A.
Phagocytosis Phagocytic
cells:
1. Neutrophils circulate in the blood
and migrate quickly in response to local invasion by
microorganisms.
2. Monocytes are derived from bone
marrow stem cells and circulate in the blood. They
migrate to the tissues, where they differentiate into
macrophages, which reside in all body tissues (e.g.,
Kupffer cells of the liver).
Phagocytic process:
1. Ameboid movement.
2.
Chemotaxis.
3.
Ingestion.
4.
Digestion.
Lysosomal granules:
The contents
of lysosomal granules include two mechanisms for
destroying foreign particles:
1. Certain proteins kill
microorganisms by oxygen-independent mechanisms.
a. Proteinases
b.
Cationic
proteins
c.
Lysozyme
d.
Lactoferrin
2. Other microbicidal
compounds are generated by oxygen-dependent
mechanisms, including:
a. Myeloperoxidase
b.
Hydrogen
peroxide
c.
Superoxide
anion
d.
Single
oxygen
e.
Hydroxyl
radical
Two kinds of
lysosomal granules:
1. Primary granules,
azurophilic granules.
2.
Secondary
or specific granules, alkaline phosphatase,
lactoferrin, and lysozyme.
Opsonization. Phagocytosis can be
remarkably enhanced in the presence of blood serum or
plasma, because of opsonization, which is the process of
enhancing phagocytoses via the presence of opsonins.
Molecules attached to particles serve as ligands for
specific phagocyte receptors:
1. The C3b split product
of the complement cascade
2.
C5a and C5b67
3.
Antibodies
4.
Fibronectin
5.
Leukotrienes
6.
Tuftsin
B.
Complement
The complement
system plays a major role in host defense and the
inflammatory process. Complement consists of a complex
series of at least 15 plasma proteins that normally are
functionally inactive. Activation of the complement
system results in:
Opsonic function,
complement components coat pathogenic organisms or immune
complexes, facilitating the process of phagocytosis.
Inflammatory
function, induction of histamine release from mast cells
and basophils and stimulation of the inflammatory
response.
Cytotoxicity by
membrane attack of target cells (e.g., bacteria and tumor
cells) leading to cell lysis.
B1.
THE CLASSICAL PATHWAY OF ACTIVATION
Complement is
activated sequentially in a cascading manner, with a
protein activated only by the protein that directly
preceded it in the sequence. Activation may occur via two
pathways, the classical and the alternative pathways. The
classical pathway require the interaction of all nine
major complement components.
Activation via
antigen-antibody complexes or by aggregated
immunoglobulins.
1. IgG (mainly IgG1,
IgG2,
and IgG3)
and IgM are most efficient in reacting with
complement.
2. Classically, an antigen-antibody
complex is designated by EA, where E is the antigen
and A is the antibody. EA might represent immune
complexes or antibody-coated bacteria, tumor cells,
or lymphocytes. Complement components bind to EA in
an orderly sequence to form a macromolecular complex,
EAC 1,4,2,3,5,6,7,8,9.
The classical
pathway involves the following components and steps:
1. C1: When IgG or IgM reacts
with antigen, the Fc fragment of the immunoglobulin
provides a C1 binding site. C1, also called the
recognition unit, contains three polypeptides-C1q, C1r
and C1s-that are held together by calcium ions. With
the removal of the calcium, C1 breaks down into its
three subunits.
a. C1q, the portion of
the molecule that attaches first to
immunoglobulin and initiates complement
activation, has six binding sites. Because of its
multivalency it can cross-link multiple
immunoglobulin molecules.
b. C1q binding leads
to activation of C1r proenzymes.
c. Activated C1r
cleaves the proenzyme C1s. The latter acquires
esterase activity and is referred to as C1
esterase (C1s). Calcium ions are essential for
activation of C1.
2. C4: C1s mediates cleavage
of native C4, the next component in the complement
cascade, into C4a and C4b. One molecule of C1s can
cleave several C4 molecules, thus serving as one of
the sites in the amplification process.
a. C4a, one of three
anaphylatoxins, is released into the fluid phase.
b. C4b can bind to
cell membranes, but rather inefficiently.
