Complement Activity
Lesson objectives
At the end of this lesson student will be able to:
List down the important steps in the complement
system
Recognize the importance of chemotaxis in the
complement system
Appreciate the role of the membrane attack
system
in immune response
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Complement Activity
Complement activation is a cascade of events
The Complement cascade consists of a series of
protein proenzymes, present in the blood, that
are converted to active enzymes by interaction
with pathogens and with each other
The Complement cascade has a number of very
important roles in combating infection and in
inflammatory disease.
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Complement Activity...
There are two major ways of activating the
Complement cascade (are three)
by the “classical pathway” or the “alternate” pathway.
The initial components of the classical pathway
(C1,C4,C2) are activated by the Fc regions of IgG or IgM
antibody, but only after antibody has bound to antigen
(usually to a pathogen).
The initial components of the alternate pathway (Factor
B, D and P) are directly activated by certain pathogens
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Complement Activity...
Either pathway can be partly activated by blood coagulation.
There are four major outcomes of activating this cascade:
1. local vasodilation and increase in vascular permeability
2. attraction of immune cells, especially phagocytes
(chemotaxis)
3. opsonization (or tagging) of foreign organisms for
phagocytosis
4. destruction of invading organisms by the membrane attack
complex (MAC attack)
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Complement Activity...
Although all the molecules in the cascade are
important, the third component, called C3 protein,
stands out as of special interest because both the
classical and alternate pathways converge on C3.
It is an enzyme that is split into two fragments (C3a
and C3b) by components of either the classical
pathway (C4b, C2a) or the alternative pathway
(factor Bb, C3b, and P).
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Complement Activity...
C3a is primarily a very powerful activator of mast cells
to release mediators which cause local vascular
permeability and vasodilation.
C3a is also a chemotactic factor for phagocytic
immune cells.
C3b, on the other hand, binds directly to the surface of
foreign organisms (or immune complexes) and acts to
'tag' these for destruction by phagocytic cells like
macrophages and neutrophils which have specific
receptors for C3b.
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Complement Activity...
• C3b also reacts directly with the other
components
of the Complement cascade (C5-9) to produce
the MAC attack.
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Complement Activity...
• Complement is triggered by antibodies or invading
organisms, and that the end result of the pathway
becoming activated is:
1. vasodilation and increased vascular permeability
2. attraction of immune cells to the site
3. opsonization for phagocytosis
4. interacting with other components of Complement
to initiate the formation of the MAC attack
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Complement Activity...
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C1qr2s2 COMPLEX
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Complement-Induced Vasodilation and
Increased Vascular Permeability
The Complement components responsible for
vasodilation, and vascular permeability, are the "a"
subunits of C3, C4 and C5 (C3a, C4a and C5a).
These three proteins are called anaphylatoxins.
There are receptors for these proteins on the
surface of mast cells and basophils.
Binding of the anaphylatoxins to their receptors
induces the cell to degranulate, releasing
mediators like histamine.
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Complement-Induced Vasodilation and
Increased Vascular Permeability...
• Anaphylatoxin is a substance composed of the C3
and C5 components of the complements which
cause vasoactive mediators to be released from
mast cells, promoting vascular permability and
the formation of edema during an anaphylatic
reaction
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Complement-Induced Vasodilation and
Increased Vascular Permeability...
Such mediators induce smooth-muscle dilation on
the arteriolar side in the vasculature making the
blood vessels wider and allowing more blood to
flow to the capillaries in the area.
They also result in an increase in the permeability
of the endothelial lining of the blood vessels.
Increased vascular permeability allows fluid as
well as macromolecules like IgG and Complement
to flow out of the blood plasma into the tissue.
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Complement-Induced Vasodilation and
Increased Vascular Permeability...
Vasodilation results in redness and increased
permeability results in swelling (edema).
The advantage of these functions of the
Complement system is to increase blood flow to the
site of infection and allow antibodies, more
Complement and immune cells to enter the tissues
to "scale up" the attack.
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Chemotaxis
Chemotaxis is the process whereby an immune cell
is attracted to, and moves toward, a soluble factor.
Usually the cell being attracted is a phagocyte (like
a neutrophil).
One of the most potent chemotactic agents is C5a.
C5a is formed when the Complement component
C5 is cleaved to form C5a and C5b.
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Chemotaxis
Other substances that have chemotactic ability
are secreted by immune cells and released during
inflammatory responses.
A number of these are small protein molecules
called “chemokines”.
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Chemotaxis...
Chemotaxis of phagocytic cells is important
because it "recruits" the cells to the tissue where
they are needed to ingest the invading organism
or antigens or debris from the inflammation.
It is important to remember that immune cells do
not “leak out” of the blood because of increased
vascular permeability like plasma proteins and
water do.
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Chemotaxis...
