Cells and organs of the immune system

By, K. P. Komal, Asst. Prof. In Biochemistry, GSC, CTA
1 Cells and Organs of the Immune system
Lecture notes in Immunology
Topic: Cells and Organs of the immune system, Antigen and Antibody
By,
Mrs. K. P. Komal
Assistant professor in Biochemistry
Government Science College, Chitradurga
Karnataka. 577501

Introduction:
Immunology is a branch of biology that covers the study of immune systems. The immune
system is a host defense system comprising many biological structures and processes within
an organism that protects against disease. To function properly, an immune system must detect a
wide variety of agents, known as pathogens, from viruses to parasitic worms, and distinguish
them from the organism's own healthy tissue. In many species, the immune system can be
classified into subsystems, such as the innate immune system versus the adaptive immune
system, or humoral immunity versus cell-mediated immunity. In humans, the blood–brain
barrier, blood–cerebrospinal fluid barrier, and similar fluid–brain barriers separate the peripheral
immune system from the neuroimmune system which protects the brain.
Pathogens can rapidly evolve and adapt, and thereby avoid detection and neutralization by the
immune system; however, multiple defense mechanisms have also evolved to recognize and
neutralize pathogens. Even simple unicellular organisms such as bacteria possess a rudimentary
immune system, in the form of enzymes that protect against bacteriophage infections. Other basic
immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants,
such as plants and invertebrates. These mechanisms include phagocytosis, antimicrobial peptides
called defensins, and the complement system. Adaptive (or acquired) immunity creates
immunological memory after an initial response to a specific pathogen, leading to an enhanced
response to subsequent encounters with that same pathogen. This process of acquired immunity
is the basis of vaccination.
Disorders of the immune system can result in autoimmune diseases, inflammatory diseases
and cancer. Immunodeficiency occurs when the immune system is less active than normal,
resulting in recurring and life-threatening infections. In humans, immunodeficiency can either be
the result of a genetic disease such as severe combined immunodeficiency, acquired conditions
such as HIV/AIDS, or the use of immunosuppressive medication. In contrast, autoimmunity
results from a hyperactive immune system attacking normal tissues as if they were foreign
organisms. Common autoimmune diseases include Hashimoto's thyroiditis, rheumatoid
arthritis, diabetes mellitus type 1, and systemic lupus erythematosus.

Innate immune system
The innate immune system, also known as the non-specific immune system or in-born
immunity system, is an important subsystem of the overall immune system that comprises the
cells and mechanisms that defend the host from infection by other organisms. The cells of the
innate system recognize and respond to pathogens in a generic way, but, unlike the adaptive
immune system, the system does not provide long-lasting immunity to the host. Innate immune
systems provide immediate defense against infection, and are found in all classes
of plant and animal life.
The major functions of the vertebrate innate immune system include:
• Recruiting immune cells to sites of infection, through the production of chemical
factors, including specialized chemical mediators, called cytokines
• Activation of the complement cascade to identify bacteria, activate cells, and
promote clearance of antibody complexes or dead cells
• Identification and removal of foreign substances present in organs, tissues, blood and
lymph, by specialized white blood cells

• Activation of the adaptive immune system through a process known as antigen
presentation
• Acting as a physical and chemical barrier to infectious agents.
Anatomical barriers
Anatomical barriers
include physical, chemical
and biological barriers. The
epithelial surfaces form a
physical barrier that is
impermeable to most
infectious agents, acting as
the first line of defense
against invading
organisms. Desquamation
of skin epithelium also
helps remove bacteria and
other infectious agents that
have adhered to the
epithelial surfaces. Lack of
blood vessels and inability of the epidermis to retain moisture, presence of sebaceous glands in
the dermis provides an environment unsuitable for the survival of microbes. In the
gastrointestinal and respiratory tract, movement due to peristalsis or cilia, respectively, helps
remove infectious agents. Also, mucus traps infectious agents. The gut flora can prevent the
colonization of pathogenic bacteria by secreting toxic substances or by competing with pathogenic
bacteria for nutrients or attachment to cell surfaces. The flushing action of tears and saliva helps
prevent infection of the eyes and mouth.
Inflammation/ Inflammatory barriers:
Inflammation is one of the first responses of the immune system to infection or irritation.
Inflammation is stimulated by chemical factors released by injured cells and serves to establish a
Anatomical
barrier
Additional defense mechanisms
Skin Sweat, desquamation, flushing, organic acids
Gastrointestinal
tract
Peristalsis, gastric acid, bile acids, digestive
enzyme, flushing, thiocyanate, defensins, gut
flora
Respiratory
airways and lungs
Mucociliary elevator, surfactant, defensins
Nasopharynx Mucus, saliva, lysozyme
Eyes Tears

physical barrier against the spread of infection, and to promote healing of any damaged tissue
following the clearance of pathogens.
The process of acute inflammation is initiated by cells already present in all tissues, mainly
resident macrophages, dendritic cells, histiocytes, Kupffer cells, and mastocytes. These cells
present receptors contained on the surface or within the cell, named pattern recognition receptors
(PRRs), which recognize molecules that are broadly shared by pathogens but distinguishable
from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs).
At the onset of an infection, burn, or other injuries, these cells undergo activation (one of their
PRRs recognizes a PAMP) and release inflammatory mediators responsible for the clinical signs of
inflammation.
Chemical factors produced during inflammation (histamine, bradykinin, serotonin, leukotrienes,
and prostaglandins) sensitize pain receptors, cause local vasodilation of the blood vessels, and
attract phagocytes, especially neutrophils. Neutrophils then trigger other parts of the immune
system by releasing factors that summon additional leukocytes and lymphocytes.
Cytokines produced by macrophages and other cells of the innate immune system mediate the
inflammatory response. These cytokines include TNF and IL-1.
The inflammatory response is characterized by the following symptoms:
• redness of the skin, due to locally increased blood circulation;
• heat, either increased local temperature, such as a warm feeling around a localized
infection, or a systemic fever;
• swelling of affected tissues, such as the upper throat during the common cold or joints
affected by rheumatoid arthritis;
• increased production of mucus, which can cause symptoms like a runny nose or
a productive cough;
• pain, either local pain, such as painful joints or a sore throat, or affecting the whole
body, such as body aches; and
• Possible dysfunction of the organs or tissues involved.

