Rev. sci. tech. Off. int. Epiz., 1998,17 (1), 43-70
Immunoglobulin diversity, B-cell end antibody
repertoire development in large farm animals
J.E. Butler
D e p a r t m e n t o f M i c r o b i o l o g y a n d I n t e r d i s c i p l i n a r y I m m u n o l o g y P r o g r a m , 51 N e w t o n R o a d , B o w e n S c i e n c e
B u i l d i n g , U n i v e r s i t y o f l o w a , l o w a City, I A 5 2 2 4 2 - 1 1 0 9 , U n i t e d S t a t e s o f A m e r i c a
Summary
The B-cell lineage, the antibodies produced by these cells and the diversification
of the antibody repertoire are essential for the health and survival of all mammals.
Cattle, sheep, swine and horses, unlike rodents and primates, develop their
antibody repertoire from a relatively small number of V (variable heavy) genes of
one or several families and cattle, sheep and horses use almost exclusively f l i g h t
chains. These large farm animals appear to use templated mutation (gene
conversion) in addition to untemplated (point) mutation in repertoire development;
this may occur predominantly in the ileal Peyer's patches. Whether B-cell
lymphogenesis is continuous throughout life - as in rodents and primates - or
whether B cells are largely of the B-1 lineage and develop only in foetal and
neonatal life, is uncertain. The fact that immunoglobulin D (IgD) is totally absent
from swine and ruminants may be significant, as IgD is expressed weakly on
rodent B-1 cells.
Information on IgG subclass diversity in large farm animals is incomplete, except
for sheep and cattle, and no information is available for any large farm animal to
show whether T helper 1 (Th1 ) and Th2 responses correlate with the expression of
any subclass antibody response, as is the case in rodents. All large farm animals
exclusively use the mammary gland to transfer immunity to offspring, although the
receptor involved in the transport of IgG into colostrum and milk has not been
characterised.
Efforts to standardise the nomenclature and measurements of antibodies and
immunoglobulins in large farm animals are discussed, and a proposal currently
under review concerning the standardisation of the nomenclature for bovine
immunoglobulins is presented as a model.
H
Keywords
B cells - Cattle - Genetics - Horses - Nomenclature - Passive immunity - Repertoire
development-Swine.
Introduction: antibodies in
animal health
Lessons from experiments of nature
The importance of a process or an element to the overall
well-being of the organism often becomes obvious when that
element or process is absent. While this is the rationale for the
use of knock-out transgenic mice in scientific research, there
are also naturally-occurring genetic deficiencies. In horses,
mice and humans, failure of the rearrangement of the genes
which encode antibodies leads to severe combined immune
deficiency (SC1D). In SCID there are no antibodies, so victims
of the disease, such as Arabian foals ( 9 8 , 165), succumb to a
wasting disease once the passive antibodies obtained from the
mare have been exhausted. Since the normal offspring of
common large farm animals, i.e., calves, piglets, lambs and
foals, possess only small amounts of 'natural' antibodies at
birth, they depend on passive antibodies obtained through
colostrum and milk ( 2 6 ) . Thus, failure of such transfer to
occur also leads to death by wasting disease (99). In the case
of hyper immunoglobulin M (IgM) syndrome, the T-cell
co-stimulatory molecule CD40 (cluster of differentiation
44
antigen 4 0 ) ligand is absent, so that affected individuals are
only able to produce IgM antibodies (3). Since most bacterial
and viral antigens are T-dependent, the inability of such
individuals to produce antibodies other than IgM leaves them
highly susceptible to bacterial infection in particular.
The above examples illustrate the consequences of the loss of
all or a portion of the antibody repertoire. There are additional
examples of holes in the repertoire, in which individuals are
unable to make antibodies to certain antigens or antibodies of
a protective isotype. These experiments of nature illustrate the
essential role played by antibodies and development of the
antibody repertoire in maintaining animal health.
The biological role of secreted antibodies
Antibodies rarely act alone in providing protection against
infectious agents, but rather act together with various
accessory cells of the immune system. IgG antibodies which
recognise bacteria are themselves recognised by macrophages
or neutrophils bearing so-called Fc-receptors. Once the
antibody has recognised the target, the phagocyte can
recognise the target-antibody complex. Killing is generally the
result of digestion of the bacterium-antibody complex in a
phagolysosome. Antibodies can also activate a cascade of
enzymes called complement which can perform the following
duties:
a) generate a membrane attack complex which lyses the
infectious organism
b) produce a series of mediators, such as anaphylatoxins,
•which produce an inflammatory response capable of killing
the pathogen
c) attach an enzyme derivative called C3b to the pathogen,
which then allows the pathogen to be recognised by
phagocytic cells in much the same manner as if coated with
antibody.
Examples of antibodies capable of direct intervention include
those which block virion penetration of cells, antitoxins which
prevent toxins from binding to their cell receptors and
antibodies such as IgA, which can block colonisation of an
epithelium by interfering with bacterial adhesion.
Secreted antibodies also play an important initial role in the
processing of foreign antigen. Namely, the soluble complexes
which they form with antigens are
subsequently
phagocytosed by accessory cells, which degrade them to
peptides and present them with major histocompatibility
complex (MHC) surface proteins to T cells. Recognition of the
foreign peptides in the context of the MHC by the T-cell
receptor (TCR) results in stimulation of the T cell. This
T-dependent event initiates a series of events which leads to
further specific antibody production (Th2 response) or
generates inflammatory T cells ( T h l response). Both of these
responses can resolve the infection.
Rev. sci. tech. Off. int. Epiz., 17 ( 1 |
The role of antibodies as B-cell receptors
The discussion so far has focused on the role of the secreted
form of antibodies in animal health. The secreted form of
antibodies is the product of terminally differentiated B cells
called plasma cells (Fig. 1). Plasma cells have elaborate
synthetic and secretory machinery and a relatively short
half-life (five to seven days). The membrane form of an
antibody is called the B-cell receptor (BCR), i.e., it is the
receptor on B cells which recognises antigen. Secreted
antibodies differ from those embedded in the membranes of
B cells in that the former have exchanged hydrophobic
transmembrane anchors for a sequence which promotes their
secretion as soluble proteins. The transmembrane tail of the
membrane antibody molecule is associated with two other
transmembrane proteins known as Igoc and Ig(3. These
accessory molecules are intricately involved in the process of
signal transduction. Signal transduction occurs when the BCR
encounters antigen and initiates a metabolic pathway which
informs the transcriptional machinery in the B-cell nucleus
that it has encountered an antigen and that new proteins must
be transcribed (or the transcription of others suppressed) to
permit the stimulated B cell to process the antigen, proliferate
and/or differentiate to a plasma cell.
Antigen encounter by B cells results in intemalisation of the
BCR-antigen complex and the subsequent digestion of the
complex to peptides (called antigen processing). The
processed peptides are then displayed on the B-cell surface in
the context of class II MHC molecules to be recognised by
T cells in much the same manner as was described above
when phagocytic cells ingest and digest the antigen-antibody
complexes they have phagocytosed. Thus the B cell, like the
phagocytic accessory cell, is called an antigen-presenting cell.
Upon receiving a second signal from the T cell to which it had
effectively presented antigen, the B cell not only proliferates
and differentiates into a plasma cell which secretes antibodies
specific for the antigen originally encountered by the BCR, but
the encounter also sets in motion a process for diversifying the
original BCR. This diversification depends on somatic
mutation of the genes encoding the original BCR. The rate of
somatic mutation of the genes, which controls the specificity
of the BCR, is 1 0 greater than for normal eukaryotic genes.
This mutational event is one of the mechanisms responsible
for expanding and diversifying the antibody repertoire.
7
Antibody structure, genes and
synthesis
The structure of antibodies
Figure 2 illustrates the sequential stages of the synthesis of an
antibody beginning with rearrangement of various gene
segments (2a, top) and ending with a diagram of the antibody
encoded by these various gene segments (2a, bottom). All
antibodies are monomers or multimers of the four
polypeptide chains unit illustrated at the bottom in Figure 2a.
45
Rev. sci. tech. Off. int. Epiz., 17 (1)
Fig. 1
Lymphocyte development
B-cell development takes place in foetal spleen or bone marrow while T cells develop in the thymus. Both are derived from lymphoid precusors which are derived
from foetal liver. During development, each proceeds through a stage in which a surrogate light chain (B cells) or surrogate a-chain (T cells) is displayed as part
of the pre-B-cell receptor and pre-T-cell receptor, respectively. Both immature T and B cells are subjected to negative selection by self-antigens and T cells must
also be positively selected for cluster of differentiation antigen 8 (CD8) and CD4 on thymic stromal cells which express major histocompatibility complex (MHC)
class I and MHC class II. B cells may also be positively selected. An estimated < 5% of all lymphoid cells survive the selection process
IgG, the most abundant serum antibody, is composed of a
single unit, i.e., the structure shown at the bottom of Figure 2a
is composed of two light and two heavy chains. The light
chain/heavy chain association at the N-terminal region of
these polypeptides forms the 'Fab' portion of the molecule
which is composed of the V + C domains of the light chain
and the V + C 1 domains of the heavy chain (Fig. 2a). Any
one antibody unit is composed of two such identical Fabs.
Since the Fab contains the antigen binding site, each
monomelic antibody unit is divalent. Some antibodies, such
as IgM, are pentamers of the basic unit and thus have ten
identical binding sites.
L
H
L
H
The C 2 and C 3 domains of the paired heavy chains at the
C-terminal end of the molecule comprise the so-called 'Fc'
portion. The Fc portion of an antibody endows it with unique
biological activities which include activation of complement,
transport across epithelial cells, recognition by Fc-receptors
on macrophages, neutrophils and mast cells, and recognition
by the lg-binding proteins produced by various bacteria.
Since different isotypes of antibodies have different Fc regions
H
H
(Table I), each isotype is able to initiate different biological
activities in protective immunity.
Both the heavy and light chains of an antibody are composed
of constant ( C , C ) and variable ( V , V ) regions. The
N-terminal portion of both heavy and light chains ( V + V )
encodes the Fab and thus determines antibody specificity.
Constant regions are encoded by only a few different genes
which give the antibody its isotypic or 'class' (or subclass)
character. For example, one gene which has been found so far
in all mammals encodes the constant region of IgM (called p),
and usually just one gene encodes the constant regions of IgA
(a-) and IgE (8-), but often multiple genes encode the
subclasses of IgG (y-) (Table I). Due to the limited number of
genes encoding the constant portion of antibodies and the fact
that these are not subject to somatic hypermutation, they are
considered 'constant'.