3. C2: C1s in the presence of
C4b cleaves the next component, C2, into C2a and C2b.
a. C2a remains linked
to the cell-bound C4b, thus forming the
bimolecular complex C4b2a. Magnesium ions are
required for formation of the C4b2a complex. C4b2a
has enzymatic activity and is referred to a
classical pathway C3 convertase.
b. C2b is released
into the fluid phase.
4. C3: The substrate for C3
convertase is C3. Circulating C3 binds to the C4b2a
complex and is cleaved into two fragments, C3a and C3b.
a. C3a is an
anaphylatoxin and remains unbound.
b. C3b is bound to the
membrane in the vicinity of the C4b2a complex (C3
convertase), forming a trimolecular complex, C4b2a3b,
which has enzymatic activity. The C4b2a3b complex
is also referred to as C5 convertase, which acts
on C5, the first complement component of the
membrane attack pathway.
B2.
THE ALTERNATIVE PATHWAY OF ACTIVATION
The alternative
pathway, also referred to as the properdin pathway, is
considered to be a primitive defense system, a bypass
mechanism that does not require C1, C4, and C2
interaction.
Activation can be
triggered immunologically (e.g., by IgA and some IgG) and
non immunologically (e.g., by certain microbial cell
surfaces, complex polysaccharides, and bacterial
lipopolysaccharides).
The alternative
pathway involves the following components and steps:
1. C3: The initial
recognition event necessary for alternative pathway
activation is the presence of C3, specifically C3b,
which is probably continuously generated in small
amounts in the circulation.
a. Factor B. C3b
interacts with factor B, also called the C3
proactivator, to form C3b, B, which is a
magnesium ion-dependent complex.
b. Factor D. The
complex C3b, B is susceptible to enzymatic
cleavage by factor D, also called C3 proactivator
convertase, into two fragments, Ba and Bb. The Ba
fragment is released. An active site is exposed
in the Bb fragment, which remains bound to C3b,
forming th C3b, Bb complex, also called
amplification C3 convertase. When stabilized by
the binding of properdin (P), which slows the
dissociation of Bb, the C3b, Bb complex becomes a
C3 convertase that cleaves C3 and generates more
C3b. C3b then fixes to the activator surface so
that more factor B binding sites are exposed, and
the amplification loop amplifies the initial
recognition event.
As more C3b is
generated, the complex expands (C3bn,
Bb; where n > 1) and becomes a C5 convertase capable
of cleaving C5 into C5a and C5b and initiating the
membrane attack pathway.
Cobra venom factor
(CVF) contained in cobra venom has the interesting
property of activating the alternative complement pathway.
Like C3b, it complexes with Bb to form CVF, Bb which is
resistant to factors H and I and capable of continuously
activating the C3 -to-C3b conversion leading to
complement depletion.
B3.
THE (COMMON) MEMBRANE ATTACK PATHWAY
The pathway of
membrane attack, also called the common pathway, is
marked by the convergence of the classical and
alternative pathways at the point of C5 activation.
Activation of the membrane attack complex is initiated by
C5 convertase (i.e., C4b2a3b in the classical pathway and
C3bnBb
in the alternative pathway). This is the only component
in the attack complex that has enzymatic activity, with
cleavage occurring only once. All other components bind
spontaneously.
The membrane
attack complex involves the following components and
steps.
1. C5 is cleaved by C5
convertase into a smaller C5a fragment and a larger C5b
fragment.
a. C5a, an
anaphylatoxin, is released into the surrounding
fluid medium.
b. C5b is the first
component of the membrane attack complex. It is
the receptor for the C6 and C7 components.
2. C6 and C7: Unstable C5b binds to C6,
forming a stable C5b67 complex that is bound to the
target cell membrane.
3. C8 attaches to the
membrane-bound C5b67 complex and membrane leakage
begins. Cell lysis can occur by the C5b678 complex in
the absence of C9.
4. C9 attachment to the C5b678
complex serves a primary function of greatly
accelerating cytolysis by the production of circular
lesions in the membrane.
5, Cell lysis.