Cellular barriers/ phagocytic barriers:
Leukocytes (white blood cells) act like independent, single-celled organisms and are the second
arm of the innate immune system. The innate leukocytes include the phagocytes (macrophages,
neutrophils, and dendritic cells), innate lymphoid cells, mast cells, eosinophils, basophils, and
natural killer cells. These cells identify and eliminate pathogens, either by attacking larger
pathogens through contact or by engulfing and then killing microorganisms. Innate cells are also
important mediators in lymphoid organ development and the activation of the adaptive immune
system.
Phagocytosis is an important feature of cellular innate immunity performed by cells called
'phagocytes' that engulf, or eat, pathogens or particles. Phagocytes generally patrol the body
searching for pathogens, but can be called to specific locations by cytokines. Once a pathogen has
been engulfed by a phagocyte, it becomes trapped in an intracellular vesicle called a phagosome,
which subsequently fuses with another vesicle called a lysosome to form a phagolysosome. The
pathogen is killed by the activity of digestive enzymes or following a respiratory burst that
releases free radicals into the phagolysosome. Phagocytosis evolved as a means of acquiring
nutrients, but this role was extended in phagocytes to include engulfment of pathogens as a
defense mechanism. Phagocytosis probably represents the oldest form of host defense, as
phagocytes have been identified in both vertebrate and invertebrate animals.
Neutrophils and macrophages are phagocytes that travel throughout the body in pursuit of
invading pathogens. Neutrophils are normally found in the bloodstream and are the most
abundant type of phagocyte, normally representing 50% to 60% of the total circulating
leukocytes. During the acute phase of inflammation, particularly as a result of bacterial infection,
neutrophils migrate toward the site of inflammation in a process called chemotaxis, and are
usually the first cells to arrive at the scene of infection. Macrophages are versatile cells that reside
within tissues and:
(i) produce a wide array of chemicals including enzymes, complement proteins, and
cytokines, while they can also
(ii) act as scavengers that rid the body of worn-out cells and other debris, and as antigen-
presenting cells that activate the adaptive immune system.

Dendritic cells (DC) are phagocytes in tissues that are in contact with the external environment;
therefore, they are located mainly in the skin, nose, lungs, stomach, and intestines. They are
named for their resemblance to neuronal dendrites, as both have many spine-like projections, but
dendritic cells are in no way connected to the nervous system. Dendritic cells serve as a link
between the bodily tissues and the innate and adaptive immune systems, as they present
antigens to T cells, one of the key cell types of the adaptive immune system.
Mast cells reside in connective tissues and mucous membranes, and regulate the inflammatory
response. They are most often associated with allergy and anaphylaxis. Basophils and eosinophils
are related to neutrophils. They secrete chemical mediators that are involved in defending
against parasites and play a role in allergic reactions, such as asthma. Natural killer (NK cells) cells
are leukocytes that attack and destroy tumor cells, or cells that have been infected by viruses.
Physiological barriers: These include body temperature; pH and other body secretions that
prevent growth of any microorganisms entering into the body. For example, body temperature i.e.,
fever, prevents the growth of pathogens and acidic atmosphere in the stomach do the same.
Moreover, lysozymes, which is present in tears digest bacterial cell wall and kill them. And when
viruses attack the body tissues, interferons present in cells protect the body tissues and
complement sysytem.
Adaptive immune system
The adaptive immune system, also known as the acquired immune system or, more rarely, as
the specific immune system, is a subsystem of the overall immune system that is composed of
highly specialized, systemic cells and processes that eliminate pathogens or prevent their growth.
Adaptive immunity creates immunological memory after an initial response to a specific pathogen,
and leads to an enhanced response to subsequent encounters with that pathogen. This process of
acquired immunity is the basis of vaccination. Like the innate system, the adaptive system
includes both humoral immunity components and cell-mediated immunity components.
Unlike the innate immune system, the adaptive immune system is highly specific to a particular
pathogen. Adaptive immunity can also provide long-lasting protection; for example, someone who
recovers from measles is now protected against measles for their lifetime. In other cases it does
not provide lifetime protection; for example, chickenpox. The adaptive system response destroys

invading pathogens and any toxic molecules they produce. Sometimes the adaptive system is
unable to distinguish harmful from harmless foreign molecules; the effects of this may be hay
fever, asthma or any other allergy. Antigens are any substances that elicit the adaptive immune
response. The cells that carry out the adaptive immune response are white blood cells known as
lymphocytes. Two main broad classes—antibody responses and cell mediated immune
response—are also carried by two different lymphocytes (B cells and T cells). In antibody
responses, B cells are activated to secrete antibodies, which are proteins also known as
immunoglobulins. Antibodies travel through the bloodstream and bind to the foreign antigen
causing it to inactivate, which does not allow the antigen to bind to the host.
In acquired immunity, pathogen-specific receptors are "acquired" during the lifetime of the
organism. The acquired response is called "adaptive" because it prepares the body's immune
system for future challenges.
The system is highly adaptable because of somatic hypermutation (a process of accelerated
somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen
receptor gene segments). This mechanism allows a small number of genes to generate a vast
number of different antigen receptors, which are then uniquely expressed on each
individual lymphocyte. Since the gene rearrangement leads to an irreversible change in the DNA of
each cell, all progeny (offspring) of that cell inherit genes that encode the same receptor
specificity, including the memory B cells and memory T cells that are the keys to long-lived
specific immunity.
Functions
Adaptive immunity is triggered in vertebrates when a pathogen evades the innate immune system
and (1) generates a threshold level of antigen and
(2) generates "stranger" or "danger" signals activating dendritic cells.
The major functions of the adaptive immune system include:
• Recognition of specific "non-self" antigens in the presence of "self", during the process
of antigen presentation.
• Generation of responses that are tailored to maximally eliminate specific pathogens or
pathogen-infected cells.