H
L
H
L
L
H
Unlike the constant region, many genes encode the 'variable
regions' of heavy and light chains (the number of variable
gene segments in the variable heavy chain locus is indicated in
Rev. sci. tech. Off. int. Epiz., 17 (1)
46
Fig. 2b
Fig. 2a
Germline D N A
Somatic
recombination
DJ-joined D N A
Somatic
recombination
VDJ-joined
rearranged DNA
Transcription
Primary
transcript RNA
Splicing
mRNA
Translation
Heavy chain
mRNA : messenger ribonucleic acid
Fig. 2
Immunoglobulin gene rearrangement and synthesis of an immunoglobulin
Modified from Janeway and Travers (78)
Fig. 2a: Progressive rearrangement events beginning with germline DNA encoding in humans 91 V segments, 30 D segments, 6 J segments and 10 different
constant Ig gene segments (c) linked on chromosome 14q. During rearrangement, a single V, D, and J segment are joined ('VDJ-joined rearranged DNA') in the
process called 'combinatorial joining'. Since the VDJ joints can add or subtract nucleotides, 'junctional diversity' also arises. Eventually an mRNA encoding the
VDJ and constant region is synthesised and subsequently translated into the heavy polypeptide chain of the Ig the darkened area. The second heavy chain of the
completed Ig molecule is identical to the first
Fig. 2b: The polypeptide structure of the variable heavy chain region of an Ig. The N and C terminals are indicated. The darkened region depicts the so-called
(hypervariable) W or (complementary-determining regions) 'CDR' loops which join the anti-parallel 6-pleated sheets together and also comprises the tip of the
variable region which interacts with antigen
Fig. 2c: A folded version of Fig. 2b showing how the three HV regions come together to form the binding site. These regions are called HV because they show
the greatest variability among antibodies and CDRs because they are complementary to the conformation of the antigens they bind
parenthesis in Figure 2a). In humans, mice and chickens,
there are about 100 genes encoding the variable heavy chain
genes and a similar number which encode the V of the
K-light chains. The light chains of antibodies are encoded at
two separate loci, kappa ( K ) and lambda (X). Any one
antibody bears either K - or A.-light chains. As far as is known,
any of the many variable region genes can be expressed with
any particular constant region gene (one is depicted as being
used in Fig. 2a). Since the specificity of the antibody is
encoded by the variable region genes, the existence of IgM,
IgG, IgA and IgE antibodies with identical variable regions,
and therefore identical antigen specificity, is possible.
L
As shown in Figure 2a, the eventual synthesis of an antibody
molecule involves the rearrangement and joining of various
gene segments, i.e., one V , J , D and C are selected for the
heavy chain and one V , J and C for the light chain. This
process of variable gene segment selection and joining,
together with somatic hypermutation, generates the repertoire
of antibodies capable of recognising so many different
H
L
H
L
H
H
L
47
Rev. sci. tech. Off. int. Epiz., 17 (1)
Table I
Immunoglobulin diversity among animals (33)
C
H
C g e n e s (a)
genes
v„ a n d
L
V
L
families
GOD
Species
IgM
IgD
IgG
Mouse
1
1
4
1
1
3
(5%)
1
(95%)
Human
1
1
4
1
2
7
(40%)
1
(60%)
Bovine
1
0
3
1
1
4 (?) ( > 9 8 % )
Sheep
1
0
2(7)
1
1 (?)
1
0
1
1
13
Swine
1
0
>6(?)
1
1
1 (?) ( 4 0 % )
Horse
1
?
1(7)
4
1
0
4(?)
•| W)
1
Chicken
(?)(">
K?)
1
Rabbit
.
•
IgA
igE
X
K
> 1 (?) ( > 9 5 % )
8
(93%)
(100%)
, b l
K
3
4
SM
7
1 (0
7
7
SM
2(7)
?
S M , CVS
1 Ici
6
3
SM
(90%)
1
?
7
S M , CVS
1 I?) ( 6 0 % )
1
7
S M , CVS (?)
1
?
?
1 (cl
?
7
1
1
0
C V S , S M (?)
1
(< 2%)
1 (?) ( < 5 % )
2
(10%)
X
H
0
(7%)
14
a) Values in parentheses indicate the proportion of each light chain type expressed
b) GOD: generation of diversity. Since all species use both combinatorial joining and junctional diversity, these mechanisms are not listed
c) Only one family appears to be expressed
d) Chicken IgY appears to share homology and function with both mammalian IgG and IgE
Ig : Immunoglobulin
SM : untemplated somatic hypermutation
CVS : templated somatic mutation (gene conversion)
(?) : the exact number is unknown but may be the number indicated
?
: unknown
antigens: the failure of this process in SCID results in an
agammaglobulinaemic condition.
Antibody and immunoglobulin genes
Figure 2, as well as illustrating the general structure of an
antibody and the gene segments encoding the antibody, also
illustrates how these segments are rearranged to yield the
complete antibody molecule. The rearrangement process is at
the basis of repertoire development. For example, the human
has 9 1 V segments, 3 0 D segments and 6 J segments, thus
there are more than 1 6 , 0 0 0 different possibilities for
generating the variable regions of the heavy chain. Since there
are 4 0 V genes and five J segments (there are no D segments
in the light chain loci) there are 2 0 0 possibilities for the K-light
chain variable region. Use of the X-light chain locus results in
somewhat fewer possibilities. The frequency of K - or X-chain
usage is species-dependent, as summarised in Table I. Using
the K locus as an example results in more than 3.2 x 1 0
different Fab fragments which could translate into as many
different antibody specificities. This method of generating a
repertoire is called combinatorial joining (CJ). In addition to
CJ, the actual joints formed between V , D and J segments
can involve the addition or removal of nucleotides. This
process leads to junctional diversity and is believed to account
for at least 1,000 additional possibilities for the variable heavy
chain segment alone.
H
K
H
H
K
6
H
H
in Fig. 2b and 2 c ) . Somatic hypermutation of this sort is
believed to account for at least another 1,000 variants. If such
hypermutation occurs in both the variable heavy and variable
light chain regions, the theoretical antibody repertoire would
be greater than 1 0 . Using synthetic variable gene
modulation, antibody-phage libraries with more than 1 0
specificities have been generated in vitro (63).
1 2
1 1
Two different forms of somatic hypermutation have been
described for introducing additional diversity into rearranged
VDJs or VJs. Untemplated mutations occur merely as point
mutations which are primarily clustered in the CDR regions.
By contrast, species such as the chicken and the rabbit use a
separate form of somatic hypermutation which, in effect,
appears to involve translocation of shon segments of
upstream variable region genes to the most 3 ' V gene which
will then be transcribed as a type of hybrid. This process, also
known as somatic gene conversion, seems to be a mechanism
favoured in species which either have, or only use, a small
number of highly homologous variable region genes to
generate their antibody repertoire (31, 3 2 ) .
H
H
Finally, the rearranged variable region gene complex VDJ
(heavy chain) or VJ (for light chains) is acted upon by an
undefined mutase or some error-prone polymerase or repair
enzyme to generate mutations at a rate 1 0 greater than that
found in conventional eukaryotic genes. These mutations
tend to be clustered in hypervariable (HV) regions which
encode the complementary determining regions (CDRs) of
antibodies (Fig. 2 b ) . The CDRs are those regions which
directly contact antigen in the folded Fab (the shaded regions
B-cell lymphogenesis
In mammals such as mice, haematopoiesis begins in the foetal
liver or yolk sac, with precursor lymphoid stem cells
developing into lymphocytes. Some of these lymphocytes
migrate to the developing thymus and there mature into
predorninandy a/p T cells (Fig. 1 ) , while others remain
behind in the foetal liver to rearrange their antibody genes
(Fig. 2a) and become pre-B cells or become Y/À T cells.
7
The gene rearrangement events depicted in Figure 2 are not
always productive; non-productive rearrangements typically
encode out-of-frame sequences and may occur more
frequendy than productive rearrangements (Fig. 1). In mice,
pre-B cells with productive rearrangements transcribe and
48
Rev. sci. tech. Off. int. Epiz., 17 (1)
translate their productivity rearranged V(D)Js into membrane
immunoglobulins which serve as BCRs. Initially, the heavy
chain variable regions of BCRs of at least conventional (B-2)
cells are expressed together with light chain-like segments (X,5
and VpreB) which serve as developmental surrogates ( 1 0 5 )
(Fig. 1). Later, these surrogate light chains are replaced by the
products of rearranged authentic V K or VK chains. These
immature B cells are now subjected to negative selection, i.e.,
those which recognise self-antigens are given a cell death
signal. The cells which emerge from the foetal liver and
migrate to secondary lymphoid tissues, such as the spleen and
lymph nodes, are regarded as mature but naive B cells, and are
estimated to comprise less than 3 0 % of the pre-B cell
population (118). This paradigm of B-cell development may
require modification when extended to non-rodent,
non-primate mammals, including the large farm animals
which are the subject of this review.
The factors and mechanism(s) that determine V , D and J
usage during the rearrangement process are poorly
understood, although certain patterns have emerged. Studies
in foetal mice and humans indicate that gene usage is not
random. The most 3 ' V families in the murine and human
genomes are over-expressed in foetal liver (121, 1 4 1 , 1 6 6 ) .
Rabbits use their 3 ' V gene 9 0 % of the time (89) and the
chicken has only one functional V gene and it is the most
3' V gene in the locus (162). Kraj et al found that V - 3 4 ,
V - 5 4 and V - 2 3 dominated the repertoire in pre-, immatureand mature B cells in humans, and foetal piglets use primarily
four V gene segments, two D segments and their single J
during a 3 0 - 1 1 0 day period in utero (92).
H
H
H
H
H
H
H
4
4
3
H
H
H
The productive rearrangement of VDJs, their transcription
and translation into BCRs and the subsequent selection and
expansion of B cells represent the antigen-independent phase
of B-cell differentiation (Fig. 1). While taking place
developrrientally in the liver, the process shifts to the bone
marrow in adult humans and mice and continues throughout
life. These continuously generated B cells are called
'conventional' or B-2 cells. On the other hand, certain B-cell
sub-populations appear to be generated only during foetal
and early neonatal life and tend to populate the peritoneum.
These are called B - l cells and are self-regenerating rather than
continuously differentiated from precursors as occurs with
conventional B-2 cells.
B-cell selection and germinal centres
Mature B cells migrate from the bone marrow (or foetal liver)
to populate lymph nodes and the spleen. At these sites, the
cells encounter antigens, which are recognised by their BCRs,
and begin the antigen-dependent phase of their development
(Fig. 1). Antigen recognition, combined with cognitive
interaction with helper T cells or exposure to certain
cytokines, results in activation of the B cells. T-cell-mediated
activation leads to proliferation, somatic hypermutation of
VDJs and switch recombination. This antigen- and helper
T-cell-driven process occurs in follicles of the germinal centres
(GC) of these secondary lymphoid organs. Since the T- and
B-cell systems are mature at this time, GC formation is
dependent on exposure to external antigen. In addition to
somatic mutation, secondary VDJ rearrangement (receptor
editing) can also occur (66, 8 6 ) , and may do so through the
use of heptamer signal sequences embedded in CDR3 which
were the result of the initial productive VDJ rearrangement.
Concomitantly, when in the presence of the correct cytokine
milieu, class switching occurs. Switching involves the
alignment of repetitive sequences known as switch regions
such that a particular VDJ is juxtaposed with downstream C
genes, such as those encoding IgA or IgE. The organisation of
the heavy chain locus is such that the gene encoding the IgM
heavy chain (u) is immediately 3 ' proximal to the rearranged
VDJ. Thus 'switching' involves removal of intervening DNA so
some downstream constant region gene now lies immediately
3' proximal to the rearranged V(D)J. This allows the original
or somatically-mutated BCR, which recognised the antigen, to
be expressed now with a heavy chain other than IgM. Since
the Fc portion of the constant region of the antibody heavy
chain is the part which determines the special biological role
of the cell, switching is an important event in B-cell
differentiation. As indicated below, individuals whose T cells
are genetically deficient in the co-stimulatory molecule CD40
ligand are unable to switch, and thus can produce only IgM
antibodies. If other classes of antibodies are needed to control
an infection, such repertoire-deficient individuals are highly
susceptible.
