells to which the specific Ig is bound first. Another technique for
measuring the number of antibody-secreting cells is the ELISPOT assay. In this
method, antigen is bound to the bottom of a well, antibody-secreting cells are
added, and antibodies that have been secreted and are bound to the antigen
are detected by an enzyme-linked anti-Ig antibody, as in an ELISA, in a
semisolid medium. Each spot represents the location of an antibody-secreting
cell. Single-cell assays provide a measure of the numbers of Ig-secreting cells,
but they cannot accurately quantitate the amount of Ig secreted by each cell or
by the total population.
Table 9-2. Identification of a Role of Helper T Cells in Antibody Responses to
Protein Antigens
Adoptive transfer (cells transferred into irradiated recipient)
Source of B cells
Source of T cells
Antigen
Anti-SRBC antibody-producing cells in spleen
Bone marrow cells
None
SRBC
-
None
Thoracic duct cells
SRBC
-
Bone marrow cells
Thoracic duct cells
SRBC
+
-
-
Cells cultured
Antigen
Anti-SRBC antibody-producing cells in culture
Unfractionated spleen cells
SRBC
+
Splenic B cells
SRBC
-
Splenic T cells
SRBC
-
Splenic B cells and T cells
SRBC
+
Splenic B cells and T cells
-
-
Bone marrow cells
Thoracic duct cells
Cell culture
Abbreviations: SRBC, sheep red blood cells.
Mouse B lymphocytes by themselves do not produce antibody against a T cell-dependent antigen, SRBC, in vivo (adoptive
transfer) or in vitro (cell culture). The addition of T cells allows the B cells to respond to SRBC. A response does not occur in the
absence of antigen.
Table 9-3. Heavy Chain Isotype Switching Induced by Cytokines
B cells cultured with
Ig isotype secreted (percent of total Ig produced)
Polyclonal activator
Cytokine
IgM IgG1 IgG2a
IgE
IgA
LPS
None
85 2 <1
<1
<1
LPS
IL-4
70 20 <1
5
<1
LPS
IFN-γ
80 2 10
<1
<1
LPS
TGF-β + IL-5
75 2 <1
<1
15
Addition of various cytokines to purified IgM+IgD+ mouse B cells cultured with the polyclonal activator lipopolysaccharide (LPS)
induces switching to different heavy chain isotypes. The values of the isotypes shown are approximations and do not add up to
100% because not all were measured. (Courtesy of Dr. Robert Coffman, DNAX Research Institute, Palo Alto, Calif.)
Table 9-4. Properties of Thymus-Dependent and Thymus-Independent
Antigens
Thymus-dependent antigen
Thymus-independent antigen
Affinity
maturation
Yes
Little or no
Secondary
response
(memory B
cells)
Yes
Only seen with some antigens (e.g.,
polysaccharides)
Chemical
nature
Features of
antibody
response
Isotype
switching
Ability to
Yes
induce
delayed-type
hypersensitivity
No