• Development of immunological memory, in which pathogens are "remembered"
through memory B cells and memory T cells.
Cell mediated and humoral immunity:
Cell-mediated immunity is an immune response that does not involve antibodies, but rather
involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of
various cytokines in response to an antigen.
Historically, the immune system was separated into two branches: humoral immunity, for which
the protective function of immunization could be found in the humor (cell-free bodily fluid
or serum) and cellular immunity, for which the protective function of immunization was
associated with cells. CD4 cells or helper T cells provide protection against different pathogens.
Naive T cells, mature T cells that have yet to encounter an antigen, are converted into activated
effector T cells after encountering antigen-presenting cells (APCs). These APCs, such as
macrophages, dendritic cells, and B cells in some circumstances, load antigenic peptides onto
the MHC complex of the cell, in turn presenting the peptide to receptors on T cells. The most
important of these APCs are highly specialized dendritic cells; conceivably operating solely to
ingest and present antigens.
Activated Effector T cells can be placed into three functioning classes, detecting peptide antigens
originating from various types of pathogen: The first class being Cytotoxic T cells, which kill
infected target cells by apoptosis without using cytokines, the second class being TH1 cells, which
primarily function to activate macrophages, and the third class being TH2 cells, which primarily
function to stimulate B cells into producing antibodies.
The innate immune system and the adaptive immune system each comprise both humoral and
cell-mediated components.
Cellular immunity protects the body by:
• T-cell mediated immunity or T-cell immunity : activating antigen-specific cytotoxic T
cells that are able to induce apoptosis in body cells displaying epitopes of foreign
antigen on their surface, such as virus-infected cells, cells with intracellular bacteria,
and cancer cells displaying tumor antigens;

• activating macrophages and natural killer cells, enabling them to destroy pathogens;
and
• Stimulating cells to secrete a variety of cytokines that influence the function of other
cells involved in adaptive immune responses and innate immune responses.
Cell-mediated immunity is directed primarily at microbes that survive in phagocytes and
microbes that infect non-phagocytic cells. It is most effective in removing virus-infected cells, but
also participates in defending against fungi, protozoans, cancers, and intracellular bacteria. It also
plays a major role in transplant rejection.
Humoral immunity or humoural immunity is the aspect of immunity that is mediated
by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins,
and certain antimicrobial peptides. Humoral immunity is so named because it involves substances
found in the humors, or body fluids. It contrasts with cell-mediated immunity. Its aspects involving
antibodies are often called antibody-mediated immunity.
Humoral immunity refers to antibody production and the accessory processes that accompany it,
including: Th2 activation and cytokine production, germinal center formation and isotype
switching affinity maturation and memory cell generation. It also refers to the effector functions
of antibodies, which include pathogen and toxin neutralization, classical complement activation,
and opsonin promotion of phagocytosis and pathogen elimination.

Haematopoiesis
Haematopoiesis (sometimes also haemopoiesis or hemopoiesis) is the formation of blood
cellular components. All cellular blood components are derived from haematopoietic stem
cells. In a healthy adult person, approximately 1011–1012 new blood cells are produced daily in
order to maintain steady state levels in the peripheral circulation.
Haematopoietic stem cells (HSCs) reside in the medulla of the bone (bone marrow) and have the
unique ability to give rise to all of the different mature blood cell types and tissues. HSCs are self-
renewing cells: when they proliferate, at least some of their daughter cells remain as HSCs, so the
pool of stem cells is not depleted. This phenomenon is called asymmetric division. The other
daughters of HSCs (myeloid and lymphoid progenitor cells) can follow any of the other
differentiation pathways that lead to the production of one or more specific types of blood cell, but
cannot renew themselves. The pool of progenitors is heterogeneous and can be divided into two

groups; long-term self-renewing HSC and only transiently self-renewing HSC, also called short-
terms. This is one of the main vital processes in the body.
All blood cells are divided into three lineages.
• Erythroid cells are the oxygen carrying red blood cells. Both reticulocytes and
erythrocytes are functional and are released into the blood. In fact, a reticulocyte count
estimates the rate of erythropoiesis.
• Lymphocytes are the corner stone of the adaptive immune system. They are derived from
common lymphoid progenitors. The lymphoid lineage is primarily composed of T-
cells and B-cells (types of white blood cells). This is lymphopoiesis.
• Myelocytes,whichinclude granulocytes, megakaryocytes and macrophages and are derived
from common myeloid progenitors, are involved in such diverse roles as innate
immunity, adaptive immunity, and blood clotting. This is myelopoiesis.
• Granulopoiesis (or granulocytopoiesis) is haematopoiesis of granulocytes.
• Megakaryocytopoiesis is haematopoiesis of megakaryocytes.
Comprehensive diagram that shows the development of different blood cells from haematopoietic stem cell to mature cells