They must be attracted to the site by chemotaxis
and penetrate through the endothelial lining of
the vasculature in an active process of
“transmigration”.
After they transmigrate they essentially “crawl”
through the extravascular tissue to the site of the
infection or inflammation
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Opsonization
Opsonization is the process where particles such
as microorganisms become coated with
molecules which bind to specific receptors on
phagocytes.
IgG and Complement proteins like C3b can
opsonize and are therefore referred to as
"opsonins“.
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Opsonization ...
The Complement fragment C3b non specifically
binds to foreign organisms or immune complexes
during Complement activation.
Since phagocytes have receptors for C3b on their
surface (CR3) the binding of C3b to a microorganism
or an immune complex tags it for ingestion and
degradation by the phagocytes
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Membrane Attack Complex -MAC
Complement activation by either the classical or
alternative pathway will result in the formation of
the membrane attack complex or MAC.
The last steps in the Complement cascade, after
the activation of C3b, involve C5b, C6, C7 C8 and
C9 which complex to form the MAC.
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Membrane Attack Complex -MAC
Briefly, C5 is broken down into C5a and C5b after
binding to C5 convertase, an enzyme formed by
other fragmented Complement proteins.
C5a diffuses away and has anaphylatoxin and
chemotactic activity
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Membrane Attack Complex –MAC..
The C5b fragment binds directly to the target
organism and becomes the binding site for other
Complement components.
C5b will quickly degrade unless C6 binds to it
creating C5b6, to which C7 will then bind.
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Membrane Attack Complex –MAC..
The C5b fragment binds directly to the target
organism and becomes the binding site for other
Complement components.
C5b will quickly degrade unless C6 binds to it
creating C5b6, to which C7 will then bind.
The addition of C7 changes the conformation of
the proteins so that they are able to insert into
lipid membranes.
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Membrane Attack Complex –MAC..
Next, C8 and finally C9 bind to the complex
creating the MAC.
C5b678 will bind up to 16 molecules of C9.
The MAC looks like a tube with a pore in the
centre (like a very short straw)
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Membrane Attack Complex –MAC..
Because of its structure and hydrophobic nature,
the MAC inserts into the membrane of the
organism or cell and allows ions, water and other
small molecules to freely pass through the pore
(the MAC attack).
As a result, the organism will not be able to
maintain osmotic integrity and will quickly die.
Cells without a cell wall will often burst (lyse).
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They Fight Back
On a closing note it is important to recognize that
the immune response to pathogens is like a war
and we don’t always win easily.
The pathogens have ways of avoiding our
defenses. Some microbes evade destruction by
having surfaces that interfere with opsonization by
C3b or insertion of the MAC.
Others inhibit Complement activation (they have
proteins that bind IgGFc such as protein A and
protein G).
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They Fight Back
Still others cause Complement degradation by
elaborating enzymes that degrade Complement
components.
Not surprisingly, such microbes are pathogenic
(meaning they cause disease).
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T Cells
It is very difficult to discuss T cell activation
without knowledge of the presentation of antigen
by protein molecules coded for by the genes in
the
major histocompatibility complex.
These molecules, MHC Class I and MHC Class II
are pivotal for T cell activation.
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T cells..
MHC stands for major histocompatibility complex.
In humans the same proteins are often referred to
as human leukocyte antigens or HLA.
Since MHC is a more specific term (there are lots
of antigens on human leukocytes, not all of them
are MHC antigens) it will be used in this resource.
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MHC-Class I
All cells in the body except red blood cells have
MHC class I protein on the surface.
Your finger cells, your liver cells, your B cells, your
gut cells - they all have MHC class I on the
surface. Everyone has MHC class I proteins but
only identical twins will have identical MHC class
I.
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MHC-Class I
The genes for the MHC proteins show considerable
diversity between people.
Class I MHC is a complex of the Class I protein and
another protein called β2 microglobulin
Since everyone has a different MHC, this allows your
body to tell what cells belong to it and what cells are
foreign.
The function of MHC-I is to sample the internal contents
of the cell and show them to the immune system.
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MHC-Class II
Class II MHC is a complex of proteins only
expressed by antigen presenting cells (APC).
The function of these proteins is to present an
antigen to T helper cells to activate an immune
response which will provide both humoral
(antibody) and cell mediated immunity.
The class II MHC consists of an alpha and a beta
chain with a transmembrane segment to hold them
on the surface of the cell.
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MHC-Class II
At the end farthest from the cell is a cleft where
the processed antigen sits.
The processed antigen consists of a small peptide
of about 13-16 amino acids.
MHC-II picks up the antigen that has been
ingested (phagocytosed) by the APC.
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MHC structure
Peptide binding cleft Peptide binding cleft
2 1 1 1
3 2-microglobin 2 2
Class I MHC Class II MHC
MHC classes I and II have an almost identical 3-D structure.
