COMPLEMENT

SYSTEM
Dr. Nizar M. Mhaidat
Assistant Professor
Clinical Oncology & Immunology, PhD
[email protected]
 Definitions
Historically; complement (C) was used to 
refer to a heat labile serum component that
was able to lyse bacteria. However,
complement is now known to contribute to
.host defenses
Functions

.Opsonize bacteria for enhanced phagocytosis 

Recruit and activate various cells including 

,PMNs and macrophages
Participate in regulation of antibody responses 

Aid in the clearance of immune complexes and 

.apoptotic cells
Complement contributes to inflammation and 
.tissue damage and it can trigger anaphylaxis
Functions
Components of complement
:Over than 20 serum proteins produced by 
,Hepatocytes (mainly).1
,Macrophages.2
.Gut epithelial cells.3

Some complement proteins bind to immunoglobulins

.or to membrane components of cells
Others are proenzymes that when activated cleave
one or more other complement proteins. Upon
cleavage some of the complement proteins yield
fragments that activate cells, increase vascular
.permeability or opsonize bacteria
 Components of complement
• C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9
• Factors B, D, H and I, properdin (P)
• Mannose binding lectin (MBL), associated
serine proteases (MASP-1 MASP-2)
• C1 inhibitor (C1-INH, serpin), C4-binding
protein (C4-BP), decay accelerating factor
(DAF), Complement receptor 1 (CR1), protein-
S (vitronectin)
 Definitions
C-activation: alteration of C proteins such that they 
.interact with the next component
C-fixation: utilization of C by Ag-Ab complexes 

Hemolytic units (CH50): dilution of serum which 

lyses 50% of a standardized suspension of Ab-
.coated RBC
C-inactivation: denaturation (usually by heat) of an 
early C-component resulting in loss of hemolytic
.activity
Convertase/esterase: altered C-protein which acts 
.as a proteolytic enzyme for another C-component
 Definitions
Activated component are usually over-lined: e.g.
C1qrs
When enzymatically cleaved, the larger moiety,
binds to the activation complex or membrane
and the smaller peptide is released in the
microenvironment
Letter “b” is usually added to the larger,
membrane-binding peptide and “a” to the smaller
peptide (e.g., C3b/C3a, C4b/C4a, C5b/C5a),
EXCEPT C2 (the larger, membrane-binding
moiety is C2a; the smaller one is C2b)
 Pathways of Complement
Activation
CLASSICAL LECTIN ALTERNATIVE
PATHWAY PATHWAY PATHWAY

antibody antibody
dependent independent

Activation of C3 and
generation of C5 convertase

activation
of C5

LYTIC ATTACK
PATHWAY
 C1r C1s
Ca++
C1q
C2 C3 C4

C1 complex
C4a C1r C1s
Ca++

C4 b
C1q
 Classical Pathway
C1 a multi-subunit protein containing three 
different proteins, C1q, C1r and C1s, binds to
the Fc region of IgG and IgM antibody
.molecules that have interacted with antigen
C1 binding does not occur to antibodies that 
have not complexed with antigen and binding
requires calcium and magnesium ions. (N.B.
In some cases C1 can bind to aggregated
immunoglobulin [e.g. aggregated IgG] or to
certain pathogen surfaces in the absence of
.antibody)
 Classical Pathway
Activated “C1qrs” also cleaves C2 into C2a and C2b. 
C2a binds to the membrane in association with C4b
and C2b is released into the microenvironment. The
resulting C4bC2a complex is a C3 convertase,
which cleaves C3 into C3a and C3b. C3b, a “sticky”
fragment of C3 that readily binds to
surfaces (of cells, microbes, or particles and
works as highly effective opsonin) binds to the
membrane in association with C4b and C2a and C3a
is released into the microenvironment. The resulting
C4bC2aC3b is a C5 convertase. The generation of
.C5 convertase is the end of the classical pathway
Generation of C3 convertase in the classical
pathway
Generation of C5 convertase in the classical
pathway
Classical Pathway
Several of the products of the classical pathway have 
potent biological activities that contribute to host
defenses. Table 2 summarizes the biological activities
.of classical pathway components
 Biological Activity of classical pathway products