Cells of immune system
The response to pathogens is orchestrated by the complex interactions and activities of the large
number of diverse cell types involved in the immune response. The innate immune response is the
first line of defense and occurs soon after pathogen exposure. It is carried out by phagocytic cells
such as neutrophils and macrophages, cytotoxic natural killer (NK) cells, and granulocytes. The
subsequent adaptive immune response includes antigen-specific defense mechanisms and may
take days to develop. Cell types with critical roles in adaptive immunity are antigen-presenting
cells including macrophages and dendritic cells. Antigen-dependent stimulation of various cell
types including T cell subsets, B cells, and macrophages all play critical roles in host defense.
Lymphocytes
The cells of the adaptive immune system are T and B lymphocytes; lymphocytes are a subset
of leukocyte. B cells and T cells are the major types of lymphocytes. The human body has about 2
trillion lymphocytes, constituting 20–40% of white blood cells (WBCs); their total mass is about
the same as the brain or liver. The peripheral blood contains 2% of circulating lymphocytes; the
rest move within the tissues and lymphatic system.
B cells and T cells are derived from the same multipotent hematopoietic stem cells, and are
morphologically indistinguishable from one another until after they are activated. B cells play a
large role in the humoral immune response, whereas T cells are intimately involved in cell-
mediated immune responses. In all vertebrates except Agnatha, B cells and T cells are produced by
stem cells in the bone marrow.
T progenitors migrate from the bone marrow to the thymus where they are called thymocytes and
where they develop into T cells. In humans, approximately 1–2% of the lymphocyte pool
recirculates each hour to optimize the opportunities for antigen-specific lymphocytes to find their
specific antigen within the secondary lymphoid tissues. In an adult animal, the peripheral
lymphoid organs contain a mixture of B and T cells in at least three stages of differentiation:
• naive B and naive T cells (cells that have not matured), left the bone marrow or thymus,
have entered the lymphatic system, but have yet to encounter their cognate antigen,
• effector cells that have been activated by their cognate antigen, and are actively
involved in eliminating a pathogen.

• memory cells – the survivors of past infections.
T lymphocytes
CD8+ T lymphocytes and cytotoxicity
Cytotoxic T cells (also known as TC, killer T cell, or cytotoxic T-lymphocyte (CTL)) are a sub-
group of T cells that induce the death of cells that are infected with viruses (and other pathogens),
or are otherwise damaged or dysfunctional.
Naive cytotoxic T cells are activated when their T-cell receptor (TCR) strongly interacts with a
peptide-bound MHC class I molecule. This affinity depends on the type and orientation of the
antigen/MHC complex, and is what keeps the CTL and infected cell bound together. Once
activated, the CTL undergoes a process called clonal selection, in which it gains functions and
divides rapidly to produce an army of “armed” effector cells. Activated CTL then travels
throughout the body searching for cells that bear that unique MHC Class I + peptide.
When exposed to these infected or dysfunctional somatic cells, effector CTL release perforin and
granulysin: cytotoxins that form pores in the target cell's plasma membrane, allowing ions and
water to flow into the infected cell, and causing it to burst or lyse. CTL release granzyme, a serine
protease that enters cells via pores to induce apoptosis (cell death). To limit extensive tissue
damage during an infection, CTL activation is tightly controlled and in general requires a very
strong MHC/antigen activation signal, or additional activation signals provided by "helper" T-cells.
On resolution of the infection, most effector cells die and phagocytes clear them away—but a few
of these cells remain as memory cells. On a later encounter with the same antigen, these memory
cells quickly differentiate into effector cells, dramatically shortening the time required to mount
an effective response.

Helper T-cells
The T lymphocyte activation pathway. T cells contribute to immune defenses in two major ways: some direct and regulate immune responses;
others directly attack infected or cancerous cells.
CD4+ lymphocytes, also called "helper" or "regulatory" T cells, are immune response
mediators, and play an important role in establishing and maximizing the capabilities of the
adaptive immune response. These cells have no cytotoxic or phagocytic activity; and cannot kill
infected cells or clear pathogens, but, in essence "manage" the immune response, by directing
other cells to perform these tasks.
Helper T cells express T cell receptors (TCR) that recognize antigen bound to Class II MHC
molecules. The activation of a naive helper T-cell causes it to release cytokines, which influences
the activity of many cell types, including the APC (Antigen-Presenting Cell) that activated it. Helper

T-cells require a much milder activation stimulus than cytotoxic T cells. Helper T cells can provide
extra signals that "help" activate cytotoxic cells.
Th1 and Th2: helper T cell responses
Classically, two types of effector CD4+ T helper cell responses can be induced by a professional
APC, designated Th1 and Th2, each designed to eliminate different types of pathogens. The factors
that dictate whether an infection triggers a Th1 or Th2 type response are not fully understood, but
the response generated does play an important role in the clearance of different pathogens.
The Th1 response is characterized by the production of Interferon-gamma, which activates
the bactericidal activities of macrophages, and induces B cells to make opsonizing (coating) and
complement-fixing antibodies, and leads to cell-mediated immunity. In general, Th1 responses are
more effective against intracellular pathogens (viruses and bacteria that are inside host cells).
The Th2 response is characterized by the release of Interleukin 5, which induces eosinophils in the
clearance of parasites. Th2 also produce Interleukin 4, which facilitates B cell isotype switching. In
general, Th2 responses are more effective against extracellular bacteria, parasites including
helminths and toxins. Like cytotoxic T cells, most of the CD4+ helper cells die on resolution of
infection, with a few remaining as CD4+ memory cells.
Increasingly, there is strong evidence from mouse and human-based scientific studies of a broader
diversity in CD4+ effector T helper cell subsets. Regulatory T (Treg) cells, have been identified as
important negative regulators of adaptive immunity as they limit and suppresses the immune
system to control aberrant immune responses to self-antigens; an important mechanism in
controlling the development of autoimmune diseases. Follicular helper T (Tfh) cells are another
distinct population of effector CD4+ T cells that develop from naive T cells post-antigen activation.
Tfh cells are specialized in helping B cell humoral immunity as they are uniquely capable of
migrating to follicular B cells in secondary lymphoid organs and provide them positive paracrine
signals to enable the generation and recall production of high-quality affinity-matured antibodies.
Similar to Tregs, Tfh cells also play a role in immunological tolerance as an abnormal expansion of
Tfh cell numbers can lead to unrestricted autoreactive antibody production causing severe
systemic autoimmune disorders.