The
C5b6789 complex induces the formation of hollow
cylinders (tubules) about 15 nm long and 8-12 nm in
diameter in the lipid bilayer of the cell membrane,
allowing passage of electrolytes and water across the
membrane and leading ultimately to osmotic lysis of
the cell.
B4.
REGULATORY MECHANISMS.
Activation of the
complement components is associated with potent
biological functions that, if left unchecked, would
exhaust the complement system. Uncontrolled activation of
the complement system is prevented by several serum
proteins that bind to (inhibit) or enzymatically attack (inactivate)
complement components.
B5.
BIOLOGICAL CONSEQUENCES OF COMPLEMENT ACTIVATION.
During complement
activation, several materials with important biological
activities are generated.
C3 and C5 and
their cleavage products appear to be the most important
complement components in terms of biological function.
1. C3a and C5a are
referred to as anaphylatoxins.
a. They cause the
release of vasoactive amines (e.g., histamine)
from mast cells and basophils in a manner that
stimulates mediator release by IgE.
b. Mediator release
causes smooth muscle contraction and increases
vascular permeability, effects which can be
counteracted by antihistamines and anaphylatoxin
inactivator.
c. C5a is much more
active than C3a on a molar basis and, in addition
to anaphylatoxin activity, has a wider range of
biological activity, including the following:
-
Chemotactic factor
- Neutropenia
- Oxidative burst, and of degranulation of
neutrophils
- Production of leukotrienes
2. C3b generation and
coating on target cells through complement receptors
(CR1) on monocytes, neutrophils and B cells is
perhaps the major biological function of complement.
Its role in the activation of the alternative
complement pathway has been described. C3b also plays
an important role in opsonization. C3b-coated cells
also tend to aggregate (immune adherence), a process
that also may promote phagocytosis.
3. C3e provokes a release
of neutrophils from bone marrow causing prompt
leukocytosis.
4. C3d, another cleavage
product of C3b, can interact with receptors on
lymphocytes.
C3 nephritic
factor (C3NeF) found in the circulation of patients
with mesangiocapillary glomerulonephritis. It acts as an
antibody against the C3bm Bb complex and leads to a
marked hypocomplementemia.
C4 and its
cleavage products have certain important biological
functions:
1. The binding of C1 and C4
by a virus-antibody complex can neutralize virus
activity.
2. C4a, have anaphylatoxin
activity, causing the release of histamine from mast
cells and basophils.
3. C4b receptor sites
exist on several cell types, suggesting a role for C4b
in opsonization as seen with C3b.
C2 cleavage has
been reported to be linked to the production of a kinin-like
molecule that increases vascular permeability and
contract smooth muscle. It is thought to be involved in
the symptoms seen in hereditary angioedema, a disease
caused by uncontrolled C1 activity due to deficiency
in C1 inhibitor.
Ba and Bb. Generated exclusively by
the alternative pathway have important biological
functions.
1. Ba is chemotactic for
neutrophils.
2. Bb activates
macrophages and causes them to adhere to and spread
on surfaces.
Immunodeficiencies
result from a lack of complement and faulty complement
activation.
C.
Cytokines
Exogenous as well
as endogenous agents that induce or inhibit cytokine
production or action can modulate immunologic reactions.
Exogenous stimuli are of primary importance as inducers
of endogenous cytokines. Most cytokines are produced by
many cell types in response to noxious or physiologic
stimuli, whereas lymphokines are produced only by
lymphocytes and have largely immunoregulatory functions.
Lymphokines that
are produced largely by T cells, including IL-2, IL-4, IL-5,
and IFN g, act predominantly on
lymphoid cells and are immunologically induced regulators
of the immune response. However, IL-2, IL-4, and IL-5 can
also modulate the function of a variety of other
leukocytes such as macrophages, mast cells, and
eosinophils, respectively; IFN g also acts on a broad
spectrum of cells in addition to lymphoid cells. In
contrast, IL-3 is a lymphokine that acts as a
hematopoietic growth factor. The other cytokines that
modulate the activities of lymphoid and nonlymphoid cells
are produced by many cell types and consist of IL-1, IL-6,
IL-7, IL-8, TNF, IFN a, IFN b, and TGFb. They presumably represent
intercellular signals that enable connective tissues,
skin, nervous system, and other tissues to communicate
with the immune system.