H
The somatic mutation or receptor editing of the BCR in GCs
which follows an encounter with a T-dependent antigen leads
to affinity maturation (79). Since activation stimulates somatic
mutation, a small number of mutational events can result in
the production of BCRs which can bind the antigen with
higher affinity than the original BCR. Thus, waves of
stimulation and somatic mutation produce B cells of higher
affinity, i.e., the affinity of the response matures. This is, in
part, the rationale behind the use of booster immunisations.
However, there is much to be learned about this process,
particularly in large farm animals, in which the process has
not been studied at the molecular level. Since most studies
have been performed in mice and involve conventional
B cells, the question as to whether an analogous series of
events occurs in GCs of species which possess predominantly
(or only) B - l cells has not been vigorously addressed.
Studies by Kraj et al. suggested that selection by ligand may
explain the overrepresentation of V - 2 0 in mature B-cells
(92). Ligands need not be antigens per se but could also be
so-called 'B-cell superantigens'. These are believed to act like
T-cell superantigens, such as Staphylococcal enterotoxin (the
cause of toxic shock syndrome in women), which can lead to
the proliferation of lymphocytes expressing certain V gene
segments regardless of the actual antigen specificity of the
BCR. Stromal ligands on host cells may also act as
superantigens and this may explain the selective use of the
most 3 ' V
gene in rabbits (123). A human B-cell
3
H
H
49
Rev. sci. tech. Off. int. Epiz., 17 (1)
superantigen specifically binds framework regions encoded
by V genes belonging to the V 3 family ( 1 4 4 ) . Since all
swine V genes are members of the V 3 family ( 1 5 0 ) , this
human foetal superantigen also binds porcine IgG, and a
protein with similar properties has recently been detected in
porcine bile (G.J. Silverman, personal communication). Thus,
preferential selection and proliferation of certain BCRs by
intrinsic B-cell superantigens may explain preferential V
expression and may represent an important aspect of B-cell
ontogeny and antibody repertoire development in farm
animals.
H
H
H
H
newborn and adult mammals to face the challenge of
environmental pathogens. Since a critical balance must be
achieved so that BCRs can recognise and respond to
pathogens but not to self-antigens, understanding the
mechanism by which the antibody repertoire is developed is
of considerable significance to both human and animal health.
H
Another potential source of both B-cell superantigens and
conventional environmental antigen is the intestinal flora. A
role of the gut flora in B-cell differentiation, or at least in
repertoire diversification, has been suspected for some time
(91). The role of intestinal flora in stimulating repertoire
diversification in farm animals needs to be addressed, since
this is a factor which could theoretically be modulated by
management practices.
Diversity of antibodies and
antibody genes among animals
lg gene diversity
Table I summarises the diversity of immunoglobulins among
common homeothermic vertebrates. Diversity is apparent in
the following factors:
a) the number of heavy chain classes and subclasses
b) the expression of K - versus X-light chains
c) the number of variable and light chain gene families
d) the generator of diversity (GOD)
The proliferative capacity of lymphocyte compared to most
other eukaryotic cell types would eventually result in
transforming the organism into a giant lymph node. To
prevent this, a mechanism for eliminating unwanted
lymphocytes has evolved. The pre-B cells which fail to
produce productive V(D)J rearrangements, or the mature
B cells which encounter self-antigen during development in
bone marrow - as well as those mature B cells in lymph nodes
which do not encounter a recognisable antigen - are all
eliminated by apoptosis (programmed cell death). Thus, more
than 9 0 % of all pre-B, immature-B and mature B-cell clones
are eliminated (Fig. 1).
of antibody specificities.
For example, IgD has been found so far only in rodents and
primates, and while many species have multiple subclasses of
IgG, rabbits have only one. Interestingly, rabbits have 13 IgA
subclasses, while the number does not exceed two for any
other species studied to date. The ratio of expressed K - and
X-light chains varies enormously, from more than 9 3 %
X-expression in ruminants and the horse to a marked
predominance of K in rodents. Surprisingly, swine, an
artiodactyl, display a K/X-chain ratio that shows more
similarity to humans than to other artiodactyls. In the case of
heavy and light chain variable gene families, both rodents and
humans have many families; in contrast, all the V genes in
the rabbit, chicken and swine belong to a single family and
raminants also appear to express only one family. Actual
numbers of V genes differ widely; rodents, humans and
chickens have circa 1 0 0 , rabbits may have as many as 2 0 0
while swine, sheep and cattle appear to have far fewer. As with
K/X-ratios, phylogenetic relationships are not a reliable
indicator of Ig diversity, except perhaps in very closely-related
species such as domesticated ruminants or rats and mice. The
same is true for the GOD. Species with large numbers of V
genes and a large number of gene families rely heavily on
combinatorial joining to create diversity, whereas rabbits,
which also have a large number of V genes but only one gene
family, rely heavily on templated mutation, i.e., somatic gene
conversion. The data in Table I emphasise the danger of
assuming that what is true for mice (or humans) is equally
applicable to large farm animals. Rather, each species needs to
be studied individually and paradigms which have been
established for mice should be applied to other species only
after experimental confirmation.
H
With regard to B cells which recognise self-antigens with their
BCRs, conventional wisdom surrounding this topic suggests
that if this recognition occurs during foetal and early neonatal
life, such cells are deleted or at least rendered tolerant to such
self-antigens. However, this appears on the surface to
contradict evidence that the so-called natural antibodies of the
foetus and neonate are characterised by self reactivity (4, 5 0 ) .
Perhaps low affinity natural antibodies of the B-1 lineage ( 6 7 )
represent the products of B cells which have escaped negative
selection because they bound too poorly to self-antigens to
cause their programmed cell death. Human diseases such as
rheumatoid arthritis, multiple sclerosis, lupus erythematosus,
rheumatic fever and autoimmune haemolytic anaemia result
from the ability of B cells to recognise self-antigens. Could low
affinity natural antibodies be responsible for autoimmunity
which appears later in life? Since most antigens involved in
autoimmunity are T-dependent, the loss of T-cell tolerance or
the retention of some autoreactive T-cell clones not eliminated
during thymic development (Fig. 1) is generally considered
more important in autoimmunity.
H
H
H
Diversity in B-cell lymphogenesis
The
processes
and
events
reviewed
above
ultimately
determine the antibody repertoire which is available to
Haematopoiesis and lymphopoiesis are evolutionarily
conserved developmental events. In most species, these
Rev. sci. tech. Off. int. Epiz., 17 (1)
50
events begin in the foetal liver and shift to the bone marrow
before birth. Diversity among species is most apparent in both
T and B lymphocyte subsets. As mentioned above, two major
subsets of B cells are recognised: B - l and B-2. The former is
characterised in mice by low expression of IgD, high
expression of CD5 and early appearance during development.
Furthermore, B - l cells are especially plentiful in the
peritoneum and have the capacity of self-regeneration. In
contrast, B-2 cells are C D 5
or negative, I g D
and are
continuously generated in bone marrow. Rabbits and
chickens do not produce B cells continuously throughout life
and, unsurprisingly, all of their B cells appear to be derived
from the B - l lineage. Rabbits also lack a gene encoding IgD
(41).
l o w
h l g h
The exact role of these two B-cell sub-populations is unclear.
There has been speculation that B - l cells are less diversified,
i.e., display more germline-like V / V sequences, are
polyreactive and recognise especially common microbial
antigens (43, 6 7 ) . However, recent studies indicate that B - l
cells can also be highly diversified ( 8 4 ) . This would be
consistent with the fact that rabbits, which have only B-l cells,
undergo splendid affinity maturation following immunisation
(84).
H
L
Both B - l and B-2 cells occur in ruminants, although the CD5
population is greatly expanded in trypanosome-infected cattle
(114). The situation in swine remains unclear since
mAb b 5 3 6 7 (anti-CD5) fails to recognise swine B cells ( 4 5 ) ,
although a monoclonal antibody made against a conserved
peptide in the cytoplasmic domain of CD5, does recognise
swine B cells (2).
Species differences in B-cell lymphogenesis may be correlated
with differences in primary lymphoid tissues/organs. In
chickens, lymphoid follicles in the hindgut collectively form
in the bursa of Fabricius. B cells in the bursa diversify by
somatic gene conversion prior to migration to secondary
lymphoid organs, e.g., the spleen, shortly before hatching.
Swine, horses and ruminants have well-developed ileal Peyer's
patches (1PP) and the rabbit appendix may serve a similar
function. Primates and rodents appear to lack discrete
homologues of these hindgut-associated primary lymphoid
tissues. While the exact role of the IPP/appendix has not been
defined precisely, repertoire diversification occurs in these
tissues; although in mammals, diversification involves somatic
point mutation as well as gene conversion. Nevertheless, all
species which utilise somatic gene conversion to diversify
their primary repertoire have some type of hind-gut associated
lymphoid organ, i.e., bursa, appendix or IPP.
Species diversity in lymphogenesis also occurs in T cells:
artiodactyls have both CD4 and CD8 double negative and
double positive cells in the periphery, whereas rodents and
primates have neither in the periphery (Fig. 1). Furthermore,
y/à T cells are more prominent in large farm animals than in
humans and rodents and some of these cells may undergo
development in foetal liver in all mammals rather than in the
thymus (95).
Diversity in the passive transfer of antibodies
from mother to young
Figure 3 summarises the diversity among common mammals
in the transfer of immunity from mother to young. Most
notable is the difference between so-called Group I and Group
III mammals. In the former, virtually all IgG is transferred in
utero by means of an Fc-dependent transport receptor in the
placenta. In contrast, Group III mammals transport no
antibodies in utero before birth but use an analogous
Fc-dependent receptor on acinar epithelial cells in the
mammary gland to transport IgG into colostrum. Group II
mammals are able to do both. The significance of this
difference in the transport of maternal IgG to the offspring is
that both foetal rabbits and humans have a serum level of IgG
at birth which is equal to or greater than the maternal serum
IgG level, while Group III mammals are b o m virtually
agammaglobulinaemic.
The way in which these differences affect the development of
the antibody repertoire may depend on whether maternal IgG
influences antibody repertoire diversification. While there is
evidence that maternal IgG primarily downregulates de novo
antibody synthesis ( 7 5 , 8 8 ) , investigators in a recent study
found no evidence for an effect of maternal IgG on either de
novo synthesis of Ig or repertoire development in foetal mice
(49). However, the extrapolation of the latter finding to all
mammals may be dangerous, since considerable diversity
exists among mammals in other aspects of B-lymphogenesis
and antibody repertoire development.
Figure 3 illustrates that the offspring of large farm animals
depend on suckling and on maternal colostrum to obtain
passive antibodies from their mothers. While this has been
known to science for more than 3 0 years, herdsman have
been aware of the effect of this process for at least 4 , 0 0 0 years.
Namely, herdsmen knew that the newborn of large farm
animals which did not suckle their mothers immediately after
birth typically died of wasting disease. Thus, Figure 3 explains
why newborn infants can be reared on cows milk without
fatal consequences and also why IgG, rather than IgA,
predominates in the colostrum of large farm animals, whereas
IgA predominates in the colostrum of women and doe rabbits.
The immunoglobulins and
immunoglobulin genes of swine
Serum concentration and non-vascular
distribution
As in all mammals, IgG is the major Ig in serum (Table II)
whereas IgA predominates in most exocrine body fluids
(Fig. 4 ) . As in other hooved mammals of Group III (Fig. 3 ) ,
51
Rev. sci. tech. Off. int. Epiz., 17 (1)
Trace
IgG-specific and prolonged in rodents. Non selective,
brief to variable in carnivores
Extensive, non-selective 'closure' in 12 h
Ig : immunoglobulin
Fig. 3
Transmission of immunity from mother to young
Diagram adapted from Butler (26)
Mammals are grouped according to the method used by each in transmission of antibodies to their offspring. The size of the symbol used for the Ig indicates its
relative concentration in colostrum
IgG is the major Ig in colostrum in swine (Fig. 4 ) but is
replaced in that role by IgA after the first week of lactation.
