Transcript Membrane Receptors for Antigen

How Cells of the Immune System
“See” and Respond to Antigen
Immunoglobulin
Class I MHC
Membrane Receptors for Antigen
• B-cell surface Receptor (BCR) – anchored
Immunoglobulin
• T-cell receptor (TCR)
• Major Histocompatiblility Complex (MHC)
– Class I molecules
– Class II molecules
• NK receptors
B and T Lymphocytes
• B-cells make Immunoglobulin – both secreted
and on the surface
• T-cells are lymphocytes that play a key role in
the humoral (Ig) immune response, and also
carry out their own recognition functions
(cytotoxic lymphocytes)
• T-cells have receptors that are similar to Ig
molecules in several respects …
Similarity of Ig and TCRs :
• The TCR has two polypeptide chains, a and b that
together form the antigen-combining site (just as Ig has
the H and L chains)
• The TCR chains each have two domains, a C and V, with
folded structure that is similar to the C and V domains in
the Fab arms of Ig.
Ig and TCRs are similar:
• The TCR has two polypeptide chains, a and b that
together form the antigen-combining site (just as Ig has
the H and L chains)
• The TCR chains each have two domains, a C and V, with
folded structure that is similar to the C and V domains in
the Fab arms of Ig.
• The antigen-combining region
of the TCR are made up of 6
CDRs, three from each chain.
CDRs on both the Ig and TCR
variable regions
Immunoglobulin Fab, H+L
(one chain)
T-cell receptor,
a and b chains
Ig and TCRs are similar:
• The TCR has two polypeptide chains, a and b that
together form the antigen-combining site (just as Ig has
the H and L chains)
• The TCR chains each have two domains, a C and V, with
folded structure that is similar to the C and V domains in
the Fab arms of Ig.
• The antigen-combining region of the TCR are made up of
6 CDRs, three from each chain.
• The high degree of variability in the variable regions (and
CDRs) is mainly due to recombination in the DNA of
developing T-cells in which one of several V, J and (in the
b subunit) D regions are joined to create millions of
different possible combinations.
Gene rearrangements in different combinations of
V, D and J segments provide for a huge number of
different TCRs
Although there are these interesting similarities between
the B-cell receptor (Ig) and the TCR, there is one really
key difference:
While the B-cell receptor can recognize antigen alone,
T-cell receptors recognize and antigen only when it
is in the context of a Class I or Class II MHC molecule.
What is a MHC molecule, you ask? Well….
The Major Histocompatiblity
Complex, or MHC
• BCRs (surface Igs) and TCRs both recognize a variety of chemical
structures, but B-cells (as with the Igs they secrete) can “see”
antigens in isolation
• T cells recognize antigens when they are associated with normal
cells -- they recognize “foreign” in the context of “self”
• The “self” they see are molecules of the Major Histocompatilbity
Complex, or MHC
• They are called “histocompatiblity” antigens because they were first
recognized as provoking graft rejection, because they seen as “not
self” by T-cells, and an immune response is elicited
• If you can find an individual with a similar (or better yet, identical) set
of MHC antigens, they will be seen as “self” and not rejected
There are two kinds of MHC Molecule: Class I and Class II
• Class I molecules are expressed in nearly every cell type in the
body
– The TCRs of Cytotoxic lymphocytes see viral and tumor
antigens when they show up on normal cells: foreign Ags “ in
the context of Class I molecules “
• Class II molecules are expressed only on cells of the immune
system, mostly macrophages and B cells
– When these cells take in Ag, some of it is put back out on the
cell surface with a Class II molecule – a way of focusing
attention on this unusual bit, and encouraging an immune
response.
Class I
Class II
Both types of molecules have two polypeptides, and a
domain structure similar to that of Ig and TCR, especially
in those domains that are closest to the membrane
Class I MHC molecules
• Expressed on nearly all cells of the animal, though are more
abundant on some cells than others
• In humans there are three main
genes, located on Chromosome 6,
called HLA-A, HLA-B and HLA-C
(HLA stands for Human Leukocyte
Antigen – they were originally called
antigens because animals make
antibodies against MHC molecules
from different individual)
• These genes are polymorphic,
meaning that are many different
alleles (or versions) of each gene in
the human population.