Cytokines, in turn,
regulate each other by competition, interaction, and
mutual induction in a series of lymphokine cascades and
circuits with positive or negative feedback effects. For
example, cytokines such as IL-1 and IL-2 induce the
production of other cytokines such as TNF and g-interferon. Furthermore, IL-1
and IL-2 induce each other reciprocally. Less well known,
but perhaps equally important, are observations that even
mesenchymal growth factors such as TGFb can induce IL-1
production by macrophages.
In addition to
cytokine regulation of cytokines, neuroendocrine hormonal
peptides such as endorphins and corticosteroids, as well
as products of the lipoxygenase and cyclooxygenase
pathway, can have agonistic or antagonistic effects on
some cytokine activities. The effects of cytokines can
also be regulated at the level of cell membrane receptors.
Agents that influence cytokine receptor expression
modulate the activities of these mediators. Thus, a
complex network of endogenous ligand-receptor
interactions is involved in regulating host defense
mechanisms. The therapeutic use of cytokines is still in
its infancy. However, some disease states have already
been shown to respond to interferons and IL-2. Agonists
and antagonists of the cytokines and their receptors will
probably play an important role in the eventual therapy
of inflammatory, infectious, autoimmune, and neoplastic
diseases.
C1.
INTERLEUKIN 1 (IL-1)
Activity induced:
Proliferation or differentiation of B cells; lymphokine
release from activated T cells; growth of fibroblasts,
synovial cells, and endothelial cells; tissue catabolism;
release of prostaglandin E2,
collagenase, acute phase protein; fever; natural killer
cell activity; neutrophil, macrophage, lymphocyte
chemotaxis.
Source: Monocytes/macrophages,
dendritic cells, NKC, B-cells, T-cells, endothelial cells,
fibroblasts, astrocytes, keratinocytes
C2.
INTERLEUKIN 2 (IL-2)
Activity induced:
T-cell growth factor. Proliferation and differentiation
of T-cells; growth of activated T-cells and thymocytes;
lymphokine production by T-cells; cytotoxic T-cell
activity; NKC activity; lymphokine-activated killer cell
activity.
Source: Activated
TH1 lymphocytes, some CTLs
C3.
INTERLEUKIN 3 (IL-3)
Activity induced:
Growth factor for many hematopoietic cells.
Source: Activated
TH1 and TH2 cells, some CTLs
C4.
INTERLEUKIN 4 (IL-4)
Activity induced:
Activation and growth of B cells; IgG1 and IgE switching.
Growth and survival of T-cells, fetal thymocytes.
Increases the growth of mast cells. Inhibits macrophage
activation and helps in the formation of giant
multinucleated cells. Increases class II MCH induction.
Source: Activated
TH2 lymphocytes.
C5.
INTERLEUKIN 6 (IL-6)
Activity induced:
Growth of plasmacytomas and hybridomas; production of
acute phase proteins by hepatoma cells; increased class I
MHC expression on fibroblasts.
Source: T
lymphocytes, monocytes, macrophages, fibroblasts, certain
tumor cells.
C6.
GAMMA-INTERFERON (gIF)
Activity induced:
Decreases viral replication in cells inducing the
production of antiviral proteins that interfere with
translation of viral RNA; decreases cell growth;
increases expression of class I and class II MHC
molecules in macrophages, endothelial cells and
parenchymal cells of various organs; increases NKC
activity; increases antimicrobial and tumoricidal
activity of macrophages; enhances tumor necrosis factor
and lymphotoxin activity.
Source: T
lymphocytes and NK cells.
D.
Histocompatibility system:
Significance of the HLA Complex
The mammalian
immune system is a very complex network of cellular and
molecular components, which are specifically encoded for
by gene products. A large number of genes control
specific responses to a variety of antigens, as well as
cellular interactions and the transmission of antigenic
specificities from generation to generation. The most
important genetic component is represented by a group of
genes which encode for some molecules called major
histocompatibility complex (MHC) antigens. These
molecules, in addition to their function in the
regulation of the immune response, and in the mechanism
of antigen recognition by T cells, also have a major role
in the rejection of grafts and tumors. In this lecture we
will review some of the most important immunogenetic
aspects of tissue transplantation.