The serum levels of Igs in the sow are significantly influenced
by the reproductive cycle (Fig. 5 ) . The precipitous decline in
serum IgG levels towards the end of gestation corresponds to
the time at which the colostrum-forming gland is selectively
accumulating levels of IgG approaching 1 0 0 mg/ml (31). The
pattern seen in sows (Fig. 5) is also seen in ewes, cows and
mares, i.e., in species in which passive IgG antibody is
delivered to the neonate through the mammary gland. In
sows, changes in serum IgM levels modestly parallel those of
IgG during reproduction (Fig. 5 ) . More noteworthy is the
increase in serum IgA levels towards the end of gestation and
during early lactation. The sow mammary glands can produce
more than 3 0 g of IgA daily (9, 147), which is 30-fold higher
than human production levels. The increased serum IgA
levels may reflect uptake by the mammary gland lymphatics
of IgA produced by the abundant plasma cell population
within the gland. The changes in swine in serum Ig levels
shown in Figure 5 illustrate the significant impact which
reproduction has on the immune system of a Group III
mammal.
While the IgA of mature swine milk is produced in the gland
and in lymph nodes associated with the mammary gland (17),
the precursors of these IgA-secreting cells are believed to have
been stimulated in the gut by enteric antigen ( 1 3 ) . The
concept of a gut-mammary gland axis for the supply of IgA
anübodies to the newborn of essentially all mammals has
become well-established (27, 136). Such a pathway probably
evolved due to the fact that newborn mammals are at major
risk to pathogens entering by the oral route.
IgA is also the major Ig in nasal and tracheal secretions by
more than a 20:1 ratio to IgG (Fig. 4 ) , although the
corresponding ratio in the lower respiratory tract is lower than
1:1 (108). While over 9 5 % of the IgA is produced locally, only
a portion of the IgG is derived from local production ( 2 1 % in
nasal secretions and 6 0 % in the lower respiratory tract). Intact
IgA is also secreted in urine ( 1 8 , 122), whereas much of the
IgG in urine is fragmented. IgA-containing plasma cells are
prominent in the reproductive tract and numbers of these
cells increase during oestrus ( 7 6 ) . IgA is the predominant
Ig-containing cell in the mesenteric lymph node (MLN) and in
the various Peyer's patches along the gut after two weeks (10).
Rev. sci. tech. Off. int. Epiz., 17 (1)
52
Table II
Serum concentration of immunoglobulins in cattle, swine and horses
(33)
Cattle
|a|
Immunoglobulin
Cattle
Swine
Horses
IgM
3.0(12.8%)
2.5 ( 8 . 8 % )
1.6 ( 6 . 2 % )
IgA
0.37(1.5%)
2.0 ( 7 . 0 % )
3.2(12.3%)
21.1 ( 8 1 % )
20.4 (86%)
24.0 (84%)
lgG1
11.2 ( 4 7 % )
7
NA
lgG2
9.2 ( 3 8 % )
NA
NA
lgG2a
NA
?
NA
lgG2b
NA
?
NA
lgG3
?
?
NA
lgG4
NA
?
NA
IgGa
NA
NA
?
IgGb
NA
NA
?
IgGc
NA
NA
?
IgG(T)
NA
NA
8.2 ( 3 2 % )
NA
NA
7
?
?
7
IgG (total)
IgG(B)
igE
Swine
| b )
a) Absolute values reported by investigators depend on:
i) method of determination
ii) age of the animals
iii) sex of the animal, especially if female (see Fig. 5)
iv) breed or herd tested
The value in parenthesis indicates the proportion of total serum Igs contributed by the Ig in
question
b) Data for Shetland ponies provided by Veterinary Medical Research Diagnostics (VMRD)
NA: not applicable since an Ig by the same designation has not been reported for the species
indicated
?: concentration unknown
Horses
Biliary transport in swine is very slow in contrast to that in
rodents and rabbits; only 2 . 1 % and 2 . 2 % of intravenously
injected IgM and IgA, respectively, is transported into bile
during the first 4 0 h (60).
Characteristics of the immunoglobulins and
immunoglobulin genes of swine
The various classes of swine immunoglobulins are highly
homologous to their counterparts in other mammals as
reflected in the following aspects:
a) in the amino acid sequences of their heavy and light chains
b) in the physical and chemical properties of the intact
immunoglobulins (20)
c) by their recognition by polyclonal
cross-react among species (29, 137).
antisera
which
IgM is highly conserved, as was initially recognised in studies
with cross-reactive antibodies (29, 104). In swine, the amino
acid sequence of the p-chain is most similar to that of sheep
and cattle and the transmembrane tail of the IgM BCR is
identical in sequence in swine, sheep and cattle ( 1 1 0 , 151).
Consistent with the latter observation is the fact that
homology among species is highest at the carboxyl terminal
end of the p-chain. This 3 ' to 5 ' trend is progressive except for
Ig : immunoglobulin
BAL : bronchial alveolar lavage
Fig. 4
The relative proportion of the major immunoglobulins in the secretions
and body fluids of cattle, swine and horses
Question marks indicate that no reliable data were available. Data are
expressed as a percentage of the total Ig concentration in the particular body
fluid
the Cp.2 domain which encodes the hinge ( 1 5 2 ) . This is
compatible with homology comparisons between the heavy
chains of other Igs as well; i.e., the greatest differences are
found in the hinge region. Overall homology of the secreted
form is highest between sheep and cattle ( 6 5 % at the protein
level) and lowest with chicken and the clawed toad, Xenopus
iaevis ( 3 7 % and 3 9 % , respectively).
53
Rev. sci. tech. Off. int. Epiz., 17(1)
DNA (cDNA) and genomic DNA have revealed an even more
complex pattern. The deduced sequences of five subclasses,
including some allotypic variants, are known and as many as
eight are suspected on the basis of genomic blots using
y-chain-specific DNA probes ( 8 0 ) . Unfortunately, mono­
clonal (or polyclonal) reagents capable of distinguishing all
the IgG subclass and allotypic variants immunochemical^ are
not available, so the relative distribution of these variants in
serum and secretions remains unknown (Table II). However,
transcripts encoding IgGl occur most frequently in lymph
nodes ( 8 0 ) , which suggests that this may be the IgG subclass
in highest concentration in the body.
Swine, like cattle, sheep, mice and perhaps horses (see below)
have a single C a locus, thus there are no IgA subclasses.
However, two very interesting allelic forms of porcine IgA
occur, including the IgA allele which encodes a heavy chain
lacking four amino acids in the hinge region ( 2 2 ) . This
molecule is commonly referred to as the 'hingeless variant' of
porcine IgA since the sequence encoded by the twelve missing
nucleotides constitutes the hinge for most species IgAs. The
IgA and IgA alleles segregate as Mendelian traits and litters
of piglets from heterozygous matings display the expected
1 : 2 : 1 ratio of genotypes. So far, animals which are
homozygous for the hingeless variant are rather scarce. The
biological significance, if any, of the lack of a hinge remains to
be determined.
b
a
b
Swine have a single gene encoding IgE, and partial sequence
analysis indicates that porcine IgE shares considerable
homology with IgE in cattle and sheep. However, the degree
of sequence homology among the IgE of large farm animals is
significantly less than the homology among IgM from these
species.
Fig. 5
The concentration of the major Igs in the serum of > 1,000 sows during
the reproductive cycle
The mean values are depicted by the heavy solid line and the variations
represent standard deviations (31,33)
The swine switch p has also been cloned and sequenced:
swine is the third species (alter mice and humans) for which
an Sp sequence has been characterised. Swine switch p is
3 , 2 kb in length and contains more than 4 0 0 pentameric
repeats; gagct is the dominant repeat. Swine Sp is highly
conserved and most similar to human switch p ( 1 5 2 ) . There is
no gene encoding IgD in swine ( 4 1 ) (Table I).
Perhaps most surprising in swine is the subclass
heterogeneity. Serological studies recognise two to four
different IgG subclasses ( 1 4 , 4 7 , 8 3 , 8 5 , 1 0 6 , 1 1 7 , 1 2 9 , 1 6 0 )
and numerous allotypes ( 1 2 7 ) . Analyses of complementary
Light chains corresponding to K and X were recognised from
protein sequences ( 5 7 , 7 4 ) and serological cross-reactivity
( 1 3 7 ) . As in humans, K - and X-analogues in swine appear in
approximately equal proportions ( 7 4 , 1 5 4 ) . Genes encoding
both K - and X-light chains have been cloned and sequenced.
C K and CX regions encode 1 0 8 and 1 0 5 amino acid,
respectively, and show 3 2 % similarity to each other ( 9 3 ) . C K
and CX are highly conserved and porcine CA, is equally
homologous ( 6 8 % to 7 4 % ) to CX in humans, cattle, rabbits
and mice. Of particular interest was the discovery that the JX
and J K segments cloned in the study cited showed 8 9 % and
8 0 % homology to consensus human JX and J K . The similar
ratio of k/X in swine Igs to that in primates is noteworthy,
since the ratio is very different from that in other related
artiodactyls such as cattle, sheep and horses.
The variable heavy chain genes of swine
The consensus opinion among immunologists is that the
variable heavy chain sequence contributes most to antibody
specificity. In any case, almost nothing is known about VX- or
V K - g e n e s in swine.
54
Rev. sci. tech. Off. int. Epiz., 17 (1)
Swine, like rabbits and chickens, have VH genes which belong
to a single family, V 3 (Table I). The number of V genes in
swine is small, estimated to be less than 2 0 (150). This is a
major departure from the rabbit, which is estimated to have
more than 100 VH genes (46). Chickens have 8 0 to 100 VH
genes, though all but the most 3 ' genes are pseudogenes
(162). Although the exact number of pseudogenes in swine
remains unknown, only one has been identified so far (151).
Since swine V genes belong to a single family they are highly
homologous and their leader and FR1 regions are essentially
identical (147, 149).
H
H
H
secondary immune responses, i.e., the foetus or newborn
were previously exposed to the same antigen. The
contaminant theory is flawed since contamination by
maternal blood, either at birth or in utero, should result in a
predominance of IgG in the sera of newborn piglets rather
than IgM. Rejection of the contamination theory gives
credence to the concept of natural antibodies. Since such
antibodies are broadly specific IgM, they might be best
appreciated as an arm of the innate immune system ( 1 0 3 ) .
Recent studies have shown that the variable regions of foetal
and neonatal antibodies are encoded primarily by four V
genes, two D segments and one J , and that somatic
mutation is absent. Thus the developing foetus has a very
limited antibody repertoire. It is important to determine
whether the limited natural antibody repertoire encoded by
these VDJs does indeed provide some measure of protective,
'innate immunity' for the newborn piglet.
H
The number of D segments in swine is still unknown,
although more than 9 5 % of all foetal and neonatal piglets use
either D A or D B (41, 1 5 1 ) .
H
H
H
The occurrence of only a single J segment in swine is of
interest (41), since this is similar to chickens but unlike all
other mammals so far examined (Table I). Thus the heavy
chain variable region locus of swine appears to be much
smaller than that described for rodents, primates and the
rabbit, and this raises the possibility that the swine antibody
repertoire is also more limited.
