Humoral Immunity

The immunity to infections conferred by

proteins termed antibodies

Rolf König, Ph.D.

 Learning Objectives
After this lecture, students should be able to
• Recognize the relationship between antibody structure
and function
• Classify the biological effector functions induced by
different antibody isotypes
• Explain immunoglobulin class switching
• Explain the maturation of antibody responses
• Illustrate the generation of antibody diversity
 Which cells produce antibodies?
1. Dendritic cells
2. Leukocytes
3. T cells
4. A cells
5. Erythrocytes
6. B cells
7. Macrophages
 Where are we in the immune system?
Innate Immunity ↔ Adaptive Immunity
●Antigen receptor diversity
Immunoglobulin genes
T cell receptor
●Lymphocyte activation

T helper cells
Cellular immunity B-cells
B-cell development
Immunoglobulin isotype
Protection against intracellular infection
Immunoglobulin structure and
Protection against extracellular infection function
 Antibody Structure
• Antibodies, also called
immunoglobulins (Ig), consist of
two light chains (Mr = 23,000) and
two heavy chains (Mr = 50,000 to
70,000).
• The Fab portion of Ig is the
fragment of the molecule that is
antigen binding.
• The Fc portion of Ig is constant
within an isotype and confers
effector functions.
 Isotypes of Immunoglobulins
• IgM - pentamer; serum conc. = 1.5 mg/ml
• IgD - serum conc. = 0.03 mg/ml
• IgG - subclasses IgG1 to IgG4;
serum conc. = 0.5 (IgG4) to 9 (IgG1) mg/ml
• IgE - serum conc. = 5 x 10-5 mg/ml
• IgA - subclasses IgA1 and IgA2;
serum conc. = 0.5 (IgA2) to 3 (IgA1) mg/ml
 Which antibody isotype can cross the
placental membrane?
FcRn: neonatal Fc receptor
1. IgD
2. IgA
3. IgM
4. IgE
5. IgG
 IgM
• Membrane-bound Ig (mIgM) on the plasma membrane of
B cells or pentameric molecule secreted by plasma cells.
• mIgM and secreted form differ in Fc region.
• mIgM does not polymerize.
• mIgM binds directly as an integral membrane protein, it
does not bind to IgM Fc receptors on mononuclear cells.
 IgM (pentamer)
• Secreted IgM is a
pentameric molecule.
• Binds to antigens on the
surface of a bacteria like a
spider.
• Efficient activator of
complement.
• Agglutination.
• First isotype produced in
response to infection.
Complement Fixation

• The Fc fragments of IgM and IgG are effective activators

of the classical pathway of complement fixation.
• Complement activation leads to formation of the membrane
attack complex (MAC).
• The MAC inserts into cell membranes and destroys osmotic
integrity of target cell membranes.
 Agglutination

• Binding of antibody to toxins in the circulation and in

tissues.
• Antibody-antigen complexes are phagocytosed.
• Antibodies can also immobilize and agglutinate
infectious agents (e.g., bacteria, viruses) by binding to
surface antigens.
Indirect Coombs’ Test
 Uses of the indirect Combs’ test
• To detect very low concentrations of antibodies
present in a patient's plasma prior to a blood
transfusion.
• To test for compatibility prior to transplantation.
• To screen pregnant women for antibodies that
may cause hemolytic disease of the newborn.
• To phenotype RBCs.
 IgD

• Mostly membrane-bound Ig.

• Function: largely unknown.
• May act as antigen receptor.
• May promote antigen-specific B cell
differentiation.
• Very small amount of IgD is secreted -
serum conc. = 0.03 mg/ml; function of
secreted form unknown.
 IgG
• Most abundant antibody class in the blood (total serum conc.
= 13 mg/ml).
• Four subclasses of IgG.
• Monomeric.
• Very high affinity for antigen.
• Unlike IgM, IgG can leave the blood and enter tissues.
• Only class of antibody that can pass the placental barrier.
 IgG (continued)
• Subclass of IgG produced is dependant on the cytokines
present.
• Good at activating complement.
• Most effective Ig for opsonization using Fc receptors on
phagocytes.
• Important for neutralizing toxins from bacterial
infections.
 Opsonization
• A process in which bacteria, virus-infected cells, and
others are 'tagged' for destruction.
• The Fab portion of antibodies binds to an antigen on the
surface of the cell or organism.
• The Fc portion binds to receptors on phagocytic cells
(e.g., macrophages, neutrophils).
• The Fc receptor signals the phagocyte to engulf and
destroy the organism or cell.
 IgE
• Particularly effective at mucosal surfaces.
• Serum concentration normally very low.
• Most IgE bound to Fc receptors on mast cells and basophils.
• Responsible for Type I hypersensitivity reactions (allergic
and anaphylactic).
• Increases greatly in response to parasitic infection.
• Also involved in inflammatory reponses through its role in
mast cell degranulation.
 Mast Cell
Degranulation
IgE bound to Fc receptors
on mast cells is cross-
linked by an allergen.
 IgA
• Monomeric form in the serum.
• Most common and most active at mucosal surfaces where
it appears as a dimeric protein held together by a J chain
(not the same as the J region of the antibody gene).
• To pass through epithelial surfaces, secretory component
is transiently attached to dimeric IgA.
• Primary defense at mucosal surfaces.
Structure of the Dimeric and Secretory
Forms of IgA
Formation of
Secretory IgA
 Antibodies - Their Functions
• Agglutination of bacteria and viruses - IgM
• Opsonization of bacteria - IgG
• Neutralization of bacterial toxins -IgG
• Immobilization of bacteria
• Complement activation - IgM and IgG
• Protection of mucosal surfaces - IgA
• Mast cell degranulation and parasite expulsion -IgE
• Precipitation of soluble antigens
• Antibody-dependent cell-mediated cytotoxicity (ADCC)
 The Humoral Response
• A primary response follows the first encounter of a B cell
with antigen.
• The serum level of antibodies rises slowly and declines
quickly in the primary response.
• The first antibodies detected are usually IgM. The
primary response may also include IgG in low levels.
 B cells
• Naïve B cells leave the bone marrow and migrate to the
spleen and lymph nodes.
• B cells can recognize antigen in its native form unlike T
cells which require antigen to be degraded and presented
on the surface of an antigen-presenting cell in the context
of MHC.
• The antigen recognition molecule of the B cell is the
membrane Ig.
 B cells (continued)
• Encountering antigen will activate naïve B cells.
• After antigen recognition, a naïve B cells must contact an
activated T cell.
• T and B cell interactions result in cytokine secretion by
CD4 T helper cells.
• T cell help induces B cell proliferation and differentiation
into Ig-secreting plasma cells.
 Thymus-Dependent and -Independent
Antigens
• TD antigens require direct contact with T H cells
• Most TI-1 antigens are B cell mitogens that can activate as many as 1/3rd of all B cells at
high concentrations
• At low concentrations, TI-1 antigens stimulate only antigen-specific B cells
• TI-2 antigens cross-link BCRs