D1.
MHC GENES AND MOLECULES
The major
histocompatibility complex (MHC) was first described by
Peter Gorer in 1936 as a blood group locus that
controlled the presence of antigens on the surface of
mouse erythrocytes. It was originally defined as the
mouse blood group antigen-II, until it was realized that
red blood cell surface antigen expression was the least
significant feature of this antigenic group, and had
little to do with its function. Further studies
correlated these antigens with the strongest
histocompatibility antigens involved in the rejection of
skin and tumor grafts, and the term histocompatibility-2
(H-2) antigens was created. This gave birth to the field
of transplantation immunology. Gorer's early work
established for the first time that "normal and
neoplastic tissues contain iso-antigenic factors which
are genetically determined. Iso-antigenic factors present
in the grafted tissue and absent in the host are capable
of eliciting a response which results in the destruction
of the graft".
For many years the
correlation between MHC and graft rejection was so strong
that the MHC molecules were commonly called
transplantation antigens. It was only in the late sixties
that the MHC was linked to the genetic control of the
immune response, and as a consequence, some MHC genes
were called "immune response" or Ir genes. Out
of this came the terms I region to denote the location of
these genes in the MHC, and I antigens (Ia) to name their
molecular products. Based on structural and functional
similarities, and evolutionary homologies, the MHC loci
are divided into two types: class I and class II. Some
confusion was created with the discovery that, in several
species of vertebrates, genes coding for some complement
components are intimately associated with the MHC genes (class
III MHC). In humans, the class III region also includes
the two structural genes for steroid 21-hydroxylase. The
tumor necrosis factor genes, a and b, and lymphotoxin
gene have been located between the HLA class I and III
regions. However, it is now generally accepted that this
genetic relationship is only due to their location on the
same chromosomal segment, and has probably nothing to do
with the function of the respective molecules.
1. Mouse MHC
genes.
The availability of inbred, congenic, and recombinant
congenic strains of mice, and the production of
highly specific allo-antisera to various gene
products, permitted immunogeneticists to build very
accurate genetic maps of the mouse MHC. Recent
technology, based on DNA cloning and molecular
analysis, is revealing the DNA sequence of several
genes, allowing scientists to have a more detailed
genetic map of the H-2 complex.
Figure
1. Simplified
genetic maps of mouse and human class
I and class II MHC genes. The
position of the class III genes is
indicated, but details of their
organization have been omitted.
Figure 1 shows
a simplified genetic map of the mouse MHC; the length
of this region is approximately 1.5 centimorgans (cM)
(1 cM is equivalent to a 1% recombination frequency
per generation). The most extensive studies on the
mouse MHC have been carried out with the BALB/c
strain. Mouse class I genes are divided into two
categories: genes located in the H-2 complex itself
which encode for the K, D, and L molecules; and genes
located within the thymus leukemia antigen (Tla)
complex which encode for proteins that are
structurally related to K, D, and L products, but
differ in tissue distribution and, presumably, in
function. The Tla complex includes the loci Qa-2,3,
Tla, and Qa-1, containing more than 23 non-polymorphic
genes with only a few alleles. For many years these
genes were not considered to be a part of the MHC,
but recent evidence indicates that they play a
significant role in the immune response.
Mouse class II
genes (Figure 1) encode for proteins that control
mechanisms for cell-cell interaction (macrophages,
thymus epithelial cells, T cells, and B cells), and
the magnitude of the immune response to different
antigens. These genes were originally denoted as
immunoregulatory or Ir genes (I region). By
functional analysis, five subregions have been mapped
within the I region: I-A, I-B, I-J, I-E, and I-C.
However, only the I-A and I-E subregions contain
functional genes for the class II MHC molecules, the
others being pseudogenes (no structural products).
The composition and organization of class II genes in
other experimental animals are still unknown.