The relevance of CD4+ T helper cells is highlighted during an HIV infection. HIV is able to subvert
the immune system by specifically attacking the CD4+ T cells, precisely the cells that could drive
the clearance of the virus, but also the cells that drive immunity against all other pathogens
encountered during an organism's lifetime.
B lymphocytes and antibody production
The B lymphocyte activation pathway. B cells function to protect the host by producing antibodies
that identify and neutralize foreign objects like bacteria and viruses.
B Cells are the major cells involved in the creation of antibodies that circulate in blood plasma and
lymph, known as humoral immunity. Antibodies (also known as immunoglobulin, Ig), are large Y-
shaped proteins used by the immune system to identify and neutralize foreign objects. In
mammals, there are five types of antibody: IgA, IgD, IgE, IgG, and IgM, differing in biological
properties; each has evolved to handle different kinds of antigens. Upon activation, B cells produce
antibodies, each of which recognizing a unique antigen, and neutralizing specific pathogens.
Like the T cell, B cells express a unique B cell receptor (BCR), in this case, a membrane-bound
antibody molecule. All the BCR of any one clone of B cells recognizes and binds to only one
particular antigen. A critical difference between B cells and T cells is how each cell "sees" an

antigen. T cells recognize their cognate antigen in a processed form – as a peptide in the context of
an MHC molecule, whereas B cells recognize antigens in their native form. Once a B cell encounters
its cognate (or specific) antigen (and receives additional signals from a helper T cell
(predominately Th2 type)), it further differentiates into an effector cell, known as a plasma cell.
Plasma cells are short-lived cells (2–3 days) that secrete antibodies. These antibodies bind to
antigens, making them easier targets for phagocytes, and trigger the complement cascade. About
10% of plasma cells survive to become long-lived antigen-specific memory B cells. Already primed
to produce specific antibodies, these cells can be called upon to respond quickly if the same
pathogen re-infects the host, while the host experiences few, if any, symptoms.
Immunological memory
When B cells and T cells are activated some become memory B cells and some memory T cells.
Throughout the lifetime of an animal these memory cells form a database of effective B and T
lymphocytes. Upon interaction with a previously encountered antigen, the appropriate memory
cells are selected and activated. In this manner, the second and subsequent exposures to an
antigen produce a stronger and faster immune response. This is "adaptive" because the body's
immune system prepares itself for future challenges, but is "maladaptive" of course if the
receptors are autoimmune. Immunological memory can be in the form of either passive short-
term memory or active long-term memory.
Passive memory
Passive memory is usually short-term, lasting between a few days and several months.
Newborn infants have had no prior exposure to microbes and are particularly vulnerable to
infection. Several layers of passive protection are provided by the mother. In utero, maternal IgG is
transported directly across the placenta, so that, at birth, human babies have high levels of
antibodies, with the same range of antigen specificities as their mother. Breast milk contains
antibodies (mainly IgA) that are transferred to the gut of the infant, protecting against bacterial
infections, until the newborn can synthesize its own antibodies.
This is passive immunity because the fetus does not actually make any memory cells or antibodies:
It only borrows them. Short-term passive immunity can also be transferred artificially from one
individual to another via antibody-rich serum.

Active memory
In general, active immunity is long-term and can be acquired by infection followed by B cells and T
cells activation, or artificially acquired by vaccines, in a process called immunization.
Neutrophil
Neutrophils are the most abundant white blood cell, constituting 60-70% of the circulating
leukocytes. They defend against bacterial or fungal infection. They are usually first responders to
microbial infection; their activity and death in large numbers forms pus. They are commonly
referred to as polymorphonuclear (PMN) leukocytes, although, in the technical sense, PMN refers
to all granulocytes. They have a multi-lobed nucleus, which consists of three to five lobes
connected by slender strands. This gives the neutrophils the appearance of having multiple nuclei,
hence the name polymorphonuclear leukocyte. The cytoplasm may look transparent because of
fine granules that are pale lilac when stained. Neutrophils are active in phagocytosing bacteria and
are present in large amount in the pus of wounds. These cells are not able to renew
their lysosomes (used in digesting microbes) and die after having phagocytosed a few
pathogens. Neutrophils are the most common cell type seen in the early stages of acute
inflammation. The life span of a circulating human neutrophil is about 5.4 days.
Eosinophil
Eosinophils compose about 2-4% of the WBC total. This count fluctuates throughout the day,
seasonally, and during menstruation. It rises in response to allergies, parasitic infections, collagen
diseases, and disease of the spleen and central nervous system. They are rare in the blood, but
numerous in the mucous membranes of the respiratory, digestive, and lower urinary tracts.
They primarily deal with parasitic infections. Eosinophils are also the predominant inflammatory
cells in allergic reactions. The most important causes of eosinophilia include allergies such as
asthma, hay fever, and hives; and also parasitic infections. They secrete chemicals that destroy
these large parasites, such as hook worms and tapeworms, that are too big for any one WBC to
phagocytize. In general, their nucleus is bi-lobed. The lobes are connected by a thin strand. The
cytoplasm is full of granules that assume a characteristic pink-orange color with eosin staining.