H
Development of the antibody
repertoire in swine
Haematopoiesis begins on day 16 in foetal liver, the yolk sac
becomes non-functional by day 2 4 and the first lymphocytes
are seen in liver on day 2 8 (157). VDJ rearrangements can be
detected in approximately 5 0 % of foetal livers on day 3 0
(153), but IgM(+) cells are scarce before day 5 0 (45), thus the
rearrangements seen prior to this time must represent proand pre-B cells (Fig. 1). Lymphocytes from 4 4 day foetuses
can be induced to secrete IgM if stimulated in vitro (45). VDJ
rearrangements reach their highest frequency on day 6 0 ,
which suggests that B-cell lymphogenesis may shift to the
bone marrow after this time (153). IgM in particular can be
detected in foetal serum after this time and low levels of IgG
and IgA are also present at parturition ( 8 8 ) . These natural
antibodies are apparently similar to those in other newborn
mammals (15) since they recognise self-antigens and common
bacterial antigens such as lipopolysaccharide (LPS) (45). Such
antibodies show high connectivity, low affinity and
autoreactivity, and are believed to play a regulatory role
during immunological development (4). There have been
other suggestions that the antibodies may be important for
protection against common bacterial pathogens ( 1 5 , 167),
and Reid et al. have recently shown that such natural
antibodies are protective against endotoxin shock in neonates
(128). Despite 3 0 years of conflicting data, Y.B. Kim continues
to regard such natural antibodies as the result of
contamination of foetal and newborn piglets during natural
birth or caesarean surgery ( 8 7 ) . He has argued that most
immune responses in other newborn mammals are in fact
H
H
At birth, the major Ig-positive cells in all swine lymphoid
tissues bear surface IgM and IgM-containing cells and these
are the first to increase after birth. The IgM cells are later
followed by IgG- or IgA-containing cells (10). IgM-containing
cells also predominate in the intestinal lamina at birth but are
gradually replaced by IgA-containing cells three to four weeks
later ( 1 , 1 0 , 2 0 , 36). Thus IgM and IgA play important roles in
the pig intestine, and this statement is supported by the high
proportion of lymphocytes in efferent lymph containing IgA
(20%) and IgM ( 1 3 % ) , while few IgG cells are seen ( 7 ) .
IgA-containing cells also become predominant in the MLN in
2-week-old conventional piglets ( 1 0 ) . As indicated, IgM(+)
cells persist in the gut mucosa whereas IgG(+) cells are
present in very low numbers (10, 3 6 ) .
What is especially surprising is the occurrence of
Ig-containing cells of all major isotypes in the thymus (10), a
situation which persists even after 10 months of age.
While numerous studies, such as those cited above, have
followed the sequential appearance of antibody isotypes in
serum and Ig(+) cells in tissues, studies are only now
underway on the diversification of the antibody repertoire in
terms of V
usage, CDR3 diversification, somatic
hypermutation and somatic gene conversion ( 4 2 , 1 5 1 , 1 5 3 ) .
Since the adult repertoire is diversified to the extent that
individual germline V or D segments cannot be recognised
and no two clones have the same sequence ( 1 5 0 ) , it is
impossible to determine whether adult animals use many
more V genes than foetal and neonatal piglets or whether
they merely diversify the same small number of V genes. A
major question surrounds the causes of the diversification of
the swine antibody repertoire after birth. Is diversification
driven intrinsically or by environmental influences? This will
be further discussed below.
H
H
H
H
H
Rev. sci. tech. Off. int. Epiz., 17 (1)
The immunoglobulins
and immunoglobulin genes
of cattle
Serum concentration and non-vascular
distribution
The numerical data presented in Table II and Figure 4 are
derived from studies in cattle. Very similar data are available
for sheep and goats, which are a very closely-related species.
Thus for conceptual and space-saving considerations, the data
presented for cattle will be considered applicable to other
domesticated ruminants.
All domesticated ruminants have an I g G l which is highly
cross-reactive among species (29). IgGl is the major Ig in the
colostrum of cows, ewes and nannies and the high
concentration of IgGl in this secretion (60 mg to 100 mg/ml)
is the consequence of a selective transport mechanism
involving IgGl-specific transport receptors in the mammary
gland ( 1 9 , 5 1 ) . Similar to the situation for total IgG in sows
(Fig. 5), serum IgGl levels decrease precipitously three to four
weeks prior to parturition, during which time IgGl is being
transported
selectively into the secretion which is
accumulating in the mammary gland ( 6 5 ) (Fig. 3 ) . To date,
the receptor responsible for this highly selective transport has
not been characterised.
IgGl and IgG2 levels in normal bovine sera are equivalent
(Table II), while values for the concentration of IgG3
(formerly known as IgG2b) ( 4 0 ) are unknown as an
IgG3-specific antibody is not available for quantitative testing.
Qualitative data would suggest that IgG3 is a minor serum
component (39).
Although not transported into colostrum or milk or into any
other secretion, IgG2 is nevertheless important
in
immunological protection. IgG2 is generally ascribed as the
most important opsonin for both neutrophil and macrophage
phagocytosis.
As is the case for most other mammals, IgM and IgA comprise
a small portion of the normal serum Igs. In cattle ( 5 3 , 1 5 9 )
and swine ( 1 6 , 4 7 ) , the majority of serum IgA is dimeric, in
contrast to human serum IgA in which 8 0 % of the 2.5 mg/ml
is monomelic. Low serum levels of dimeric IgA (less than 1
mg/ml) are the rule in most non-primates although swine
serum IgA levels are higher than most (Table II).
Figure 4 gives the relative concentration of the various Igs in
the exocrine body fluid of cattle. With similarity to IgG in
swine, IgGl in cattle (and other domesticated ruminants) is
the principal Ig of colostrum. However, unlike the situation in
either swine or horses, IgA never becomes the major Ig in
mature cow milk; rather, IgGl persists in ruminants. This was
55
initially suspected to reflect a local immunodeficiency of the
mammary gland (35) but the persistance could also reflect the
efficiency of the IgGl transport mechanism; during normal
lactation (when the IgG transport mechanism is regarded to
be downregulated) the transport of IgGl from serum into
milk exceeds the amount of IgA that the gland can produce.
Even if IgA is not the predominant Ig in bovine milk, cows are
nevertheless secreting more than 2 g of IgA/day. The
predominance of IgGl, even in the milk of mature cows, may
also reflect local synthesis within the gland or in nearby
mammary lymph nodes. Such a possibility is supported by in
vitro studies on Ig synthesis by bovine tissues ( 3 5 ) and by
calculated values of 'relative occurrence' ( 3 3 ) . The ratio of
IgGl:IgG2 in many exocrine cattle body fluids supports the
notion that IgGl is synthesised locally, or is transported
selectively into such secretions by a mechanism similar to that
which is operative in the bovine mammary gland.
Like swine, but in contrast to rodents and the rabbit, biliary
transport of IgA is very inefficient in cattle ( 3 7 ) ; most IgA is
recovered as protein fragments. Despite early reports to the
contrary ( 1 1 6 , 1 4 2 ) , serum transport of IgA into the
mammary gland of both sows and cows either does not occur,
or occurs at a very low rate (37). The same is true of IgM ( 5 9 )
even though this is also recognised with high affinity by the
poly-Ig transport receptor. These findings suggest that IgA
and IgM in bovine colostrum and milk are synthesised locally
while most all IgGl in colostrum is derived selectively from
serum, although some local synthesis of IgGl persists in the
mammary gland during lactation (see above). In a pattern
similar to that seen in swine, IgA is synthesised in the thymus
of calves (35).
The ratio of IgGldgA in body fluids of swine versus cattle
(Fig. 4 ) supports the original findings from in vitro synthesis
studies in cattle which suggested that IgGl in ruminants is
produced locally ( 3 5 ) . This has led to the concept that
ruminant IgGl is a special type of ruminant Ig which is
important both systemically and locally ( 3 0 , 1 0 8 ) , which
distinguishes ruminant artiodactyls from other members of
the same family of mammals. Whether or not this difference
evolved with the development of ruminant digestion can only
be speculated.
Characterisation of ruminant immunoglobulins
and immunoglobulin genes
The Igs of catde and the genes encoding them are the most
completely characterised of the large farm animals. The
constant region of the heavy chain locus contains genes
encoding IgM, three subclasses of IgG, IgE and IgA (90). Like
swine, cattle lack IgD ( 4 1 , 1 1 2 ) . Deduced amino acid
sequences have been published for each isotype and for
allotypic variants of I g G l , IgG2 and IgG3 ( 2 3 , 7 1 , 8 2 , 1 0 9 ,
1 2 6 , 1 5 5 ) . Sequence data are also available for ovine IgM (68),
IgGl (55) and IgE (54); these are more than 7 5 % homologous
to those in cattle. Furthermore, polyclonal and monoclonal
reagents specific for the heavy chains of the bovine Igs are
56
Hev. sci. tech. Off. int. Epiz., 17 (1)
strongly cross-reactive with their ovine homologues ( 2 9 ,
118).
In the case of IgM, immunodiffusion
tests show
complete identity. For these reasons, a separate section
devoted to sheep has been omitted from this review.
Allotypic variants of bovine immunoglobulins
were
recognised early in the study of the Igs of this species ( 3 0 )
with the two allelic forms of IgG2 having been most
thoroughly studied (12, 3 8 , 7 0 , 7 1 , 8 2 ) . These allotypic
variants, IgG2 and I g G 2 , differ significantly in the hinge
region, and the CH3 domain of IgG2 contains an
immunodominant epitope which is recognised by most
polyclonal and monoclonal reagents and which can cause
IgG2 detection bias in serological assays ( 4 0 ) . While there is
no overt clinical correlation between allotype and disease
susceptibility ( 8 1 ) , there is a difference in complement
activation
(5) and
in
the
immune
response
to
Haemophilus somnus ( 4 4 ) .
a
b
a
Since IgM is the most conserved Ig among vertebrates, the
very high rate of homology among closely-related species is
not surprising. Between cattle and sheep, homology is 8 8 %
and the transmembrane tail of IgM is identical in sequence for
cattle, sheep and swine (110). Homology with swine, another
artiodactyl, ranges from 6 5 % to 7 5 % .
The discovery of IgA in cattle was initially delayed because
investigators focused on colostrum without considering that
the mode of passive immunity in Group 111 mammals was
different from that in rabbits and humans ( 1 4 9 ) . IgA was
ultimately shown to be the major Ig synthesised by mucosal
tissues in cattle ( 3 5 ) and found in exocrine secretions, except
colostrum and milk ( 1 0 1 ) (Fig. 4 ) . Serum IgA in cattle and
other domesticated ruminants is dimeric ( 5 3 , 159) and is
present in low concentration compared to humans, whereas
high levels of monomelic IgA are found (Table II). Based on
available data, humans - rather than mminants - appear to be
the exception (30).
As in other species, bovine IgA in secretions is associated with
There are two allotypes of IgG3 and apparently several
allotypes of I g G l , but these do not result in the dramatic
antigenic differences characteristic of the genetic variants of
IgG2. Both serological ( 4 8 ) and sequence evidence exist for
allotypes of bovine IgA ( 2 3 ) . The latter rests on restriction
fragment length polymorphism (RFLP) and one Brown Swiss
animal which differed from 5 0 Swedish cattle. However, this
observation is consistent with the report of an RFLP variant in
Holstein cattle (90). Although IgM is monomorphic in most
species, there is evidence from RFLP, from sequence analysis
and from the differential specificity of monoclonal anti-IgMs
that allotypic variants of IgM occur in cattle and sheep ( 6 8 ,
110,113).
a secretory component (SC) of circa 8 6 kiloDaltons (kDa) ( 3 4 ,
100). Free SC is abundant in colostrum and milk and SC was
actually first described as glycoprotein-a ( 6 4 ) . Even in the
milk of mature cows, SC levels average 2 5 0 pg/ml, none of
which appears to be proteolytically released from fat globule
membranes (125). Both IgA and SC are highly cross-reactive
among domesticated ruminants ( 1 1 9 ) . Although secretory
IgA is typically an 1 I S protein, higher aggregates (~15S) are
not uncommon (25).