2. Human MHC
genes. Since
the original description of leukocyte antibodies by
Jean Dausset in 1952, 36 years of research have
produced a considerable amount of information about
the human leukocyte antigen (HLA) system. The HLA
locus has been mapped on the short arm of chromosome
6 in the distal portion of the 6p 21.3 band; the b-2-microglobulin (b2m) gene is located on
chromosome 15. This complex shows considerable
similarity to the mouse MHC (Figure 1). The HLA
complex is approximately 1.8 cM long containing
approximately 3500 kilobase pairs, and it is also
divided into class I and class II regions (class III
region is in between these two). Class I genes code
for the classical transplantation antigens HLA-A, B
and C, and class II genes code for the
immunoregulatory molecules (Ia equivalent) HLA-DP, DQ
and DR. Unlike the detailed mapping of the mouse MHC
regions, the molecular analysis of the class I HLA
region has produced rather fragmentary maps.
3.
Polymorphism and evolution of MHC genes. The MHC locus is one
of the most highly polymorphic gene complexes known.
Exchange of DNA (gene conversion) between these genes
appears to be the most likely mechanism for the
generation of this polymorphism. Genealogical
analysis of several H-2 mutant mouse strains have
indicated that at least some, if not all, of the
interaction of the genes generating these mutations
occurred during mitotic amplification of the germ
cells. Genetic recombination among histocompatibility
genes occurring in nature could readily generate
mosaic transplantation genes containing sequences
derived from other MHC genes. Thus, it seems likely
that different forms of genetic interaction play a
major role in the diversification and ongoing
evolution of the MHC.
The MHC is a
multigene multi-allele complex coding for
glycoproteins, some of which are present on the
surface of all cells. This gene complex belongs to a
large family of genes that encode for other similar
membrane proteins, many of them related to the immune
system, e.g. immunoglobulins and T cell receptor
genes (Figure 2). The MHC antigen genes of humans (HLA),
mice (H-2), rats (RT), and rabbits (RLA) show close
homologies between them.
4. MHC
molecules. The class I and class II MHC
genes encode for cell surface glycoproteins (Figure 2)
which associate with foreign antigens to provide a
context of self-nonself recognition by T lymphocytes.
The functional structure of MHC antigens in one
individual has a very close relationship with the
specificity of his T cell receptor (TCR) molecules.
This kinship occurs during the development of
neonatal tolerance in the thymus.
Figure
2. Schematic
representation of some molecules of
the immunoglobulin family. Shaded
areas indicate domains with high
similarities in the amino acid
sequence and with low degree of
polymorphism even between species.
Domains that are distant from the
cell membrane show a high degree of
polymorphism, with differences
between species, individuals, and
cell clones.
Class I MHC
molecules represent
only about 1% of the cell surface proteins, but
higher levels of expression can be induced after
exposure to some lymphocyte products (e.g. interferon
gamma). These antigens are in very low concentration
on erythrocytes, and undetectable on mature
trophoblast and neurons. In humans, the HLA-C
antigens are expressed in lower concentration than
the HLA-A and B alleles; in some cells (e.g.
platelets) the HLA-B antigens show less expression.
The mouse and human class I molecules are comprised
of a 45 kilo-Daltons (kDa) polypeptide (a chain) which is
noncovalently associated with a lighter 12 kDa
polypeptide called b-2-microglobulin (b2m). b2m is not glycosylated,
contains no MHC allotypic determinants, and can also
be found free as a serum protein. The heavy a chain is divided into
five domains or regions: three external domains, a-1, a-2, and a-3, with 90 amino acids
(aa) each, a transmembrane domain (40 aa), and the
cytoplasmic domain (30 aa). The a-1 and a-2 domains contain the
haplotype-determining regions and the regions that
associate with antigens to interact with the TCR
molecules. The a-3 domain is invariant and
associates with the b2m. The complete amino acid
sequence of the a-3 domain and b2m have been determined,
and they show considerable homology to the constant
regions of Igs.
New
technological advances using recombinant DNA and gene
cloning, complemented with refined biochemical
analysis and X-ray crystallography, have allowed
scientists to get new insights into the organization
and regulation of the genetic and molecular structure
of the HLA complex. Figure 3 shows a top view of the
HLA-A2 molecule obtained by X-ray crystallography.