Class I MHC proteins
The TCRs of Cytotoxic lymphocytes see viral and tumor
antigens when they show up on normal cells: foreign Ags
“in the context of Class I molecule” (CD8 is also involved)
Class II MHC molecules
• Expressed on cells of the immune system,
mainly macrophages and B-cells
• In humans there are three main genes, located
on Chromosome 6, called HLA-D – there are
many genes, each with several alleles
• These are also important to match in tissue
transplantation, especially if lymphocytes are
transplanted.
• MHC Class II molecules are important in
achieving good Antibody responses to antigen
T-cell help in Antibody generation
Class II molecules are important in the humoral immune response
because they present antigen to T cells that enhance the immune
response
• Foreign antigens are engulfed by
macrophages, non-specifically
• The macrophage partially degrade the
antigen, and pieces are bound to Class II
molecules in the internal membrane
system of the cell
• These are put out on the surface: Antigen
is presented in the context of Class II MHC
molecules
• The TCR on CD4+ T cells can now “see
the foreign antigen – it is in the context of
the Class II molecule (which CD4 helps to
bind)
• The T-cell is activated….
The interaction stimulates both the
T-cell and the macrophage:
• T-cell
– Stimulated to secrete IL-2
• stimulates the T-cell to proliferate -- expansion of clones that
can see this antigen
• Macrophage
– stimulated to release IL-1
• further stimulates the T-cell
• recruits other immune cells (inflammatory response)
Antigen can also be presented by B-cells
• Antigen can be bound to the Ig (BCR) on the
surface of ‘virgin” B cells
• Binding causes endocytosis, and
“processing” (partial digestion) of the
antigen.
• Fragments of antigen bound to Class II
molecules are put out to the surface
• Antigen + Class II engages T-cells
• The T-cell clones that can see this antigen
are expanded, and secrete cytokines (IL-4
and IL-5) that stimulate the B-cells to
proliferate and differentiate into plasma cells.
Besides being just interesting, this explains why
some molecules can be good antigens and yet not
be immunogenic …
• To elicit a good Ab response, a
molecule must be able to be
presented to T-cells
• If it is not big enough to bind to the
Class II molecule and the T-cell
receptor at the same time, it may fail
to recruit T-cell help
• The B-cells that will recognize it may
be activated, but without the help, the
response will be poor.
• Small molecules, including peptides,
need to be attached to carrier
molecules in order to elicit a good
responses.
Practical Issues in Ab Production
(some may make more sense
now that we have looked at T-cell help)
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Purity and Integrity of Antigen
Form of antigen
Animals
Adjuvant
Route of injection, dose etc
Antigen
• How pure should it be? …
– For polyclonals, purified is best.
– If you can “clean it up” in the serum, you do not need
to start with a pure Ag
– Antigen-affinity column
– Adsorb “out” other antibodies
– For monoclonals it is less of an issue (more unwanted
clones, but if you can screen them out….)
Antigen, other considerations
• Native or denatured antigen (SDS-PAGE gel slices) –
but if denatured, you may not get as many “native”
epitopes
• Peptides
– Need carrier protein (T-cell help).
– Abs raised to peptides may not recognize native molecule well
Depends on how much information you have
• Particulate Ags tend to work well because they are easily
phagocytized (for Ag presentation)
Animals
• Species considerations:
– Size/cost
– Phylogenetic distance (not good to use mouse for Abs to a
mouse Ab. Chickens are sometimes used for this reason.
– Useful Ig properties, such as IgY (birds) or Ig (llamas)
– Monoclonals are usually mouse, unless phylogenetic distance
is needed. (Also mice are more limited in the varriety of Class
II molecules they have b/c they are inbred, but outbred strains
are available )
• Several animals are generally done to provide
more heterogeneity in Class II options
• (empirically animals differ in their response, so do >1
Injections
• Adjuvant
– An emulsion of mineral oil/water (Freund’s) that will
hold the Ag in place long enough for macrophages to
get there
– May contain an irritant to cause local inflammation
(“Complete” adjuvant) to recruit cells of immune
response
• Route of injection, does per injection and period
of time between boosts
– depend on the animal, whether adjuvant is used, the
nature of the antigen, and experience (empirical) …
people base their practices on history