When the bovine IgA sequence is compared to that of other
species, the greatest variation occurs in the hinge region; this
is a reoccurring theme when comparing the IgA sequences
between species ( 2 1 , 2 3 ) . The hinge of bovine IgA is
a
comparable to that in the swine IgA allotypic variant and
consists of five amino acids, three serines and two cystienes.
Bovine IgA shares 7 5 % sequence homology with porcine IgA;
sequence data for sheep and horse IgA are not currently
available (23).
Both IgA and IgM in milk tend to associate with the fat layer
(58, 7 3 , 9 4 ) . Both Igs are 5 fold more prevalent in the fat layer
:
than in whey, so that the concentrations of these two Igs is
2- to 3-fold higher in milk fat than in milk whey. This is a
selective association, and such an association is not seen with
IgGl and IgG2 (58). Whether or not this phenomenon is of
biological significance (e.g., necessary for the delivery of these
Igs to the gut) is unknown. A similar association seen with
IgM and IgA in bronchial mucus is noteworthy (28).
As early as 1 9 6 7 ( 7 4 ) , the fact that light chain distribution in
cattle and the horse was highly skewed to favour A-chain
expression had been observed (Table I). Since studies in mice
originally showed that A-chains were only expressed if
productive K-rearrangements failed to develop, the K-locus in
these farm animals was presumed defective. In the case of
horses, however, this does not appear to be the correct
explanation, and apparently functional K-chains have also
been
identified
in
cattle
(B. Osborne,
personal
communication) and sheep (W.R. Hein and L. Dudler,
personal
communication). Nevertheless, the
skewed
A/K-chain ratio among ruminants is especially interesting
since swine, another artiodactyl, do not show this feature
(Table I).
The variable heavy and light chain genes of
ruminants
In contrast to swine, information is available on both the light
chain variable region genes of domesticated ruminants and
those encoded by the heavy chain locus. As A-chains are
predominantly
expressed in domesticated
ruminants
(Table I ) , they have been the focus of studies in both sheep
(131) and cattle ( 1 2 0 , 1 4 6 ) .
Sinclair et al. grouped the VA genes into two families of which
only one was predominantly expressed ( 1 4 6 ) and Parng et al.
showed that the A-locus in cattle contains about 2 0 Vk genes,
although 14 of these appear to be pseudogenes ( 1 2 0 ) . The
bovine Vk genes appear closely-spaced and many of the
57
Rev. sci. tech. Off. int. Epiz., 17 (1)
pseudogenes are fused to JA. in the germline, which suggests
that they are unlikely to be expressed as such. The abundance
of lambda pseudogenes in cattle had been suggested on the
basis of the transcripts recovered from a cDNA library
prepared from the mammary gland ( 7 7 ) . While more than
one JA is present in the genome, only one is expressed. Studies
in sheep have estimated that there are 9 0 to 100 VA. genes
belonging to six families in this species (132). As is discussed
below, Parng et al. presented evidence for gene conversion in
bovine VA. ( 1 2 0 ) whereas Reynaud et al. reported no evidence
of this in sheep (132). Whether this difference is a
consequence of the method used, the time of sampling or
whether it represents a real species difference, remains to be
determined.
life but there is little or no evidence for lymphopoiesis in this
organ. B cells are first seen at 4 8 days ( 1 5 0 day gestation) and
occupy 2 0 % of the spleen by 7 7 days ( 1 2 4 ) . This level of
B-cell expansion in the spleen precedes or coincides with the
presence of B cells in other lymphoid tissues and occurs in the
absence of lymphopoiesis in the bone marrow ( 1 0 7 ) .
Haematopoiesis begins at about day 70 in bone marrow while
erythopoiesis predominates until day 130; lymphopoiesis is
an inconspicuous element of haematopoiesis at any time in
foetal bone marrow (139). Considering that there are few
IgM(+) cells in liver and that proliferating B cells are present at
day 6 8 in the 1PP ( 1 2 4 ) , the spleen may play an earlier role in
B-cell lymphopoiesis in ruminants than has been described in
other species.
The expressed V genes which encode bovine and ovine
antibodies are homologues of the murine Q 5 2 family, i.e.,
clan II or V 2 (8, 5 2 , 1 3 8 , 1 4 8 ) , although homologues of
other families are also present in the genome (8, 138). Despite
the fact that the number of different VH2 genes in these
ruminants is low (13 to 15) ( 5 2 , 1 3 8 ) and is reminiscent of the
situation in swine, another artiodactyl ( 1 4 7 ) , those which are
expressed do not belong to the V 3 family.
The IPP becomes quite active by day 8 5 . The lymphocytes
present are almost exclusively IgM(+) cells and if this organ is
resected, animals remain deficient in B cells for at least one
year (61). Thus the IPP is the major source of the peripheral
B-cell pool of the sheep, although less than 5% reach the
periphery and the remainder die of apoptosis ( 1 3 3 ) . Since the
IPP is the site of both proliferation and negative selection, IPP
follicles can be inferred as the major site for generation of the
pre-immune repertoire in ruminants. In sheep, cattle, swine
and horses (all species which have IPPs), the organ reaches
maximum size early in post-natal life and then involutes with
advancing age (134); this developmental pattern is
reminiscent of that of the thymus in all mammals. By the age
of 18 months, all follicular structures have gone from the IPP
of sheep ( 1 3 1 ) . That the IPPs are indeed the site of B-cell
repertoire diversification for ruminants but not B-cell
development was first shown by Reynaud et al. (131), whose
work on VA, revealed no evidence of gene conversion
(templated mutation) as had been described for chickens
(130, 1 5 6 ) but rather displayed considerable untemplated
point mutation. When germ-free, thymectomised lambs were
compared to conventional lambs, no evidence for antigen or
T-dependence of this mutational diversification was found
( 1 3 2 ) . These observations on VA, genes in the IPP have also
been confirmed for V K genes (W.R. Hein and L . Dudler,
personal communication). Thus, in contrast to results shown
for rabbits (91) and swine ( 1 5 3 ) , vigorous somatic
hypermutation appears to occur before birth in ruminants,
and is apparently driven by intrinsic factors.
H
H
H
There is currently little definitive information on the D and
J regions in domesticated ruminants but, in contrast to the
D and j regions in swine, there appear to b e more than a
single J , even though many transcripts use the same J ( 8 ) .
Those which are expressed resemble human J 4 and J 5
(138).
H
H
H
H
H
H
H
H
Antibody repertoire development in ruminants
A great deal of what is known about B-cell development and
antibody repertoire development in ruminants is an
outgrowth of the research performed by Zdenek Trnka et al.
at the Basel Institute, and Silverstein et al. at Johns Hopkins
University. The ewe, unlike the sow, allows considerable in
utero manipulation of the foetus, including the surgical
placement of indwelling catheters, so that humoral aspects of
foetal physiology can be studied kinetically. Silverstein et al.
used this technology to demonstrate that the ruminant foetus
was immunocompetent, responding to different classes of
antigens in a progressive manner during foetal development
(145). Although some differences (or discrepancies) in the
pattern of repertoire development between cattle and sheep
are known, the discussion here - perhaps oversimplified makes the assumption that the process is more similar than
different between these two ruminants, since the phylogenetic
relationship and Ig gene homology between catde and sheep
are very high.
During foetal development in lambs, haematopoiesis begins
in the yolk sac at 16 days and then moves to the liver; the yolk
sac disappears at approximately 2 7 days ( 1 3 9 ) . The liver
remains the principal organ for haematopoiesis during foetal
Studies on the development of the V repertoire in both sheep
and catde are less advanced than those on VA. in terms of
developmental changes. Goldsby et al. have nevertheless
presented evidence in support of gene conversion ( 9 6 ) , while
three other investigative groups have made interesting
observations regarding the CDR3 region: namely, CDR3 is
especially diverse and encodes 13 to 2 8 amino acids with
many encoding more than 2 0 amino acids (8, 1 3 8 , 1 4 8 ) . In
exceptional cases, CDR3s encoding more than 5 0 amino acids
have been found (8) (A. Kaushik, personal communication).
These observations contrast with those from mice, in which
CDR3 encodes 7 to 12 amino acids. Catde share both these
H
58
exceptionally long CDR3s and the occurrence of cysteine
residues within the encoded loop with the camel ( 8 , 1 1 1 , 1 3 8 ,
148). Since the cysteines allow for potential disulfide
bridging, their presence may be needed to stabilise such long
CDR3 loops.
The immunoglobulins and
immunoglobulin genes of the
horse
The concentration and distribution of
immunoglobulins in horses
Table II demonstrates that there is no established homology or
consistent nomenclature for the various IgG sub-isotypes
among the large farm animals discussed in this review. The
case for horses is the same as for swine; various IgG
sub-isotypes are recognised but data only exist for total IgG
concentrations. Since studies in mice, humans and ruminants
have demonstrated that different IgG subclasses have different
biological functions, there is much to leam about this topic in
both swine and horses.
Serum IgM and IgA levels in horses appear most similar to
those in swine (Table II). In all large farm animals, the serum
concentration of IgE is unknown. In exocrine body fluids, the
relative contribution of the major Igs parallels that seen in
ruminants and swine (Fig. 4 ) . Concentrations of IgG are very
high, but IgA levels exceed IgG by 2:1 in the milk of mature
mares; this pattern is reminiscent of IgG levels in sows. IgA
levels are also greater than IgG levels in intestinal secretions,
although the difference is not as pronounced as in swine,
resembling rather the situation in cattle (Fig. 4 ) . IgM levels are
very low in intestinal secretions. In all secretions of the
urogenital tract, IgG predominates (164). In the respiratory
tract, IgA and IgM predominate in the nasal secretions, with
little IgG present. The levels of IgA and IgM progressively
decrease in the bronchi as IgG increases. In the lung, IgG
predominates (102). Thus the same pattern as described for
cattle and swine in terms of the relative Ig content in different
parts of the respiratory tract is true for horses (Fig. 4 ) .
Characterisation of the immunoglobulins and
immunoglobulin genes of the horse
In horses, IgM and IgA have been characterised and sequence
data are available for horse IgM (see below). The landmark
studies of Rockey et al. nearly three decades ago suggested the
existence of four subclasses of IgG (135). This is supported by
more recent work with heterohybridomas which have
identified IgGa, IgGb, IgGc and IgG(T) as subclasses ( 9 7 ) .
Although sequence data are still lacking, IgG(T) is clearly a
type of IgG and not a separate Ig isotype (163), and polyclonal
Rev. sci tech. Off. int. Epiz., 17 (1)
reagents distinguish IgG(T) from IgG (62, 6 9 ) in a manner
reminiscent of the antigenic difference which has long been
recognised between ruminant IgGl and IgG2 ( 2 4 ) . Recent
data on horse IgM are consistent with similar data from other
species in that there appears to be a single p-chain gene
encoding a conserved molecule which shows highest
homology to other mammals in the Cu3 and Cp4 domains.