Basophil
Basophils are chiefly responsible for allergic and antigen response by releasing the chemical
histamine causing the dilation of blood vessels. Because they are the rarest of the white blood
cells (less than 0.5% of the total count) and share physicochemical properties with other blood
cells, they are difficult to study. They can be recognized by several coarse, dark violet granules,
giving them a blue hue. The nucleus is bi- or tri-lobed, but it is hard to see because of the number
of coarse granules that hide it.
They excrete two chemicals that aid in the body's defenses: histamine and heparin. Histamine is
responsible for widening blood vessels and increasing the flow of blood to injured tissue. It also
makes blood vessels more permeable so neutrophils and clotting proteins can get into connective
tissue more easily. Heparin is an anticoagulant that inhibits blood clotting and promotes the
movement of white blood cells into an area. Basophils can also release chemical signals that
attract eosinophils and neutrophils to an infection site.
Monocyte
Monocytes, the largest type of WBCs, share the "vacuum cleaner" (phagocytosis) function of
neutrophils, but are much longer lived as they have an extra role: they present pieces
of pathogens to T cells so that the pathogens may be recognized again and killed. This causes an
antibody response to be mounted. Monocytes eventually leave the bloodstream and become
tissue macrophages, which remove dead cell debris as well as attack microorganisms. Neither
dead cell debris nor attacking microorganisms can be dealt with effectively by the neutrophils.
Unlike neutrophils, monocytes are able to replace their lysosomal contents and are thought to
have a much longer active life. They have the kidney shaped nucleus and are typically agranulated.
They also possess abundant cytoplasm.

Fixed leucocytes
HSC=Hematopoietic stem cell, Progenitor=Progenitor cell, L-blast=Lymphoblast, Lymphocyte, Mo-
blast=Monoblast, Monocyte, Myeloblast, Pro-M=Promyelocyte, Myelocyte, Meta-M =
Metamyelocyte, Neutrophil, Eosinophil, Basophil, Pro-E = Proerythroblast, Baso-E=Basophilic
erythroblast, poly-E=Polychromatic erythroblast, Ortho-E = Orthochromatic erythroblast,
Erythrocyte, Promegakaryocyte, Megakaryocyte, Platelet
Some leucocytes migrate into the tissues of the body to take up a permanent residence at that
location rather than remaining in the blood. Often these cells have specific names depending upon
which tissue they settle in, such as fixed macrophages in the liver, which become known
as Kupffer cells. These cells still serve a role in the immune system.
• Histiocytes
• Dendritic cells (Although these will often migrate to local lymph nodes upon ingesting
antigens)
• Mast cells

• Microglia
A histiocyte is an animal cell that is part of the mononuclear phagocyte system (also known as the
reticuloendothelial system or lymphoreticular system). The mononuclear phagocytic system is
part of the organism's immune system. The histiocyte is a tissuemacrophage or a dendritic cell
(histio, diminutive of histo, meaning tissue, and cyte, meaning cell).
Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the
mammalian immune system. Their main function is to process antigen material and present it on
the cell surface to the T cells of the immune system. They act as messengers between the innate
and the adaptive immune systems.
Dendritic cells are present in those tissues that are in contact with the external environment, such
as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and the
inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature
state in the blood. Once activated, they migrate to the lymph nodes where they interact with T
cells and B cells to initiate and shape the adaptive immune response. At certain development
stages they grow branched projections, the dendrites that give the cell its name. While similar in
appearance, these are structures distinct from the dendrites of neurons. Immature dendritic cells
are also called veiled cells, as they possess large cytoplasmic 'veils' rather than dendrites.
A mast cell (also known as a mastocyte or a labrocyte) is a type of white blood cell. Specifically,
it is a type of granulocyte derived from the myeloid stem cell that is a part of the immune and
neuroimmune systems and contains many granules rich in histamine and heparin. Although best
known for their role in allergy and anaphylaxis, mast cells play an important protective role as
well, being intimately involved in wound healing, angiogenesis, immune tolerance, defense against
pathogens, and blood–brain barrier function.
The mast cell is very similar in both appearance and function to the basophil, another type
of white blood cell. Although mast cells were once thought to be tissue resident basophils, it has
been shown that the two cells develop from different hematopoietic lineages and thus cannot be
the same cells.
Microglia are a type of glial cell located throughout the brain and spinal cord. Microglia account
for 10–15% of all cells found within the brain. As the resident macrophage cells, they act as the

first and main form of active immune defense in the central nervous system (CNS). Microglia (and
other glia including astrocytes) are distributed in large non-overlapping regions throughout the
CNS. Microglia are key cells in overall brain maintenance—they are constantly scavenging the CNS
for plaques, damaged or unnecessary neurons and synapses, and infectious agents. Since these
processes must be efficient to prevent potentially fatal damage, microglia are extremely sensitive
to even small pathological changes in the CNS. This sensitivity is achieved in part by the presence
of unique potassium channels that respond to even small changes in extracellular potassium.
The brain and spinal cord, which make up the CNS, are not usually accessed directly by pathogenic
factors in the body's circulation due to a series of endothelial cells known as the blood–brain
barrier, or BBB. The BBB prevents most infections from reaching the vulnerable nervous tissue. In
the case where infectious agents are directly introduced to the brain or cross the blood–brain
barrier, microglial cells must react quickly to decrease inflammation and destroy the infectious
agents before they damage the sensitive neural tissue. Due to the unavailability of antibodies from
the rest of the body (few antibodies are small enough to cross the blood–brain barrier), microglia
must be able to recognize foreign bodies, swallow them, and act as antigen-presenting
cells activating T-cells.
Organs of the immune system
The immune system includes primary lymphoid organs, secondary lymphatic tissues and various
cells in the innate and adaptive immune systems.
The organs of the immune system are positioned throughout the body. They are called lymphoid
organs because they are home to lymphocytes, small white blood cells that are the key players in
the immune system.
There are two groups of immune system organs.
• Primary (central)--organs where immature lymphocytes develop
Thymus
Bone marrow

