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CHAPTER 13
T-CELL/B-CELL COOPERATION IN HUMORAL IMMUNITY
See APPENDIX (12) PLAQUE-FORMING CELL ASSAY
Adoptive transfer experiments illustrate the use of antibodies to cell surface
markers in distinguishing the roles of different cell types in the CELL/CELL
COOPERATION required for humoral immune responses. While the THYMUS is not
itself a site of immune reactivity, it is the source of T-CELLS (“helper” TH-cells) which
are required to cooperate with B-CELLS (the precursors of antibody-forming cells) to
generate antibody responses to THYMUS-DEPENDENT (TD) ANTIGENS, as well as
those T-cells (“effector T-cells") responsible for CELL-MEDIATED IMMUNITY.
Humoral responses to THYMUS-INDEPENDENT (TI) ANTIGENS, however, do not
require T-cell help. While B-cells in mammals are produced in the bone-marrow, a
distinct organ exists for their production in birds (the BURSA). One widely utilized
animal model for immunodeficiency is the athymic or NUDE MOUSE. The congenital
absence of a thymus in these mice results in the absence of T-cells, and consequently an
almost complete lack of cell-mediated immunity and of humoral responses to TD
antigens.
THE THYMUS IS REQUIRED FOR THE DEVELOPMENT OF IMMUNE
RESPONSIVENESS
The thymus is an organ containing large numbers of lymphocytes, which in humans
surrounds the top part of the heart. Until the 1950’s nothing was known of its function,
although its histology clearly made it part of the lymphoid system. Classical kinds of
experiments to determine its function by surgical removal in adult animals gave no clear
results – no physiological defects became apparent and the organ was apparently not missed.
One unusual feature which has been recognized since ancient times is that the thymus starts
out as a fairly large organ in very young animals (including humans) which continues to grow
through early life, but then undergoes a process of involution or progressive degeneration and
decrease in size, beginning at about the time of puberty. In older adult animals, the thymus is
often little more than a small bit of connective tissue.
Our present understanding of thymic function developed only around 1960 through the
experiments of J.F.A.P. Miller, who showed that the thymus was critical for the development
of immune competence. For unrelated reasons, he had removed the thymus from mice within
24 hours of birth (neonatal thymectomy), and found that such mice grew poorly, suffered
from a continuous series of viral and other infections, and often died before reaching
adulthood, a condition known as runting syndrome. However, runting only developed if
thymectomy was carried out within the first 24 hours of birth -- thymectomy in older mice
had little or no effect, consistent with the results of the earlier experiments referred to above.
The cause of runting in these neonatally thymectomized mice was shown to be due to the fact
that they are severely deficient in their ability to mount immune responses both to infectious
agents (which accounts for their susceptibility to infections) and to experimental antigens.
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This immunodeficiency includes both humoral immunity (poor antibody response to some,
but not all, antigens), and cell-mediated immunity (lack of ability to reject skin grafts) and
can be corrected by a thymus transplant. Thus, while the thymus is not required for immune
responses, it is necessary for the development of immune responsiveness. We will see later
that this is because T-cells are produced in the thymus and exported to the peripheral
lymphoid tissues, and it is only in the periphery that they normally respond to antigen
challenge.
SYNERGY IN IMMUNE RESPONSES: ANTIBODY FORMATION
REQUIRES T-CELLS AND B-CELLS
One researcher who followed up on Miller's findings was Henry Claman, who in 1966
published the results of his studies on the role of the thymus in humoral antibody responses.
He used an adoptive transfer system enabling him to mix cells from different sources to
examine the antibody response to SRBC. He transferred thymus cells, bone marrow cells, or
both, together with the antigen SRBC, into lethally irradiated recipients.
900R
THY cells
B.M. cells
SRBC
TEST SPLEEN
FOR PFCs
ON DAY 7
ADOPTIVE TRANSFER DEMONSTRATING T/B CELL COOPERATION
Figure 13-1
He measured the response not by assaying circulating antibody, but by removing the spleen
and determining the number of antibody-forming cells it contained, using the newlydeveloped hemolytic Plaque-Forming Cell (PFC) assay (see APPENDIX 12). An example
of his results is shown below:
Lymphoid
Cells transferred
PFC's per spleen
none
thymus cells
bone marrow cells
thymus + bone marrow
20
50
60
550
These results show very little response with either thymus cells or bone marrow cells alone (a
normal response to SRBC would yield 50,000 or more PFC's per spleen), but there was tenfold higher response with both cell populations together than with either alone. This was the
first evidence of synergy, or cell cooperation in immune responses. While Claman
concluded that the humoral response required participation of cells from both thymus and
bone marrow (which later became known as T-cells and B-cells respectively), he was unable
to determine which of the two sources gave rise to the antibody-forming cells themselves.