When compared to other species, horse. IgM was most
homologous to human and canine IgM ( 1 4 0 ) .
Wagner et al. have provided new data on the organisation of
the horse heavy chain locus indicating the occurrence of six
Cy genes (B. Wagner, personal communication). The exact
identity of each of these in relationship to the IgG
nomenclature used in Table II, and those described by Rockey
et al. (135) and Lunn et al. (97), remains to be demonstrated.
Horses possess a single gene for Ce and Co., and these are
found at the 3 ' end of the heavy chain locus just as in
primates, rodents and the rabbit (161). Equine IgE has been
cloned and sequenced and shows highest amino acid
sequence homology with sheep IgE ( 1 1 5 ) . However, when
homology comparisons are made using either sheep or bovine
IgE as the standard, horse IgE is no more similar to ruminant
IgE than dog IgE. Homology is highest in C e 3 and C e 4 ,
which perhaps imparts 'IgE-ness' to the molecule since both
Fcsl and Fcell recognise the C s 3 domain and recognition by
the Fcsl receptor is necessary for the biological activity of IgE.
Although the sequence of horse IgA is not yet available, RFLP
using the BamHI restriction enzyme suggests the presence of
allotypic variants (161). This is conceptually consistent with
studies in other mammals, including those covered in this
review, in showing that mammalian IgA is typically
polymorphic.
The ratio of A.:K in expressed horse Igs resembles that of
ruminants as a result of being heavily skewed to A-chains
(Table I). There appear to be three functional CX, genes and
one CX pseudogene (72), while there is only one G c gene in
the horse ( 5 6 ) .
Antibody repertoire development in the horse
The horse, like other large farm animals, has well-developed
IPPs which, like the thymus, appear to reach maximum
development early in life and then gradually involute ( 1 5 8 ) .
Although phylogenetically distant from swine and ruminants,
horses (Perissodactylae) are also Group III mammals with
regard to the transfer of passive immunity from mother to
young (Fig. 3 ) . One might therefore suspect that antibody
repertoire development in this species is likely to resemble
that of artiodactyls rather than that of rodents and primates.
In support of such a prediction, horse antibodies use a A-light
chain more than 9 0 % of the time, which is similar to the
pattern seen with ruminants (Table I). Usage of Ck does not
59
Rev. sci. tech. Off. int. Epiz., 17 (1)
appear to reflect the number of Vk versus V K genes since
numbers of both are nearly equal ( 5 6 , 72). According to the
V-gene rearrangement paradigm established from studies in
mice, À,-chain usage is thought to indicate that all previous
attempts at rearrangement in the kappa locus have been
non-productive. Alternatively, the mouse paradigm may not
apply universally to all species. In the case of chickens, the use
of X-light chain is due to the absence of a kappa locus. Clearly
this is not the case for horses or ruminants.
There have been no extensive examinations of the V locus or
V gene usage in horses. In the case of IgE, the expressed V
genes were clearly not of the V 3 family as in swine or rabbits,
but resembled more the V 2 family which characterises the
expressed antibody repertoire of ruminants (115). Schrenzel
et al. made similar observations when IgM transcripts were
examined ( 1 4 0 ) . These investigators also reported seven
different V genes and five distinct J segments.
H
H
H
H
H
H
H
One aspect of antibody repertoire development in horses has
been studied in Arabian foals that are unable to develop a
repertoire. Hypogammaglobulinaemia in these foals was
shown by McGuire and Poppie to be an autosomal recessive
which causes a primary combined immunodeficiency ( 9 8 ) .
More recently, Wiler et al. showed this to be the horse
equivalent of SCID (165). This is a genetic deficiency of VDJ
recombination which results from a frameshift mutation in
DNA-dependent kinase (143). The absence of the kinase
prevents both coding and signal joint formation so the SCID
foals have neither antibodies or rearranged T-cell receptors.
product rigidly before marketing; that becomes the
responsibility of the consumer. Since very few consumer
laboratories are equipped or supported to conduct such
evaluations, reagents are used 'as labelled'. The problem is
exacerbated because even if laboratories wished to conduct
such tests, suitable reference standards are unlikely to be
available for comparison.
Among the three requirements listed above, the third is most
benign and perhaps least important in collecting valid data.
Nomenclature evolves continuously as more information
becomes available and this is associated with the addition of
new names or changes to old ones, as required. Generally,
investigators and diagnostic laboratories become aware of
these changes through reviews, conferences or individual
articles. However, the nomenclature issue is related to the
issues of specificity and the availability of standards. For
example, an investigator who purchases a monoclonal
antibody specific for swine IgA needs to know whether the
antibody is specific for IgA , IgA or both. Therefore, the
Committee on the Nomenclature and Standardisation of
Immunoglobulin for Species of Veterinary Importance,
sponsored by the International Union of Immunological
Societies and the Veterinary Immunology Committee
(IU1S/VIC), has considered these three issues to be linked to
such an extent that changes or progress in any one could have
a significant impact on the other two. This section describes
the goals of this IUISATC Committee and the obstacles to
achieving these goals.
a
b
Establishment of a uniform nomenclature
International standardisation of
immunoglobulin nomenclature
and reagents for large farm
animals
The need for standardisation
Virtually all experimental studies on the immune system of
farm animals, including those on repertoire development and
clinical immunodiagnostic assays, depend on the use of
monoclonal or polyclonal antibodies specific for certain
epitopes on antibody isotypes or sub-isotypes. The validity
and usefulness of information gathered using such reagents
depends on the following factors:
a) the specificity of the reagent used
b) the availability of reference standards
c) the use of a standardised nomenclature.
The consensus opinion at the last workshop held by the
IUIS/V1C Committee was that a delay in the establishment of a
nomenclature for a particular species, until characterisation of
at least the major Igs of that species had been completed,
would be prudent (11). Since the major bovine isotypes and
sub-isotypes have been characterised at the protein and
molecular genetic level, a proposed nomenclature for this
species is currently being considered by the IUIS (Table III).
The Committee hopes to follow the pattern described for
cattle in establishing similar nomenclature for the Igs of sheep,
swine, horses and other species of veterinary importance.
Standardisation of reagents for detection and
measurement of immunoglobulins or
immunoglobulin genes
The IUIS/VIC Committee recognises the need for two types of
reagents before standardisation can become a reality. First,
there is a need for a purified standard for each isotype or
sub-isotype of Ig. In some cases, such as the IgA and IgA
allotypes of swine or the IgG2 and I g G 2 allotypes of cattle,
Igs purified from animal sera can be used as standards at least
initially. In most cases, a particular Ig isotype cannot be
obtained in this manner so hybridoma or heterohybridoma
products must be used. The advantage of such products is
a
3
Unlike the pharmaceutical and vaccine fields, there are no
national or international regulatory agencies to insure the
quality of reagents in the field of immunodiagnostics.
Suppliers of specific antibodies have no obligation to test the
b
b
Rev. sci. tech. Off. int. Epiz., 17 (1)
60
Table III
Bovine immunoglobulin heavy chain class
Proposed
Current
designation
lgG1
lgG2a(A1)
lgG2a(A2)
lgG2b/lgG3
IgA
IgM
.
a
igGi
a
y1
igGi
b
y1»
lgG2
a
y2
Locus
Allotype
G1
G1*01
Hinge, Arg218; Thr226
G1
G1*02
Hinge; Thr218, Pro224, Pro226
a
G2
G2
a
b
allotypes, subclasses, etc.
CH3; intradomain loop h e p t a p e p t i d e ; A r g 4 1 9
M i d d l e hinge, CH3, Glu419
igG2
b
y2
b
G2
G2
igG3
a
y3
a
G3
G3*01
37 a m i n o acid hinge
igG3
b
y3
b
G3
G3*02
6 amino acid substitutions; 84bp INV3 insertion
igA
a
a
a
A
A*A1
Pst I RFLP
igA
b
a
b
A
A*A2
Pst I RFLP
IgM
IgE
Location and m a j o r f e a t u r e of
Chain
designation
igE
igE
Vi
a
b
M
6
a
E
E*E1
Pst I RFLP
E
b
E
E*E2
Pst I RFLP
RFLP : restriction length fragment polymorphism
Ig
: immunoglobulin
that the transcript (as cDNA) encoding the in vitro product
can be sequenced to ensure the exact identity of the
synthesised Ig, and the sequence is recorded in GenBank. In
the case of swine and horses (or lgG3 in cattle), for which IgG
subclass proteins cannot readily be purified free of other
subclass IgGs, there is currendy no reasonable alternative to
reliance on in vitro synthesised standards, in vitro synthesised
standards must also be relied on for IgE in all species. The
strategy for preparing such 'gold standards' is outlined in
Figure 6.
The second requirement is the availability of antibodies for
each isotype, sub-isotype, L-chain type and the major
allotypic variants of a species. In this situation, only
monoclonal antibodies are considered: firstly, because the
cells which make the antibodies are theoretically immortal
and rarely mutate; secondly, because the goats and rabbits
used to prepare polyclonal antibodies die and the specificity
of their antibodies changes during the course of blood
collection; and thirdly, because monoclonal antibodies offer
the best opportunity
for recognising subclass- or
allotype-specific epitopes. A critical process in this scheme is
the testing required to confirm the specificity and usefulness
of any monoclonal reagent. Unfortunately, proper testing
requires that the reference standards described in Figure 6
first be available.
Obstacles to standardisation
Perhaps the major obstacle to the standardisation process is
the (as yet unproven) need for such measures because making
a better vaccine for livestock may not directly depend on
standardisation, and animal health economics, not basic
science, drives the funding of research. Perhaps for
companion animals, such as the horse, whose medical care is
more likely to be administered as if horses were humans, the
cost of fine-tuned immunodiagnostic kits may be tolerated by
the owner of the animal.
A second obstacle is that national or international funding
agencies view the type of research needed to achieve the goals
outlined above as purely technical and there is little
enthusiasm for the support of such work. This means that the
laboratories which must perform such work are almost
certainly federal or state laboratories which do not depend on
the 'soft money' provided by competitive research grants.
A third obstacle to standardisation is the resistance from the
private sector, i.e., the reagent suppliers. Given the current
situation that suppliers are under no obligation to prove the
quality of their reagents, what would happen if a vendor were
to supply a reference centre with a monoclonal antibody
which was widely used by investigators who found out that
the antibody did not have the same performance as
advertised? This is equivalent to allowing a geneticist into a
famous dog kennel to deliberately search for genetic defects
among the founder animals!
Thus, the success of the effort to standardise reagents as
envisioned by the IUIS/VIC Committee will depend on the
interaction of many factors at the level of the bench scientists,
federal administrators, the reagent suppliers and the
consumers.
61
Rev. sci. tech. Off. int. Epiz., 17 (1 )
S o u r c e o f m o n o c l o n a l I m m u n o g l o b u l i n s (Igs)
Heterohybridomas
True hybridomas
Supernatant harvest
Cell harvest
Transfectomas
(engineered antibodies)
IgGs
Ascites fluid
Other
Total RNA
Igs
Grown in
serum-free
Standard
media
media
1st strand c D N A
Biochemical and
Protein-G
affiny column
Anti-Fab
chromatographic
affinity column
purification
Sequence analysis
C h a r a c t e r i s a t i o n o f p u r i f i e d Ig b y
S D S - P A G E , IEF, e t c .