Component Biological Activity

C2b Prokinin; cleaved by plasmin to yield kinin, which

results in edema

C3a Anaphylotoxin; can activate basophils and mast

cells to degranulate resulting in increased vascular
permeability and contraction of smooth muscle
cells, which may lead to anaphylaxis

C3b Opsonin
Activation of phagocytic cells

C4a Anaphylaotoxin

C4b Opsonin
 Regulation of the Classical
Pathway
Component Regulation

All C1-inhibitor (C1-INH); dissociates C1r and C1s

from C1q
C3a C3a-inactivator (C3a-INA; Carboxypeptidase B)

C3b Factors H and I; Factor H facilitates the degradation

of C3b by Factor I
C4a C3a-INH
C4b C4 binding protein (C4-BP) and Factor I; C4-BP
facilitates degradation of C4b by Factor I; C4-BP
also prevents the association of C2a with C4b thus
blocking formation of C3 convertase
C1-inhibitor deficiency:
hereditary angioedema
Lectin Pathway:
Component
C4
MASP2

MBL C2 MASP1

mannose binding lectin (MBL)

MASP-1 and MASP-2 (MBL-associated serine proteases).
 Lectin Pathway
The lectin pathway is initiated by the binding of mannose binding 
lectin (MBL) to bacterial surfaces with mannose-containing
polysaccharides and results in the association of two serine
proteases, MASP-1 and MASP-2 (MBL-associated serine
.proteases)
MASP-1 and MASP-2 are similar to C1r and C1s, respectively 
.and MBL is similar to C1q
Formation of the MBL/MASP-1/MASP-2 tri-molecular complex 
results in the activation of the MASPs and subsequent cleavage
.of C4 into C4a and C4b
The C4b fragment binds to the membrane and the C4a fragment 
.is released into the microenvironment
Activated MASPs also cleave C2 into C2a and C2b. C2a binds to 
the membrane in association with C4b and C2b is released into
.the microenvironment
 Mannose-binding lectin
pathway
_____
C4a C2b
C4b2a is C3 convertase; it
will lead to the generation of
C5 convertase
MASP1 C4b
C4
MASP2 2a
CC2
MBL C4b C2a
 Lectin Pathway
The resulting C4bC2a complex is a C3 
.convertase, which cleaves C3 into C3a and C3b
C3b binds to the membrane in association with C4b 
and C2a and C3a is released into the
microenvironment. The resulting C4bC2aC3b is a
.C5 convertase
The generation of C5 convertase is the end of the 
.lectin pathway
The biological activities and the regulatory proteins 
of the lectin pathway are the same as those of the
classical pathway
 Alternative Pathway
In serum there is low level of 
spontaneous hydrolysis of C3 to
produce C3i. Factor B binds to C3i
and becomes susceptible to Factor
D, which cleaves Factor B into Bb.
The C3iBb complex acts as a C3
convertase and cleaves C3 into
C3a and C3b. Once C3b is formed,
Factor B will bind to it and
becomes susceptible to cleavage
by Factor D. The resulting C3bBb
complex is a C3 convertase that
will continue to generate more C3b,
.thus amplifying C3b production
Alternative Pathway

If this process continues unchecked the 

result would be the consumption of all C3 in
the serum. Thus, the spontaneous
.production of C3b is tightly controlled
As spontaneously produced C3b binds to 
autologous host membranes it interacts with
DAF (decay accelerating factor), which
blocks the association of Factor B with C3b
thereby preventing the formation of
.additional C3 convertase
Regulation of activated C3 by DAF