Limited
 Activating Signals

• TI-1 antigen can induce both signals required for activation

• TD antigen induces only the BCR-mediated signal; 2nd signal provided via
interaction with T helper cell
 Plasma Cells

• B cells are about 6µm in diameter and have a thin rim of

cytoplasm around the nucleus. They look very much like
other lymphocytes, especially T cells.
• Plasma cells are unique. They are about 15 µm in
diameter and contain abundant endoplasmic reticulum in
the cytoplasm. This ER is necessary for the production of
antibodies.
 B cell Memory
• Antigen stimulation and T cell-derived cytokines
together induce maturation of naïve B cells into
antibody-secreting plasma cells.
• Some of the B cells that recognize the antigen
differentiate into B memory cells.
• B cell memory allows the secondary response to be much
faster than the primary response.
• The secondary response is also much greater, more
sensitive, and usually comprised of IgG.
Concentration and Isotype of Serum
Antibody After Immunization
 Antibody Isotype Switching
• Isotype switching allows B cells to change the class of
antibody they produce while retaining the same antigen
specificity.
• The isotype is defined by the Fc portion of the heavy chain.
Isotype switching rearranges this area of the Ig gene.
• Class switching occurs after B cell activation and
proliferation. It is controlled by cytokines such as
interleukin-4 (IL-4) and IL-5, and interferon-gamma.
Deficiency in Class Switching
 Genetic Model Compatible with Ig
Structure
• Diversity of Antibody Specificities
– Vertebrates can respond to an apparently limitless array of
antigens
• Presence of Variable and Constant Regions
– Every antibody contains a unique amino acid sequence in its V
region, but only one of a limited number of invariable
sequences in its C region
• Existence of Isotypes with Identical Antigenic
Specificity
 Two-Gene Model

• Dryer and Bennett 1965

• Variable and constant region are independently
encoded
• Contradicts the one gene-one protein principle
• Without precedent
Heavy Chain Gene Arrangement in the
Germ Line Facilitates Isotype Switching
 Affinity Maturation

• Affinity maturation is the gradual increase in the affinity of

antibodies synthesized by a B cell clone.
• Affinity maturation results in antibodies with 100 to
10,000 times higher affinity for the antigen and improves
efficiency of the secondary response.
• Somatic hypermutation in the Ig genes (rate = 10 -3).
• Location: germinal centers of primary follicles.
 Antibody Diversity

• The immune system can respond to an apparently limitless

array of foreign antigens.
• Capable of generating more than 1013 specificities.
• Ig sequence data show that virtually every Ig molecule
contains a unique amino acid sequence in its variable
region, but only one of a limited number of invariant
sequences in its constant region.
Multigene Organization
 The Genetic Basis
• Germ-line DNA contains multiple gene segments encoding a single
Ig heavy or light chain.
• Gene segments cannot be transcribed and translated into heavy and
light chains until they are arranged into functional genes.
• During B-cell differentiation in the bone marrow, gene segments are
randomly shuffled by a dynamic genetic system.
• Ordered progression of immunoglobulin-gene rearrangements
during B-cell differentiation.
• Immunocompetent B cells contain a single, functional variable-
region DNA sequence for its heavy chain, and a single, functional
variable-region DNA sequence for its light chain.
 Generation of Antibody Diversity

• Multiple germ line V genes

• V-J and V-D-J recombinations
• Recombinatorial inaccuracies
• Independent recombination of heavy and light chain genes
• Somatic point mutations
 B cell Malignancies
• B cell malignancies or neoplasms can arise in all
lymphoid tissues where B cells are normally produced.
• Most common B cell neoplasms involve bone marrow or
lymph nodes.
• Diagnosis is based on morphologic criteria,
immunophenotyping, and the presence of monoclonal
immunoglobulins.
• Examples are multiple myeloma, and Burkitt's, follicular
cell, and non-Hodgkin’s B cell lymphomas.
 Aspirate of a Reactive Lymph Node

Morphology: Polymorphous
lymphoid population with
sustentacular cells and
macrophages with tangible
bodies (H-E; x400).
Diagnosis: Polyclonal
lymphoid population
Aspirate of a Follicular Cell Lymphoma

Morphology: A monotonous
population of small
lymphocytes with clumped
chromatin (H-E; x1,000).