MICROBIOLOGY

Humoral Immunity
Dr. Palacpac
HUMORAL IMMUNITY CD4 + T cells  express CD40L  secrete cytokines  activate B cells
 Your CD4 + T cells will integrate into a specific kind of receptor and
this would be your CD40L which secretes cytokines. Cytokines
activate further the proliferation of B cells

Polysaccharides and lipids  stimulate secretion mainly of IgM

 The immunoglobulin involved in the linkages of peptides and
polypeptides would be IgM

Protein antigens  induce production of antibodies of different classes or

isotypes (IgG, IgA, or IgE)

ANTIBODIES AS ANTIGENS
 Antibodies are proteins that contain carbohydrates (glycoproteins)
 Can also serve as strong antigens and immunogens
 Make up about 20% of the protein in blood plasma
Example:
Human IgG injected into rabbits  rabbit start producing an
Picture above: immune response  produce Ab vs. human IgG
It all started from the hematopoietic stem cell or pluripotential stem cell.  Antibody molecules with antigenic determinants  classified into
From the pluripotential stem cell it becomes the myeloid and lymphoid three types:
organs. Our lymphoid organs are usually associated with the formation of B 1. Isotypic
cells and the other one is the T cells that travels along through the thymus. 2. Allotypic
3. Idiotypic

Isotypic Epitopes/Determinants
 Constant region determinants
 Antibody from one species induce an antibody response to the
isotypic determinants in another species
 Used to determine the class or subclass of serum antibody

Allotypic Epitopes
 Allelic variations in Ig-specifying genes  variant forms of an Ig
molecule produced
 In humans, allotypes have been characterized for all four IgG
subclasses, for one IgA subclass, and for the κ light chain
 Found in constant region of heavy and light chains
Example:
Kappa chain  with allotypic region called Km  Km(1),
Km(2), Km(3)
Picture above:
o IgG from Km(1)-bearing individual  injected to
In retrospect, we also have what we call the Antigen Presenting cells (APC).
person with Km (2) allotype  Km(2)-bearing
One of our APC would be the macrophages. One function of our antibody
individual produce anti-Km(1)
(coming from B cells) would be in terms of its phagocytic activity. Antibodies
allow the engulfment of foreign materials by the macrophages by putting
“eat-me markers”. After the macrophage phagocytose the organism it will be Idiotypic Epitopes
elaborated by the helper T cells which then forms the cytokines. Cytokines  Found in variable regions of heavy and light chains
particularly the interleukins (e.g. IL-4, IL-5,IL-6) would take a role in the  Antibodies produced called anti-idiotypic Abs  involved in
formation or proliferation of B cells which in turn will proliferate further to controlling an individual’s immune response against a specific
form the Plasma cells which then creates antibodies. Cytokines also help in antigen
the proliferation of more T cells.  Each individual’s antigenic determinant of the variable region is
 Adaptive Immunity is highly specific. It has two types, it could be referred to as an idiotope
Cell-Mediated or Antibody-Mediated  In some cases an idiotope may be:
 the actual antigen-binding site
Globular protein antigens  ingested and processed by B cells  peptides  variable-region sequences outside of the antigen-binding
bound to MHC molecules  present to CD4+ T cells
site
 We have antigen-presenting cells and these are your globular
Example:
proteins antigens. It will be ingested and processed by the B cells o IgG3 produced vs. mumps virus – with a characteristic
which then form polypeptides or peptide linkages making it bound to variable region specific for mumps virus
MHC molecules (MHC class II). The MHC molecules would then o IgG3 produced vs. tetanus toxoid – with variable
present it to mucosal surfaces through the CD 4 + T cells region specific for tetanus toxoid

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Humoral Immunity
Dr. Palacpac
Idiotypic antigens continued…..
 IgG3 molecules in same individual will have different idiotypic 5. ADCC (Antibody-dependent Cell-mediated Cytotoxicity)
determinants although isotypic & allotypic determinants may be  Attachment of Ab to viral proteins  interaction of Ab-
identical coated cells with NK cells  cell lysis

B CELL RECEPTOR FOR ANTIGENS (BCRs) 6. Inhibition of attachment to mucosal cells – IgA
 Specific  respond to only one Ag or a closely related group of 7. Induction of inflammation through complement system
antigens
 Ag binds to surface Ig via the BCR  initial step in antibody formation
o BCR-Ag complex internalized by the B cell  degradation
of Ag  peptides brought to surface by class II MHC
molecules
 MHC class II: peptide complex recognized by CD4 + T cells  release
of cytokines (IL-4, IL-5, IL-6)  induction of B cell proliferation
 Activated B cells migrate into follicles  form germinal centers  B
cells differentiate into either plasma cells or memory B cells