Both classes of MHC are polygenic (each cell has many MHC genes) and
polymorphic (there are many alleles for each locus), but the MHC genes do
not undergo recombination.
Note: Human MHC are called HLA (human leukocyte antigen).
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T cells
T cells are lymphocytes which develop in the
thymus.
T cells have antigen recognition molecules (T cell
receptor, TcR) on the surface which are composed
of two different polypeptide chains; alpha and
beta chains.
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T cells
The alpha and beta chains are associated with a
group of five linked proteins collectively referred
to as CD3.
A minor population of T cells has two different
chains, a gamma chain and a delta chain
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T cells...
Little is known of the activities of these gamma-
delta T cells.
Like antibody, the TcR has both a variable and a
constant region.
As described for antibodies, the variable region of
the TcR is created by gene rearrangement.
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T cells...
The “T cell receptor complex” includes the alpha
and beta chain of the TcR and the associated CD3
proteins.
The recognition of antigen-MHC by the TcR-CD3
complex does not require any other molecules.
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T cells...
However, other proteins on T cells are important
in the interaction leading to T cell activation.
These "accessory " or “co-stimulatory” molecules
on T cells bind to ligands (surface molecules
which function as a lock and key binding pair) on
antigen presenting cells to give the 'second
signal' required for T cell activation.
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T cells...
The TcR will only recognize antigen as a peptide
in the groove of MHC.
This is because part of the TcR actually binds to
part of the MHC molecule during recognition.
The TcR will not bind to free antigen.
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T cells...
Recent evidence also suggests that some T cells can
recognize antigen presented by a molecule called CD1.
This molecule is similar in general shape to Class I MHC
but is capable of presenting lipid and carbohydrate
antigen to T cells.
CD1 presentation is particularly important in the
activation of gamma/delta T cells but there is evidence
that alpha/beta T cells can also be activated in this
manner
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T cells development
For a T cell to be functional it needs to have a functional
T cell receptor on its surface.
That T cell receptor must be able to recognize antigen as
a peptide in the groove of self MHC.
The T cell receptor must be appropriately linked to
CD3 for proper signal transduction.
Finally, you don’t want the T cell to be self-reactive. The
development of the T cell receptor and the testing of the
reaction of the receptor occur in the thymus.
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T cells development...
Pre-T cells (like pre-B cells) arise from lymphoid
stem cell precursors in the bone marrow.
They leave the bone marrow and transit, via the
blood stream, to the thymus where they develop
and undergo “thymic education”.
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T cells development...
The first thing to happen is that the β chain of the
TcR undergoes gene rearrangement.
Just like in surface antibody development in B
cells this β chain in T cells has to be tested for
functional activity before the α chain is
rearranged.
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T cells development...
The β chain is brought to the surface with a
surrogate (pseudo) α chain and tested.
If it links properly with the signal transduction
components then the T cell goes on to rearrange
α chain genes and put a mature T cell receptor on
its surface.
At this stage the T cell is both CD4+ and CD8+
(called a double positive cell).
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TCR genetics: Similar to Ig genetics
V regions (70-80) J regions (61) C region (1)
chain
V regions (52) D1 (1) J1 (6) C1 (1) D2 (1) J2 (7) C2 (1)
chain
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T cells development...
Putting a T cell receptor on the surface is only the first
stage.
T cells are very specific in that not only do they have to
recognize specific antigen, they have to recognize it in
a specific context, as a peptide in the groove of self MHC.
Therefore, the next step is to make sure the T cell can
recognize self MHC.
The T cells interact with MHC bearing cells in the thymus
and if the TcR binds weakly to them the cell goes on to
further development
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T cells development...
If the T cell gives the MHC bearing cells the cold shoulder
then it does not receive the life prolonging signals that
come from this interaction and it dies.
Most of the T cells in the thymus will fail this test and die.
If they pass this test they then have to be tested for self
reactivity.
If they interact with MHC bearing cells very strongly,
indicating that the self peptide and the self MHC is their
chosen antigen to respond to, then they will be killed.
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T cells development...
If they pass both of these tests they will become mature
double positive T cells.
These cells will then lose either CD4 or CD8 to become naive
CD4+ cells or CD8+ cells.
The cells with a CD4 marker are called helper T cells (Th
cells).
The CD8 positive cells that develop are cytotoxic T cells (Tc
cells).
Th and Tc cells both have a TcR, but they perform very
different functions in the immune system.
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Th cell activation
T cell activation is, in general, similar for both Th and Tc
cells but each are considered separately for clarity.
Th cell activation is initiated by the interaction of TcR-
CD3 complex with antigen-MHC class II molecules on
the surface of an antigen presenting cell.
This interaction initiates a cascade of biochemical
events in the T cell that eventually results in growth and
proliferation of the T cell.