Preservation
[20% glycerol, - 7 0 ° C ]
S i m u l a t e d ELISA
Fab
cDNA
ig
SDS-PAGE
IEF
EtISA
SRIg
Capture A b
performance
monovalent antibody fragment produced by papain digestion
complementary DNA
immunoglobulin
sodium dodecyl sulphate polyacrylamide gel electrophoresis
isoelecric focusing
enzyme-linked immunosorbent assay
standard reference immunoglobulin
Fig. 6
Establishment of standard reference Igs for animals of veterinary importance
SRIgs should be monoclonal and should be supported by sequence data. Since these are derived from hybridoma cell lines (or engineered) their deduced
sequences can be determined from their cDNAs. The SRIgs are purified from the culture supernatants (or ascites) and their protein integrity and characteristics
determined. These SRIgs will be used to prepare reference standard sera for world-wide distribution and for testing the specificity of monoclonal antibodies to
Igs inappropriate ELISA tests
Future studies on antibody
repertoire development in farm
animals
A general perspective
Antibodies, the cells which secrete antibodies and the B cells
which display these as BCRs are an essential arm of specific
adaptive immunity. Those encoded by foetal genes as
so-called 'natural antibodies' may also function as a part of
innate immunity, perhaps in a similar manner to that
proposed for NK cells and ylb cells ( 6 ) .
In addition to intrinsically-encoded natural antibodies, the
mammalian immune system has an extraordinary capacity to
diversify its antigen receptors through combinatorial joining,
junctional diversity and somatic mutation. Since large farm
animals appear to have a smaller and less diverse variable gene
genome than humans and mice, the mechanisms which are
62
Rev. sci. tech. Off. int. Epiz., 17 (1)
used by these animals to develop adult repertoires may not
follow
the
paradigm
established
in
laboratory
mice.
Furthermore, the intrinsic, maternal or environmental factors
which influence this process may also differ from rodents
because of differences in the passive transfer of immunity
(Fig. 3 ) .
Although antibody diversity and repertoire development are
basic science issues, these processes are no doubt an
important ingredient in animal health. Clearly, the ability to
design effective vaccines will depend on an understanding of
antibody repertoire development. This knowledge will be
more important in the future as biotechnology develops more
sophisticated approaches to problem solving, especially in the
area of vaccination o r improving animal health. Proper
repertoire development may also depend on nutritional
factors which can influence the growth of the microbial gut
flora which colonises the gastrointestinal tract of the newborn.
If the need to better understand this process is accepted, then
what priority should be given to research in this area of
immunology? The author discusses his perception of this
issue below.
Complete the characterisation of the
immunoglobulin genome
A problem which has plagued veterinary immunology for
thirty years is that only 'bits and pieces' of information have
been available on the immunoglobulins of large farm animals.
Since only partial information is available, an appreciation of
how the immune systems of large farm animals differ from
those of well-studied laboratory animals has been difficult to
achieve, and therefore the task of justifying research on these
species per se has also been harder. The lack of information
has also meant that reagents were not available to characterise
humoral immune responses carefully in these species. While
knowledge of the Igs of sheep, cattle, horses and swine were
on a par with that known in humans, mice and rabbits in the
late 1960s, information about the Igs of the latter species
rapidly expanded in the next two decades while very few
advances occurred for large farm animals. Thus, significant
gaps exist in current knowledge of the potential repertoire of
the large farm animals. These gaps include the following:
a) the number and biological activities of the IgG subclasses,
especially in swine but also in horses
b) the heavy chain variable region genes of horses and the Vk
and V K genes of swine. Reasonable progress is apparently
being made in characterising these regions of the genome in
the other large farm animals
c) the circumstances and cytokine milieu which determine
whether T 1 and T 2-like responses occur in large farm
animals as in rodents and humans and the IgG subclass
responses which serve as indicators of T 1 and T 2 responses
in different farm animals.
H
H
H
H
Characterisation of B-cell development and
repertoire development in large farm animals
Investigators working with laboratory animals and humans
have ' available panels of monoclonal antibodies which
recognise CD markers of pro-, pre- and B cells, thus allowing
the careful examination of B-cell ontogeny. Such reagents may
be applied directly to tissues, to leucocyte population
recovered from animals or may be used in immobilised form
to purify and sort the various cell populations. The
unavailability of such reagents for large farm animals has
seriously hindered progress. For example, even though some
DNA probes are available for use in situ, there are no reagents
available to identify the cell type which may specifically
hybridise with these probes.
In addition to the need for monoclonal antibodies specific to
CD markers and other antigens of the B-cell lineage, there is
also a need for more DNA probes which can specifically
hybridise with variable region genes, or gene segments,
transcripts of regulatory enzymes and polymerases as well as
promoter and enhancer sequences.
Both of these categories of reagents will be needed for use in
tandem before definitive answers to many of the important
questions on B-cell and repertoire development in large farm
animals can be given. Assuming that these technological
advances will be made in due course, there are several
important issues regarding repertoire development in large
farm animals which have a major impact on animal health. A
few are listed below:
a) What determines variable region gene usage during foetal
life, and are the 'natural antibodies' encoded by these foetal
V(D)Js important for survival of the neonate?
b) Is colonisation of the gastrointestinal tract of the newborn
necessary for survival and/or is this responsible for
diversification of the antibody repertoire?
c) Are maternal factors that are transmitted in milk or
colostrum to the offspring of large farm animals instrumental
in directing (or suppressing) the development of the antibody
repertoire in neonates? Is this activation (or suppression)
important to animal health?
d) Are swine, horses and cattle dependent on the proper
development of the IPP to the same degree as sheep?
Furthermore, if this organ is the principal site of B-cell
repertoire diversification in all large farm animals, what
mechanism(s) regulate this diversification?
e) Are the B cells of all large farm animals the equivalent of
the B-1 cell population in mice, or do farm animals also have a
conventional (B-2) cell population?
International standardisation of nomenclature
and reagents
The previous section of this article reviewed the objectives,
progress and obstacles being encountered in the attempt to
63
Rev. sci. tech. Off. int. Epiz., 17(1)
standardise the nomenclature of Igs of animals of veterinary
importance. It is too early to tell whether this effort will be
successful, since that will really depend on the priority given
to standardisation in the budgets of institutes and funding
agencies and whether or not such work is viewed as
favourable to reagent suppliers or rather something which
would curb profits. Should such an effort be successful, future
standardisation could then focus more on the standardisation
of DNA probes. This might be a far simpler procedure than
standardisation of the Ig proteins and monoclonal reagents.
Conclusion
The introduction indicated that holes' in the antibody
repertoire exist in some cases and can determine life and
death. Like an immunodeficiency, there are many holes' in
current understanding of Igs and Ig genes of large farm
animals which can be critical for proper management and
veterinary practice in dealing with large farm animals.
•
Diversité des immunoglobulines, développement des cellules B
et du répertoire des anticorps chez les grands animaux d'élevage
J.E. Butler
Résumé
La lignée des lymphocytes B, les anticorps produits par ces cellules et la
diversification du répertoire des anticorps sont essentiels à la santé et à la survie
de tous les mammifères. Les bovins, ovins, porcins et équins, contrairement aux
rongeurs et aux primates, développent leur répertoire d'anticorps à partir d'un
nombre relativement faible de gènes V (variable heavy) d'une ou de plusieurs
familles, et les bovins, ovins et équins recourent presque exclusivement aux
chaînes légères À. Ces grands animaux domestiques semblent utiliser des
conversions géniques en sus des mutations ponctuelles dans le développement
du répertoire; ce processus pourrait essentiellement se produire dans les
plaques de Peyer de l'iléon. On ignore encore si la lymphogénèse des cellules B
se poursuit to ut au long de la vie - comme chez les rongeurs et les primates - ou si
les cellules B appartiennent en grande partie à la lignée B-1 et se développent
uniquement au cours des stades foetal et néonatal. Le fait que l'immunoglobuline
D (IgD) soit totalement absente chez les porcins et les ruminants pourrait
constituer un élément significatif, dans la mesure où l'IgD est faiblement exprimée
dans les cellules B-1 des rongeurs.
La diversité de la sous-classe d'IgG chez les grands animaux d'élevage n'est
complètement connue que pour les ovins et les bovins, et il n'y a pas de données
pour aucune grande espèce d'élevage sur l'existence d'une corrélation entre les
réponses des lymphocytes « T helper » (Th1) et Th2 et l'expression d'une
sous-classe particulière d'anticorps, comme c'est le cas chez les rongeurs. Dans
toutes les espèces animales considérées, la transmission de l'immunité à la
descendance se fait exclusivement par la glande mammaire, bien que le
récepteur intervenant dans le transfert de l'IgG au colostrum et au lait n'ait pas
été caractérisé.
Les actions visant à standardiser la nomenclature et les titrages d'anticorps et
d'immunoglobulines chez les animaux sus-visés font l'objet de la discussion et un
projet en cours, portant sur la standardisation de la nomenclature des
immunoglobulines bovines, est présenté à titre de modèle.
H
Mots-clés
Bovins - Cellules B - Développement du système immunitaire - Equins - Génétique Immunité passive - Nomenclature - Porcins.
•
64
Rev. sci. tech. Off. int. Epiz.. 17 (1)
Diversidad de inmunoglobulinas, linfocitos B y desarrollo del
repertorio de anticuerpos en los mamíferos de granja
J.E. Butler
Resumen
La diferenciación de los linfocitos B, la producción de inmunoglobulinas por parte
de estas células y la diversificación del repertorio de anticuerpos son procesos
fundamentales para la salud y la supervivencia de cualquier mamífero. A
diferencia de lo que ocurre en roedores y primates, el repertorio de anticuerpos
de los bovinos, ovinos, porcinos y equinos se desarrolla a partir de un número
relativamente pequeño de genes V (variable heavy) de una o varias familias. Es
más, los bovinos, ovinos y equinos utilizan para ello casi exclusivamente las
cadenas ligeras. En la creación del repertorio de anticuerpos de eses grandes
animales parecen intervenir, además de mutaciones "no emparejadas"
(puntuales), mutaciones "emparejadas" (por conversión génica), proceso que
podría tener lugar principalmente en las placas de Peyer del íleon. No se sabe
con certeza si la linfogénesis de células B es un proceso continuo a lo largo del
ciclo vital -como ocurre en roedores y primates- o si dichas células descienden
en su mayoría del linaje B-1 y se desarrollan sólo durante las etapas fetal y
neonatal de la vida. El hecho de que los porcinos y los rumiantes carezcan por
completo de inmunoglobulinas D (IgD) podría ser significativo, dado que estos
anticuerpos se expresan débilmente en las células B-1 de los roedores.
La información existente sobre la diversidad de IgG en el ganado es incompleta,
excepto en lo que atañe a ovinos y bovinos. No hay datos que permitan saber si,
en los grandes animales la respuesta del linfocito T coadyuvante (helper) 1 (Th1 ) y
la del Th2 están correlacionadas con la expresión de la respuesta de alguna
subclase de anticuerpos, como ocurre en los roedores. En todos estos animales,
la glándula mamaria representa la única vía por la que se transmite inmunidad a
la descendencia, aunque hasta ahora no ha podido caracterizarse el receptor
implicado en el transporte de la IgG al calostro y la leche.
Se examinan aquí las tentativas realizadas para estandarizar la nomenclatura y la
medida de anticuerpos e inmunoglobulinas en las ya mencionadas especies, y se
presenta como modelo una propuesta (actualmente objeto de revisión) para
estandarizar la nomenclatura de las inmunoglobulinas bovinas.
H
Palabras clave
Bovinos - Desarrollo del repertorio de anticuerpos - Equinos - Genética - Inmunidad
pasiva - Linfocitos B - Nomenclatura - Porcinos.
•
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-
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