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B-CELLS BECOME ANTIBODY-FORMING CELLS -- T-CELLS ARE "HELPERS"
Mitchell and Miller were soon able to answer this question by developing a modified and
more complex adoptive transfer system, and by using anti-H-2 antibodies to distinguish the
different cell types. Instead of doing a short-term transfer into irradiated hosts, they injected
bone marrow cells into mice that had previously been thymectomized as adults and lethally
irradiated. The injected cells rescued the animals from radiation death by completely
restoring their hemopoietic system, except for T-cells, which could not be regenerated in the
absence of the thymus. After three weeks (to allow hemopoietic restoration to take place),
the animals were immunologically equivalent to neonatally thymectomized animals -- they
had the same immunological deficiency, which could be corrected by transplanting thymus
tissue, or, as we will see below, by providing another source of competent thymus-derived
cells. Such animals are known as ATxBM mice (for Adult Thymectomized, Bone-Marrow
restored).
The second improvement Mitchell and Miller were able to make was to use lymphocytes
from the thoracic duct lymph (the major lymphatic vessel) instead of thymus cells. These
thoracic duct lymphocytes (TDL's) contained more mature thymus-derived cells than
thymus tissue itself, and resulted in a much higher adoptive response. Their adoptive transfer
system is illustrated below:
900R
TDL + SRBC
B.M. cells
7 days
3wks
TEST SPLEEN
FOR PFCs
"ATxBM"
"ATx"
ADOPTIVE TRANSFER IN STABLE CHIMERAS
Figure 13-2
Using this new adoptive transfer system, and measuring the number of PFC's seven days after
immunization, they obtained the kinds of results shown below:
Treatment of ATxBM Hosts
Spleen PFC's
SRBC alone
200
TDL + SRBC
11,000 (!!)
While the ATxBM animals responded almost not at all to SRBC, addition of TDL's restored
their response to levels comparable (although not quite equal) to those of normal, intact
animals.
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The third advantage of this system turned out to be the fact that they could get this high level
of responsiveness using cells derived from genetically different donors. They constructed an
adoptive transfer with the following MHC types:
Strain
ATxBM HOST
BONE MARROW
TDL
H-2 Haplotype
C57Bl/6
(C57Bl/6 X BALB/c)F1
C57Bl/6
b
(H-2 )
b/d
(H-2 )
b
(H-2 )
Note that in this particular combination all cells bear H-2 antigens of the H-2b haplotype, but
only the bone-marrow cells (and therefore their progeny) bear H-2d antigens.
In order to exploit the MHC difference between the two cell populations, Mitchell and Miller
added one additional step to the assay procedure: after removing the spleen cells for assay,
but before placing them in the PFC assay system, they treated the cells with anti-H-2 antisera
plus complement. This treatment killed any cells bearing those H-2 antigens recognized by
the antibody, and such cells would therefore not be seen in the PFC assay. They used two
antisera, one detecting H-2b, the other H-2d. If the antibody-forming cells are derived from
bone-marrow, they should be killed by both antisera; if they are derived from TDL, they
should be killed only by anti-H-2b. The results they actually obtained are illustrated below:
PFC per spleen
Treatment of cells
none
anti-H-2b
anti-H-2d
11,000
550
450
These results show that these antibody forming cells can be killed by both anti-H-2 antisera,
and therefore the antibody-forming cells must have been derived from the bone marrow, and
not from the TDL's. Thus, they demonstrated that while both "B cells" (bone-marrow
derived) and "T cells" (thymus-derived) are required to generate a humoral response to
SRBC, only the B-cells develop into antibody-forming cells, while the T-cells perform a
"helper" function. The nature of this "helper" function will be discussed in Chapter 15.