• Secondary (peripheral)--tissues where antigen is localized so that it can be effectively
exposed to mature lymphocytes
Lymph nodes
Appendix
Peyer's Patches (of GI tract)
Tonsils
Adenoids
Spleen
MALT (Mucosal-Associated Lymphoid Tissue)
GALT (Gut-Associated Lymphoid Tissue)
BALT (Bronchial/Tracheal-Associated Lymphoid Tissue)
NALT (Nose-Associated Lymphoid Tissue)
VALT (Vulvovaginal-Associated Lymphoid Tissue)
Thymus
The thymus (in Greek it means warty outgrowth) is the site of T cell maturation. T cells become
immunocompetent here; that is, they develop their ability to mount an effective immune response
against foreign invaders without attacking the host's own tissues. The thymus lies just above the
heart in the mediastinum. It is largest in childhood, and it begins to shrink significantly as a person
ages. The organ itself contains two lobes, and each lobe contains numerous lobules, separated
from each other by connective tissue septa known as trabeculae. Each lobule is separated into an
inner medulla (with few immature thymocytes) and an outer cortex (with large numbers of
immature thymocytes). In the thymus, the many different T cells are exposed to MHC/Ag and
MHC/self-Ag. If they do not react to MHC/Ag, they are destroyed due to their ineffectiveness
(positive selection). On the other hand, if they do react to MHC/self-AG, they are destroyed in
order to stop them from becoming traitorous autoimmune responses against the body's own
tissue (negative selection). Only 1 out of 20 immature thymocytes will pass successfully through
this vetting process and become functional T cells. Dendritic cells, macrophages, and epithelial
cells are interspersed throughout both the medulla and cortex; special epithelial nurse cells
surround clusters of thymocytes in the cortex.

Myasthenia gravis is an autoimmune disease where the body creates antibodies against
acetylcholine receptors at post-synaptic neuromuscular junctions. In up to 25% of MG cases, there
is a tumor of the thymus present (thymoma); the exact reasons for this tumor development is
unknown. Additionally, removal of the thymoma will halt the disease's progress; this deserves
further study as antibodies are released from plasma B cells rather than T cells, yet T helper cells
may mediate the autoimmunity.
DiGeorge's Syndrome is a congenital lack of a thymus, and causes in increase in infections and a
depressed immune system (especially the cell-mediated response is retarded by the lack of a
thymus).
Bone Marrow
Bone marrow (medulla ossea) is the site of B cell maturation in mice and humans. B cells undergo
both positive and negative selection, similar to T cell maturation in the thymus. Bone marrow is
also the site of hematopoiesis, the development of the myriad blood cells from progenitor cells.
The site of B cell maturation in birds is the bursa of Fabricius, after which B cells are named. The
tissue of bone marrow where leukocytes, red blood cells, and platelets develop (i.e., the site of
hematopoiesis) is known as myeloid tissue.
Leukemia is a cancer of the bone marrow that causes abnormal production of leukocytes (WBCs).
Acute leukemia is the biggest killer of children in the US; it results from the overgrowth
of immature leukocytes in the bone marrow, retarding the production of functional
WBCs, RBCs, and platelets.
Chronic leukemia is the overgrowth of relatively mature (but abnormal) leukocytes in
the bone marrow.
Lymphocytic leukemia is the overgrowth of lymphoid cells (T cells, B cells, Natural
Killer Cells, dendritic cells).
Myeloid leukemia is the overgrowth of myeloid cells (all blood cell types that are not
lymphocytic are produced from myeloid progenitor stem cells).

Locations
In developing embryos, blood formation occurs in aggregates of blood cells in the yolk sac,
called blood islands. As development progresses, blood formation occurs in
the spleen, liver and lymph nodes. When bone marrow develops, it eventually assumes the task of
forming most of the blood cells for the entire organism. However, maturation, activation, and
some proliferation of lymphoid cells occurs in the spleen, thymus, and lymph nodes. In children,
haematopoiesis occurs in the marrow of the long bones such as the femur and tibia. In adults, it
occurs mainly in the pelvis, cranium, vertebrae, and sternum.
Lymph Nodes
Extracellular fluid flows from capillary beds into tissue; from this tissue it enters lymphatic
capillaries that are "pumped" along with the movement of skeletal muscle towards lymph nodes.
The paracortical areas of the nodes contain T cells, and the central areas contain germinal
centers, where B cells are contained. APCs and antigen are sent from the tissue into the
lymphatics, eventually reaching the lymph nodes where they can be exposed to the T and B cell
populations. This allows a faster response, as the many combinations of T and B cell specificities
are able to reside in several locations throughout the body (the lymph nodes) rather than relying
on random meetings of antigen and lymphocytes throughout the tissues themselves.
Spleen
The spleen acts as a site of hematopoiesis during the second and third trimesters of development,
before the long bones have fully developed. In the adult, the spleen acts as a site for breakdown of
dying red blood cells (lifespan 120 days). For this reason, enlargement of the spleen
(splenomegaly) can occur in sickle cell anemia or in certain infections. White pulp, near the
arteriolar entry points into the spleen, is where lymphocytes reside and are degraded. The central
red pulp is the site of RBCs breakdown. The white pulp region has a central part, with the T cells
residing in the PALS, or Peri Arteriolar Lymphoid Sheath and a B cell ring (or corona)
surrounding the PALS.