This occurs primarily through an increase in IL-2
secretion by the T cell and an increase in IL-2 receptors
on the T cell [Link] Immunology
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Th cell activation...
IL-2 is a potent T cell growth cytokine which, in T cell
activation, acts in an autocrine (self-stimulating) fashion to
promote the growth, clonal proliferation and differentiation
of the T cell recently stimulated by antigen.
The T cell receptor is an antigen recognition molecule and
therefore the T cell that best responds to the antigen presented
is the one that gets turned on.
All its progeny will recognize the same “T cell epitope” of the
original antigen, since they will all have identical T cell
receptors.
Naive, bystander T cells have no IL-2 receptors and thus cannot
be involved.
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Th cell activation...
The activation takes place through the T cell
receptor complex and is aided by the CD4 molecule
on Th cells as well as other accessory molecules,
such as CD45, CD28 and CD2.
The APC also provides a co-stimulatory signal
through molecules such as B7 and by the secretion
of co-stimulatory cytokines (such as IL-1 or IL-12).
Activated Th cells then continue to become effector
cells whose role includes B cell help and activation
of effector cells such as macrophages.
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Antigen Presenting Cells (APC)
Antigen presenting cells are a functionally defined
group of cells which are able to take up antigens
and present them to T lymphocytes in a
recognizable form (in the groove of an MHC class
II molecule).
Although many cells can do this, the cells which
are most efficient, the so-called "professional
antigen presenting cells", are dendritic cells.
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Antigen Presenting Cells (APC)...
These are professional cells because they are
highly effective at producing the "second signal"
required for naive T cell activation.
The APC first internalizes the antigen (maybe in
the form of a bacteria or bacterial product),
processes it (breaks it down into antigenic
peptides by digesting it with enzymes
(lysosyme)and then expresses the antigen
fragment (peptide) on its surface in the groove of
an MHC class II molecule. Now the antigen is in
the form recognizable by T cells
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Antigen Presenting Cells (APC)...
Antigen presentation by APC is a required first
step in Th cell activation.
Presentation to naive T cells like this happens
almost exclusively in the lymph nodes and
spleen.
This means the dendritic cells have to leave the
site of infection and travel to the local draining
node to accomplish this.
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Antigen Presenting Cells (APC)...
Dendritic cells are the only cells that can present
antigen to naive T cells but macrophages and B
cells can present antigen to activated or memory
T cells.
It is thought that antigen presentation to memory
T cells by macrophages is very important the
second time you see an infection since this can
happen at the site of infection
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T Cell Help -Th Cells
The generation of an effective immune response
depends on the activation of Th cells.
Both humoral and cell-mediated responses are
initiated or amplified by Th cells. The importance
of these CD4+ cells to a functioning immune
system is clearly demonstrated by the impact of
the HIV virus.
This virus preferentially kills CD4+ T cells and the
devastating immune deficiency syndrome resulting
from this is the disease known as AIDS.
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T Cell Help -Th Cells...
Mature B cells that have already seen antigen
require contact with a T cell in order to become
plasma cells or memory cells (T-B cell
interaction).
The T cells provide signals to the B cell through
contact of the TcR complex with processed
antigen presented on Class II MHC by the B cell.
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T Cell Help -Th Cells...
In addition, the activated T cell produces
cytokines which stimulate B cell proliferation and
the differentiation of these cells into antibody
secreting plasma cells.
Th cell cytokines are required for “class switch”
so that isotypes other than IgM can be produced.
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T Helper Cell Subsets
Our understanding of how and why T cells behave
in an immune response has markedly increased
with the discovery that there are at least two
subsets of T helper cells.
The two subsets look the same and have the same
T cell markers and receptors. However, they
secrete very different cytokines upon activation.
In addition to other cytokines, the Th1 subset
produces abundant IL-2 and IFN gamma.
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T Helper Cell Subsets...
These cytokines are particularly important in cell-
mediated immunity.
The Th2 subset is very good at providing B cell help
by secreting abundant IL- 4.
Because the various cytokines have different effects
in an immune response, sometimes activation of one
subset may be preferable to the other.
For example, virus infection is best combated with
cytotoxic T cells
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T Helper Cell Subsets...
This response is more likely to be stimulated by
Th1 cytokines.
Although there is evidence that these subsets are
cross-regulatory (activation of one downregulates
the other) it is likely that they work together in
normal immune responses.
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T Helper Cell Subsets...
To use the example above, viral infection is also
marked by anti-viral antibody.
There are, however, some instances where only
Th1 immunity is useful, as in the case of
tuberculosis infection, where few antibodies are
made to the pathogen and they have no role in
dealing with the infection.
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Reading assignment
Read about
Regulation of the complement activity
Disorders of the complement system
Differentiate among the classical, alternate and
Lectin binding path ways
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Next class will be
effecter cells of
the immune sytem
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