As spontaneously produced C3b binds to 

autologous host membranes it interacts
with DAF (decay accelerating factor),
which blocks the association of Factor B
with C3b thereby preventing the formation
.of additional C3 convertase
Regulation of activated C3 by
DAF
In addition, DAF 
accelerates the
dissociation of Bb
from C3b in C3
convertase that
has already
formed, thereby
stop the production
of additional C3b.
Some cells
possess
complement
.receptor 1 (CR1)
 Control of the amplification
loop
Binding of C3b to CR1 facilitates 
the enzymatic degradation of C3b
by Factor I. In addition binding of
C3 convertase (C3bBb) to CR1
also dissociates Bb from the
.complex
Factor H can bind to C3b and 
facilitate the enzymatic
.degradation of C3b by Factor I
Thus, the amplification loop is 
:controlled by
Blocking C3 formation 
,convertase
,Dissociating C3 convertase 
.Enzymatic digestion of C3b 
 Generation of C5 convertase
C3b generated by C3 convertase on the activator surface 
associates with the C3bBb complex to form a C3bBbC3b
.complex (C5 convertase)
Generation of C5 convertase is the end of the alternative 
.pathway
The alternative pathway can be activated by many Gram- 
negative (most significantly, Neisseria meningitidis and N.
gonorrhoea), some Gram-positive bacteria and certain viruses
and parasites, and results in the lysis of these organisms. A
deficiency of C3 results in an increased susceptibility to these
.organisms
Thus, the alternative pathway of C activation provides another 
means of protection against certain pathogens before an
.antibody response is mounted
 Membrane attack pathway
C5 convertase from the 
classical (C4b2a3b), lectin
(C4b2a3b) or alternative
(C3bBb3b) pathway cleaves
C5 into C5a and C5b. C5a
remains in the fluid phase and
the C5b rapidly associates with
C6 and C7 and inserts into the
membrane. The C5b67
complex is referred to as the
membrane attack complex
.(MAC)
Subsequently C8 binds, 
followed by several molecules
.of C9
The C9 molecules form a pore 
in the membrane through which
the cellular contents leak and
.lysis occurs
Membrane attack pathway
C5a generated in the lytic pathway has 
:several potent biological activities
.Potent anaphylotoxin.1
.Chemotactic factor for neutrophils.2
Stimulates the respiratory burst in .3
.neutrophils
Stimulates inflammatory cytokine .4
.by macrophagesproduction

Activities of C5a are controlled by inactivation

.by carboxypeptidase B (C3-INA)
Membrane attack pathway

Some C5b67 complex formed can 

dissociate from the membrane and
enter the fluid phase and bind to other
.nearby cells leading to their lysis
This damage is prevented by Protein 
S (vitronectin). Protein S binds to
soluble C5b67 and prevents its
.binding to other cells
 Complement Deficiencies and Disease
Classical Pathway

Pathway Component Disease Mechanism

C1INH Hereditary Overproduction of C2b

Angioedema (prokinin)

C1, C2, C4 Predisposition Opsonization of immune

to SLE complexes help keep
them soluble, deficiency
results in increased
precipitation in tissues
and inflammation
Complement Deficiencies and Disease
Classical Pathway

Pathway
Disease Mechanism
Component

MBL Susceptibility to Inability to initiate

bacterial infections lectin pathway
in infants or
immunosuppresse
d
 Complement Deficiencies and Disease
Classical Pathway
Pathway/Component Disease Mechanism

Factors B or D Susceptibility Lack of sufficient

to pyogenic opsonization of bacteria
(pus-forming)
bacterial
infections
C3 Susceptibility Lack of opsonization and
to bacterial inability to utilize the
infections membrane attack pathway
C5, C6, C7 C8, or C9 Susceptibility Inability to attack the outer
to Gram- membrane of Gram-negative
negative bacteria
infections
 Complement Deficiencies and Disease
Classical Pathway