THYMUS-DEPENDENT VERSUS THYMUS-INDEPENDENT ANTIGENS
Not all humoral responses are absent in neonatally thymectomized animals; T-cell deprived
animals can respond quite well to a number of antigens, which is the basis for distinguishing
between thymus-dependent and thymus-independent antigens.
Thymus-dependent (TD) antigens generally include most protein antigens and most
cell surface antigens (which are commonly glycoproteins). Such molecules are
generally capable of inducing not only primary (IgM) responses, but also memory
responses, i.e. class switching to IgG, IgA and IgE isotypes.
Thymus-independent (TI) antigens include polysaccharides with highly repetitive
epitopes; since many antigens in bacterial cell walls (and other microorganisms) bear
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such structures, this is clearly a biologically significant category. These antigens
commonly induce only primary (IgM) responses with little or no memory or class
switching. (See Chapter 15 for more detail on TI antigens.)
THYMUS AND BURSA IN BIRDS:
SEPARATE PRIMARY LYMPHOID ORGANS FOR T CELLS AND B CELLS
In addition to having a thymus analogous in structure and function to that of mammals,
members of the Class Aves (birds) have another lymphoid organ called the Bursa of
Fabricius, located near the cloaca. Removal of the embryonic thymus before hatching results
in chickens with no cellular responsiveness (they cannot reject skin grafts), and deficient
humoral responses to many, but not all antigens; this is similar to what we have seen
following neonatal thymectomy in mice, and in congenitally athymic (“nude”) mice (see
below). Removal of the embryonic Bursa, on the other hand, results in a lack of ability to
make any humoral response, while the ability to reject grafts (and carry out other cellmediated immune responses) is unaffected.
Both the Thymus and Bursa are considered to be Central Lymphoid Organs in birds; Tcells mature in the thymus and B-cells in the Bursa, although they actually carry out their
immune responses in other (peripheral) organs. There is no distinct organ in mammals
homologous to the Bursa of birds, and mammalian B-cells are produced largely within the
bone marrow itself.
[It is interesting to note that the chicken was the first system in which the dichotomy between
humoral and cellular responsiveness was made clear, and the term B-cell, in fact, was
originally coined to refer to "Bursa-derived" cells.]
GENETIC THYMIC DEFICIENCY: THE "NUDE" MOUSE
Surgical removal of the neonatal thymus provided the earliest demonstration of that organ's
importance in the development of the immune system. Soon afterwards, however, a genetic
animal model of thymic insufficiency was discovered, namely mice homozygous for the
recessive mutation nu (for "nude").
"Nude" mice, whose genotype is nu/nu, lack body hair (hence the name), and also lack a
functional thymus. They show all the features of neonatally thymectomized mice, i.e.,
"runting" and poor general health, inability to make humoral responses to many (but not all)
antigens, and lack of ability to reject skin grafts and carry out other cell-mediated immune
reactions. They lack functional T-cells in the blood and peripheral lymphoid tissues.
Implanting a fetal thymus (e.g. under the kidney capsule) completely restores the normal
level of T-cells as well as the ability to generate all normal immune responses.
The nude (nu/nu) mouse has been widely used for studies on T-cell function; it is a more
reliable model than neonatally thymectomized animals, since this surgery is difficult to carry
out and may result in a small amount of thymic tissue being left behind. The nude mouse has
also been a valuable tool for the study of heterologous tumors, especially human tumors. In
the absence of cell-mediated immunity, the foreign tumors are not rejected, as they would be
in a normal animal, and they can be grown and transferred from mouse to mouse. Decades of
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studies in tumor biology have relied on the ability of human tumor cells to grow in nude mice
as a defining feature of their oncogenic potential. The human counterpart of the mouse nu
gene has recently be shown to be the whn gene, and homozygosity for rare mutations at this
locus result in a combination of hairlessness and immunodeficiency very similar to the
condition seen in the nu/nu mouse
CHAPTER 13, STUDY QUESTIONS:
1.
How was the role of the THYMUS in immunity first discovered?
2.
Describe the experiments of Mitchell and Miller which defined the roles of T and B
cells, and state succinctly what their conclusions were.
3.
Define TI versus TD antigens. Give some examples of each which are of significance
in people.
4.
Why are there so many NUDE (nu/nu) MICE in the world?
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