ANTIBODY STRUCTURE

PROTECTIVE MECHANISMS OF HUMORAL IMMUNITY

1. Enhanced Phagocytosis
 Mechanisms:
a) Opsonization
IgG- in the presence or absence of complement
IgM- promote opsonization only through C3b
receptors after complement activation

b) Capsular Ag + Ab complex → increased

susceptibility to phagocytosis
2. Virus Neutralization
 Ab vs specific viral proteins  bind virus  inhibit  Heavy & Light Chains
attachment to cellular receptor o The terms “heavy” and “light” refers to molecular weight
Example: H Ag of influenza virus o Two identical light chains (23kD) and two identical heavy chains
(50-70kD)
3. Toxin Neutralization o Two classes of L chain: either κ (kappa) or λ (lambda)
 Antibodies to exotoxins  prevent binding to cellular  The 2 L chains of a single Ig are of the same type, either
receptors kappa or lambda
 Toxin + antitoxin  form immune complex  removed by o 5 classes of H chain: γ (gamma), μ (mu), α (alpha), δ (delta),
phagocytic cells ε (epsilon)

4. Complement-Mediated Lysis
 Attachment of Ab to viral proteins  activation of
complement system  formation of MAC (membrane
attack complex)  cell lysis

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Humoral Immunity
Dr. Palacpac
 Variable (V) & Constant (C) Regions  Fc Fragment
o Composed of 3-dimensionally folded repeating segments called o Fragment crystalline
domains o Mediates effector functions such as ADCC & complement-
o Light chain dependent cytotoxicity
 One VL (110 amino acids); one CL (110 amino acids)
 L chain consists of 1 variable (VL) and 1 constant (CL)
domain
o Heavy chain
 One VH (amino acids); three CH (330 – 440 amino acids)
 Most H chains consist of 1 variable (VH) and 3 constant
(CH1,CH2,CH3) domains; IgM and IgE with extra constant
region (CH4)
o Variable regions found in the amino terminal and contain the
antigen binding sites
 Antigen binding sites = VL + VH
o Constant region found in carboxy-terminal and are responsible for
various biological functions such as:
 Complement activation Ig GENES
 Binding to cell surface receptors  Separate pool of genes on different chromosomes that code for the
chains
 Disulfide Bonds o λ – chromosome 22
o Two types: o κ – chromosome 2
1. Inter-chain disulfide bonds o Heavy chain family – chromosome 14
Holds together two heavy chains and heavy chains  VL – encoded by V & J gene segments
to light chains  VH – encoded by V, D, & J gene segments
 Segments united into one functional V-variable gene by DNA
2. Intra-chain disulfide bonds rearrangement
Within each of the polypeptide chain
PHYSICAL & BIOLOGICAL PROPERTIES OF ANTIBODIES
 Fab Fragment
o Fragment antigen
binding
o Monovalent
o Created by both
VL and VH

 Hinge Region IgD

o Region at which the arms  Serves as surface marker or membrane for B cells
of the antibody molecule  No known biologic function
forms a Y  Less than 1% of serum Igs
o With some flexibility  185 kDa molecular mass
 Monomer

IgM
 Largest immunoglobulin
 First Ab produced versus antigen challenge (primary immune
response)
 Predominant in acute inflammation → disappears from the serum
after the acute stage of infection
 Can be produced in T-independent manner
 5-10% of total Igs in the adult
 Half-life is 5 days

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Humoral Immunity
Dr. Palacpac
IgM continued…… IgA
 Pentameric  theoretically with 10 Ag binding sites  5-15% of all Igs in the adult; 2nd most abundant in circulation
 Binds complement the best  Half-life of 6 days
 Act as antigen receptor on B cells  Molecular mass of 160 kDa, basic 4-chain monomer, BUT can occur
 Cannot go from blood to tissue because of size as dimers, trimers or multimers thru the J chain
 Secretory IgA = appears in body secretions for local immunity (sweat,
saliva, milk, urine, tears, mucosal surfaces, etc.)
 Production requires T-cell help and mucosal stimulation by
adjuvants like cholera toxin and attenuated salmonella
 Secreted from mucosal epithelia
 About 2 gms of IgA/day secreted by adults
 Appears in colostrum, intestinal and respiratory secretions, saliva,
tears and other secretions
 IgA-deficient persons have more frequent respiratory tract
infections
 Monomer in serum but dimer in external secretions; α chain