Pathway Component Disease Mechanism

Properdin (X-linked) Susceptibility Lack of opsonization of

meningococcal bacteria
meningitis

Factors H or I C3 deficiency Uncontrolled activation of

and C3 via alternative
susceptibility to pathway resulting in
bacterial depletion of C3
infections
Structure of Class I MHC
Four regions 

A highly conserved α3 
domain to which CD8
.binds
A highly polymorphic 
peptide binding region
formed from the α1 and
.α2 domains
Β2-microglobulin helps 
.stabilize the conformation
Structure of Class I MHC
.Variability map of Class 1 MHC α Chain; the peptide binding region
Structure of Class I MHC
Ag-Binding Groove
Groove composed of 
two α helix on two
opposite walls and eight
β-pleated sheets
.forming the floor
Residues lining the 
groove are most
.polymorphic
Groove accomodates 
(binds) peptides of 8-10
.amino acids long
From Janeway et al., Immunobiology 6th Ed.
 Structure of Class I MHC
Ag Binding Groove
Many different peptides can bind different MHC 
.I molecules since they are polymorphic
Specific amino acid on peptide required for 
.“anchor site” or binding in groove
.Vaccine development 
Class I Gene Polymorphism

Within the MHC there are 6 genes 

that encode class I molecules HLA-A,
HLA –B, HLA-C, HLA-E, HLA-F and
.HLA-G
Among these HLA-A, HLA –B, and 
HLA-C are the most important and are
.most polymorphic
 Number of alleles
Locus )allotypes(
HLA - A 218
HLA - B 439
HLA - C 96
There are also HLA - E, HLA - F Relatively few alleles
and HLA - G
 Structure of Class II MHC
Two polypeptide 
chains,α and β, of
.roughly of equal length
:Both have four regions 
Cytoplasmic region 
containing sites for
phosporylation and
binding to cytoskeletal
elements
Structure of Class II MHC
Four regions 
Transmembrane region 
containing hydrophobic
amino acids (anchoring)
A highly conserved α2 
and a highly conserved
β2 domains to which
CD4 binds
A highly polymorphic 
peptide binding region
formed from the α1 and
.β1 domains
Structure of Class II MHC
Variability map of Class2 MHC β Chain
Structure of Class I MHC
Ag-Binding Groove
Groove composed of 
two α helix on two
opposite walls and eight
β-pleated sheets
forming the floor
Both the α1 and β1 
domains make up the
groove
Residues lining the 
groove are most
polymorphic
From Janeway et al., Immunobiology 6th Ed.
Structure of Class I MHC
Ag-Binding Groove
Groove is open and 
accomodates
peptides of 13-25
amino acids long,
some of which are
ouside of the groove
Anchor site rules 
.apply
From Janeway et al., Immunobiology 6th Ed.
Class II Gene Polymorphism

Within the MHC there are 5 loci that 

encode class II molecules, each of
which containa gene for an α chain and
at least one gene for a β chain. The loci
are designated as HLA-DP, HLA –DQ,
HLA-DR, HLA-DM, and HLA-DO Among
these, HLA-DP, HLA –DQ, and HLA-DR
are the most important and are most
polymorphic. Table 2 shows the degree
.of polymorphism at each of these loci
 Class II polymorphism
Number of alleles
Locus )allotypes(
HLA - DPA 12
HLA - DPB 88
HLA - DQA 17
HLA - DQB 42
HLA - DRA 2
HLA - DRB1 269
HLA – DRB3 30
HLA – DRB4 7
12
HLA – DRB5