The domains a-1 and a-2 form a platform
composed of a single b-pleated sheet topped by a-helices with a long
groove between the helices. This groove is the area
where the polymorphic amino acids of class I
molecules are clustered, and is the recognition site
for processed foreign antigens. This site would also
be the part of the molecule that carries the
allotypic determinants that mediate graft rejection.
Class II MHC
molecules are
designated IA and IE in mice, and DP, DQ, and DR in
humans. They are normally expressed on dendritic
cells, some macrophages, and B cells; they are also
expressed on activated human T cells. The I-E and I-A
molecules are homologous to the DR and DQ molecules,
respectively. The H-2 and HLA class II molecules are
all composed of two glycosylated polypeptides, a and b chains, noncovalently
bound (Figure 2). The a chains weigh about 33 kDa, and
the b chains weigh about 28
kDa. Two carbohydrate units are bound to the a chain, and one to the b chain. Both chains
contain two external domains each with 90 aa length (a-1, a-2, b-1, and b-2 respectively), a
transmembrane domain of 30 aa, and a cytoplasmic
domain of 10-15 aa. The allelic polymorphism is
centered in the a-1 and b-1 domains. HLA-class
II molecules are associated intracellularly with
another transmembrane, non polymorphic, 30 kDa
glycosylated polypeptide (Ii), coded by a gene in
chromosome 5.
D2.
HISTOCOMPATIBILITY TESTING
In order to
determine the histocompatibility between two individuals,
several in vivo and in vitro techniques have been
designed. In transplantation research, these techniques
have made it possible to study the complex immunogenetic
mechanisms involved in graft rejection. In the clinical
field, the goal is to find a tissue donor with the
closest histocompatibility with the recipient, namely, a
phenotype with the least antigenic differences with the
recipient, whose tissue can survive for a long period of
time with minimal immunosuppression.
1. In vitro
tests. Peripheral
leukocytes are the cells of choice for in vitro
histocompatibility testing. They carry
transplantation antigens, as well as reacting with
them. When two allogeneic lymphocyte populations are
mixed in the same culture (mixed leukocyte reaction
or MLR), important morphological changes occur: the
nucleus of the cells become euchromatic (light
reticular staining) and one or more nucleoli appear,
both the nucleus and the cytoplasm become larger, and
the cytoplasm takes a basophilic staining quality.
Under the electron microscope, the cytoplasm shows a
large Golgi apparatus, abundant ribosomes and
endoplasmic reticulum. The lymphocytes are now called
lymphoblasts, being able to divide and proliferate as
long as the stimulus and the culture conditions are
appropriate.
In the
original MLR tests it was difficult to study the
reactivity of only one lymphocyte set, since both
sets responded to each other (two way stimulation).
An important improvement of this method was obtained
when one cell population was prevented from
replicating, without losing its capacity to stimulate
the other population (one-way reaction). This was
achieved by pretreatment of the stimulating
population with agents that alter its DNA structure,
thus suppressing replication; e.g. gamma or X-ray
irradiation, nitrogen mustard, mitomycin C.
Lymphocyte stimulation can be measured by changes in
the cell morphology, sizing and counting of the
responding cells, incorporation of 14C-thymidine,
3H
thymidine, or by the detection of lymphocyte hormones
(lymphokines) released to the culture medium.
2. MHC typing.
Originally,
class I MHC antigens were serologically identified,
whereas class II antigens were recognized by MLR
tests. All the tests for histocompatibility described
above have been largely replaced by MHC typing
techniques which use highly specific antisera (antibody
typing) or lymphocytes (cellular typing) to determine
the antigenic constitution of an individual. Cellular
typing of the MHC remains important in the detection
of HLA determinants that are still not identified by
serology. Antibody and cellular MHC typing techniques
are being used to assess compatibility between donors
and recipients of organ and bone marrow allografts.
The identification of alleles encoded within the HLA
region is also used for HLA-disease association
studies, paternity testing, and as a measure of
immune responsiveness.
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