IgE
 Also known as reaginic antibody
 Monomer; epsilon chain
 Less than 1% of total serum Igs in adults (least Ig in concentration in
the serum under normal conditions)
 Half-life of about 2.5 days
 Most are bound to Fc receptors on mast cells  serve as receptors
for allergens and parasite Ags
IgG
 Production stimulated by TH2 helper cells
 About 85% of all Igs in the adult; Most abundant Ig in serum
 Serve as protection versus parasitic infections (helminths)
 Monomer; gamma chain
 Responsible for the manifestations of anaphylactic (Type 1)
 Half-life of 23 days (longest)
hypersensitivity
 4 subclasses: IgG 1 – 4 , different structures, relative concentration
and function CLASSES & SUBCLASSES
 Only Ig that can pass through the placenta → provide passive  5 classes: IgG, IgM, IgA, IgE, IgD
immunity to the new born  IgG and IgA with sub-classes → IgG1 , IgG2 , IgG3 , IgG4 & IgA1 , IgA2
 Production stimulated by TH1 helper cells  protective response to  Classes and sub-classes referred to as isotype
most bacteria and viruses
 Both polymeric forms of IgA and IgM with additional peptide known
 Production is T-cell dependent as the J chain → hold the monomeric sub-units together
 Provides the longest protection against infection  Dimeric IgA (in secretion) with additional component known as the
 Predominant Ig in secondary immune response (booster & secretory piece → derived from cells of the secretory epithelium
anamnestic response) and chronic infection
 Can neutralize toxins and fix complement (except IgG4) PRIMARY & SECONDARY HUMORAL RESPONSE
 An opsonin → enhance phagocytosis  Primary Immune Response
 Stimulates chemotaxis o Response on first encounter with the antigen
o Longer response time; fewer antibodies of which IgM is the
predominant
 Secondary Immune Response
o Booster response; response on re-exposure to the same
antigen
o Shorter response time; more antibodies produced of which
IgG is predominant

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Humoral Immunity
Dr. Palacpac
Additional Information from Manual:
DEVELOPMENTAL PATHWAY OF B CELLS
1. Antigen-Independent Phase
 Pro-B cell- first member of the B cell lineage
o Begins the process of Ig gene rearrangement (largely
limited to H chain genes)
 Pre-B cell- with heavy chain of IgM synthesized in the cytoplasm
 Immature B cell
o With complete L chain synthesized
o Monomeric form of IgM (sIgM) is made and inserted
into the cell’s membrane → permits the cell to
recognize and respond to antigen but contact with the
antigen leads to the shutdown and deletion of this
close
 Mature B cell
o Marked by appearance of IgD on the cell surface
o Fully capable of responding to stimulation by specific
antigen
Note: All the maturation at this point has taken place in the
Picture above: absence of specific antigens and proceeds constantly in cells
This table shows the different responses of our antibodies: Primary and derived from the pool of stem cell progenitors
Secondary responses. When a specific antigen is being introduced or exposed
to a specific antigen, it would create an initial response to an antibody, 2. Antigen-Dependent Phase
provided that a patient has a good immune system.  Cells selected specifically by reaction with antigen and appropriate
T cell help are triggered to differentiate further in one of two
An antibody titer would rise or increase upon exposure to a particular antigen ways:
usually day 14. When an antigen-antibody responses is being created, initially a) Differentiate into plasma cell
creating a primary immune response, the level of antibodies usually b) A proportion of daughter cells transform back into
decreases on the 3rd or 4th week after the exposure to a particular antigen
resting, mature B cells capable of being activated for a
subsequent and even more rapid response → memory
If there is an event that the person is exposed to the same antigen. 2nd
exposure to an antigen previously encountered would create a rise again (4- B cells
fold increase) in the antibody titer
B CELL ACTIVATION
IgM antibodies would first rise in acute infection (primary response). When 1. T-dependent B-cell Activation
IgM antibodies would start to proliferate again in a 2nd exposure to the same  Involves T-dependent antigens
antigen, it would create an anamnestic response to the previous antigen  Resting B cells bind antigens that bear epitopes complementary to
exposure (1st exposure), what would predominate now would be the IgG. IgG their cell surface Ig → antigen is endocytosed → enters endosomes
would predominate further after an acute attack of infection, and this would and lysosomes → degraded to peptides
now be your secondary immune response to the previous antigen. o Peptide loaded into class II MHC molecules and brought to
the cell surface → peptide: MHC II complex is recognized by
This principle can be seen in vaccinations. During the first injection of pre-
CD4 + T cells → signal B cell activation
formed antibodies would create antigen-antibody responses in the body
which would only be protective for a period of time which is why a booster  T cell secretes cytokines (IL-4,IL-5,IL-6, or IL-2) → regulate growth
shot is given to prolong the protective effect of the vaccine. and differentiation of the stimulated B cells
2. T-independent B-cell Activation
In other cases, such as viral infection, like dengue virus, we have what we call  Involves T-independent antigens (e.g. certain bacterial components
the dengue duo which helps in identifying on what stage the antibody is like flagella and capsule)
positive. If the person is positive for the antibody IgM and negative for IgG,  Antigen with repeating antigenic determinants → cross-link the
this means that the person is on the acute stage. If the person is negative for receptors of B cells to which they are specifically bound → stimulate
IgM and positive for IgG antibodies for dengue, this means that the person biochemical signals within the cell including tyrosine
had a dengue exposure before BUT since the person has a good immune phosphorylation & increased inositol phospholipid metabolism →
status, he was able to create an antibody for the dengue virus. This indicates
together with cytokines, cause B cell activation, growth &
that it the person is at the chronic stage.
differentiation

REFERENCES
 Microbiology Manual (2018)
 PPT
 Dr. Palacpac Recordings

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