There are also HLA - DM and HLA - DO Relatively few alleles

Important Aspects of MHC
Although there is a high degree of 
polymorphism for a species, an individual
has maximum of six different class I MHC
products and only slightly more class II
MHC products (considering only the major
.loci)
Each MHC molecule has only one binding 
site. The different peptides a given MHC
molecule can bind all bind to the same site,
.but only one at a time
 Important Aspects of MHC
Because each MHC molecule can bind 
many different peptides, binding is termed
.degenerate
MHC polymorphism is determined only in 
the germline. There are no recombinational
.mechanisms for generating diversity
MHC molecules are membrane-bound; 
recognition by T cells requires cell-cell
.contact
Important Aspects of MHC
Alleles for MHC genes are co-dominant. 
Each MHC gene product is expressed on
the cell surface of an individual nucleated
.cell
A peptide must associate with a given MHC 
of that individual, otherwise no immune
response can occur. That is one level of
.control
Important Aspects of MHC
Mature T cells must have a T cell receptor 
that recognizes the peptide associated with
.MHC. This is the second level of control
Cytokines (especially interferon-γ) increase 
.level of expression of MHC
Important Aspects of MHC
Peptides from the cytosol associate with 
class I MHC and are recognized by Tc
cells . Peptides from within vesicles
associate with class II MHC and are
.recognized by Th cells
?Why so much polymorphism 

Survival of the species 

Antigen processing and presentation
 Antigen processing and presentation

Protein catabolism occurs within either acid 

.vesicle or within the cytoplasm
Peptides generated in acid vesicles bind to 
.newly synthesized MHC class II molecules
Peptides generated in cytoplasm bind to newly 
.synthesized MHC class I molecules
Binding of peptides to MHC molecules and 
transport the complex through the cell to the cell
surface occurs by certain transporters called
.Chaperones
 Generation of MHC class II-
peptide complex
Exogenous antigens (bacteria, viruses, sheep RBCs, 
ovalbumine) are taken into cells by endocytosis (soluble
antigen) or phagocytosis (insoluble particles) by
.specialised cells such as macrophages
Internalised antigen is found in intracellular vesicle that will 
fuse with a highly acidic endosomal or lysosomal vesicles
.containing a set of proteases and peptidases
Vesicles containing an immunogenic peptide intersects with 
newly synthesized MHC class II molecules, the peptide
binds to the MHC class II clip and the complex then moves
to the cell surface in order to bind CD4 T-cell expressing the
.appropriate receptor
 The association of MHC -
class II molecules and
processed peptides is
selective to peptides
consisting of 12-20 amino
acid lengths. The amino acid
sequence of the peptide-
binding groove of MHC
molecules determines which
processed peptides are
.accommodated
 Generation of MHC class I-
peptide complex
Generally the antigenic peptide is synthesized intracellularly 
.(Virus or Parasite)
Processing of the peptide occurs in the cytoplasm via a 
giant protein complex called Proteasome (cuts the peptide
.into 8-9 amino acid long)
These fragments are transported via products of either TAP1 
.and TAP2 transporter genes into the ER
MHC class I molecule and β2-microglobulin chains 
associate with Chaperones (assist the folding of MHC class I
molecule and β2-microglobulin chains and direct the
.molecule through ER)
Binding of peptide to MHC class I molecule is selective and 
.occurs in the ER
The complex moves to Golgi apparatus and then expressed 
on the cell surface to bond to CD8 T-cell expressing the
.appropriate receptor
 MHC molecules bind
peptides derived from self-
molecules
Ribosomal and mitochondrial peptides 
might bind to MHC molecules, and
generally do not result in T-cell response
?activation
These peptides are present at very low .1
.number to activate T-cell response
.T-cell made tolerant to such peptides .2
 Structure of the T cell
Receptor
Heterodimer with one α 
and one β chain of
roughly equal length
A short cytoplamic tail 
not capable of
transducing an
activation signal
A transmembrane 
region with hydrophobic
amino acids
 Structure of the T cell
Receptor
Both α and β chains 
have a variable (V) and
constant (C) region
V regions of the α and β 
chains contain
hypervariable regions
that determine the
specificity for antigen
 Structure of the T cell
Receptor
Each T cell bears TCRs 
of only one specificity
(allelic exclusion)
Genetic Basis for Receptor
Generation

Generation of a vast array of BCRs is 

accomplished by recombination of
various V, D and J gene segments
encoded in the germline
Generation of a vast array of TCRs is 
accomplished by similar mechanisms
TCR β chain genes have V, D and J 
gene segments
TCR α chain genes have V and J gene 
segments
 Organization and Rearrangement of the T
Cell Receptor
Germline ß-Chain Gene
L Vß1 L Vß2 L Vßn Dß1 Jß11--------Jß16 Cß1 Dß2 Jß11---------------Jß17 Cß2

P P P E
D-J rearrangement

L Vß1 L Vß2 L Vßn Dß1Jß15 Cß1 Dß2 Jß11---------------Jß17 Cß2

DNA
P P P E
V-D rearrangement

L Vß1 L Vß2 Dß1Jß15 Cß1 Dß2 Jß11---------------Jß17 Cß2

DNA
P P E
Transcription

Vß2 Dß1Jß15 Cß1

RNA
 Comparison of TCR and
BCR
Property BCR (sIg) TCR
Genes
Many VDJs, Few Cs Yes Yes
VDJ rearrangement Yes Yes
V regions generate Ag-binding site Yes Yes
Allelic exclusion Yes Yes
Somatic mutation Yes No
Proteins
Transmembrane form Yes Yes
Secreted form Yes No
Isotypes with different functions Yes No
Valence 2 1
 γδ TCR
Small population of T cells express a TCR 
that contain γ and δ chains instead of α and β
chains
The Gamma/Delta T cells predominate in the 
mucosal epithelia and have a repertoire
biased toward certain bacterial and viral
antigens
Genes for the δ chains have V, D and J gene 
segments; γ chains have V and J gene
segments
Repertoire is limited 
 γδ TCR
Gamma/Delta T cells can recognize antigen 
in an MHC-independent manner
Gamma/Delta T cells play a role in responses 
to certain viral and bacerial pathogens
TCR and CD3 Complex
TCR is closely 
associated with a
group of 5 proteins
collectively called
the CD3 complex
γ chain 
δ chain 
ε chains 2 
ξ chains 2 

CD3 proteins are 

invariant
Role of CD3 Complex
CD3 complex 
necessary for cell
surface expression
of TCR during T
cell development
CD3 complex 
transduces signals
to the interior of
the cells following
interaction of Ag
with the TCR
 The “Immunological
”Synapse
The interaction between 
the TCR and MHC
molecules are not strong
Accessory molecules 
stabilize the interaction
CD4/Class II MHC or 
CD8/Class I MHC
CD2/LFA-3 

LFA-1/ICAM-1 
 The “Immunological
”Synapse
Specificity for antigen 
resides solely in the
TCR
The accessory 
molecules are invariant
Expression is increased 
in response to cytokines
 The “Immunological
”Synapse
Engagement of TCR and 
Ag/MHC is one signal needed
for activation of T cells
Second signal comes from 
costimulatory molecules
CD28 on T cells interacting 
with B7-1 (CD80) or B7-2
(CD86)
Others 
Costimulatory molecules are 
invariant
”Immunological synapse“ 
Costimulation is Necessary for T Cell Activation

Engagement of TCR and 

Ag/MHC in the absence of
costimulation can lead to anergy
Engagement of costimulatory 
molecules in the absenece of
TCR engagement results in no
response
Activation only occurs when both 
TCR and costimulatory
molecules are engaged with their
respective ligands
Downregulation occurs if CTLA-4 
interacts with B7
CTLA-4 send inhibitory signal 
Key Steps in T cell
Activation
APC must process and present peptides to T cells 

T cells must receive a costimulatory signal 

Usually from CD28/B7 

Accessory adhesion molecules help to stabilize 
binding of T cell and APC
CD4/MHC-class II or CD8/MHC class I 
LFA-1/ICAM-1 
CD2/LFA-3 
Signal from cell surface is transmitted to nucleus 

Second messengers 
Cytokines produced to help drive cell division 

IL-2 and others 