Antigen
In immunology, an antigen is a molecule capable of inducing an immune response on the part of
the host organism, though sometimes antigens can be part of the host itself. In other words, an
antigen is any substance that causes an immune system to produce antibodies against it. Each
antibody is specifically produced by the immune system to match an antigen after cells in the
immune system come into contact with it.
An antigen is a molecule that binds to Ag-specific receptors, but cannot necessarily induce an
immune response in the body by itself. Antigens are usually peptides, polysaccharides or lipids. In
general, molecules other than peptides (saccharides and lipids) qualify as antigens but not
as immunogens since they cannot elicit an immune response on their own. Furthermore, for a
peptide to induce an immune response (activation of T-cells by antigen-presenting cells) it must
be a large enough size, since peptides too small will also not elicit an immune response. The term
antigen originally described a structural molecule that binds specifically to an antibody. It
expanded to refer to any molecule or a linear molecular fragment that can be recognized by highly
variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system.
The antigen may originate from within the body ("self-antigen") or from the external environment
("non-self"). The immune system usually does not react to self-antigens under
normal homeostatic conditions due to negative selection of T cells in the thymus and is supposed
to identify and attack only "non-self" invaders from the outside world or modified/harmful
substances present in the body under distressed conditions.
Antigen presenting cells present antigens in the form of peptides on histocompatibility molecules.
The T cells of the adaptive immune system recognize the antigens. Depending on the antigen and
the type of the histocompatibility molecule, different types of T cells are activated. For T-Cell
Receptor (TCR) recognition, the peptide must be processed into small fragments inside the cell
and presented by a major histocompatibility complex (MHC). The antigen cannot elicit the
immune response without the help of an immunologic adjuvant. Similarly, the adjuvant
component of vaccines plays an essential role in the activation of the innate immune system.
An immunogen is a substance (or adduct) that is able to trigger a humoral (innate) and/or cell-
mediated immune response. It first initiates an innate immune response, which then causes the

activation of the adaptive immune response. An antigen binds the highly variable immunoreceptor
products (B-cell receptor or T-cell receptor) once these have been generated. All immunogen
molecules are also antigens, although the reverse is not true.
At the molecular level, an antigen can be characterized by its ability to bind to an
antibody's variable Fab region. Different antibodies have the potential to discriminate among
specific epitopes present on the antigen surface. A hapten is a small molecule that changes the
structure of an antigenic epitope. In order to induce an immune response, it needs to be attached
to a large carrier molecule such as a protein. Antigens are usually proteins and polysaccharides,
and less frequently, lipids. This includes parts (coats, capsules, cell walls, flagella, fimbrae, and
toxins) of bacteria, viruses, and other microorganisms. Lipids and nucleic acids are antigenic only
when combined with proteins and polysaccharides. Non-microbial non-self antigens can include
pollen, egg white and proteins from transplanted tissues and organs or on the surface of
transfused blood cells. Vaccines are examples of antigens in an immunogenic form, which are
intentionally administered to induce the memory function of adaptive immune system toward the
antigens of the pathogen invading the recipient.
Terminology
• Epitope – The distinct surface features of an antigen, its antigenic determinant.
Antigenic molecules, normally "large" biological polymers, usually present surface
features that can act as points of interaction for specific antibodies. Any such feature
constitutes an epitope. Most antigens have the potential to be bound by multiple
antibodies, each of which is specific to one of the antigen's epitopes. Using the "lock and
key" metaphor, the antigen can be seen as a string of keys (epitopes) each of which
matches a different lock (antibody). Different antibody idiotypes, each have distinctly
formed complementarity determining regions.
• Allergen – A substance capable of causing an allergic reaction. The (detrimental)
reaction may result after exposure via ingestion, inhalation, injection, or contact with
skin.
• Superantigen – A class of antigens that cause non-specific activation of T-cells,
resulting in polyclonal T cell activation and massive cytokine release.

• Tolerogen – A substance that invokes a specific immune non-responsive due to
its molecular form. If its molecular form is changed, a tolerogen can become an
immunogen.
• Immunoglobulin-binding protein – Proteins such as Protein A, protein G, and protein
L that are capable of binding to antibodies at positions outside of the antigen-binding
site. While antigens are the "target" of antibodies, immunoglobulin-binding proteins
"attack" antibodies. .
• T-dependent antigen – Antigens that require the assistance of T cells to induce the
formation of specific antibodies.
• T-independent antigen – Polysaccharides (usually) that stimulate B cells directly.
• Immunodominant antigens – Antigens that dominate (over all others from
a pathogen) in their ability to produce an immune response. T cell responses typically
are directed against a relatively few immunodominant epitopes, although in some cases
(e.g., infection with the malaria pathogen Plasmodium spp.) it is dispersed over a
relatively large number of parasite antigens.
Sources
Antigens can be classified according to their source.
Exogenous antigens
Exogenous antigens are antigens that have entered the body from the outside, for example
by inhalation, ingestion or injection. The immune system's response to exogenous antigens is
often subclinical. By endocytosis or phagocytosis, exogenous antigens are taken into the antigen-
presenting cells (APCs) and processed into fragments. APCs then present the fragments to T
helper cells (CD4+) by the use of class II histocompatibility molecules on their surface. Some T cells
are specific for the peptide:MHC complex. They become activated and start to secrete cytokines,
substances that activate cytotoxic T lymphocytes (CTL), antibody-secreting B cells, macrophages
and other particles.
Some antigens start out as exogenous, and later become endogenous (for example, intracellular
viruses). Intracellular antigens can be returned to circulation upon the destruction of the infected
cell.

Endogenous antigens
Endogenous antigens are generated within normal cells as a result of normal cell metabolism, or
because of viral or intracellular bacterial infection. The fragments are then presented on the cell
surface in the complex with MHC class I molecules. If activated cytotoxic CD8+ T cells recognize
them, the T cells secrete various toxins that cause the lysis or apoptosis of the infected cell. In
order to keep the cytotoxic cells from killing cells just for presenting self-proteins, the cytotoxic
cells (self-reactive T cells) are deleted as a result of tolerance (negative selection). Endogenous
antigens include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous)
antigens.
Autoantigens
An autoantigen is usually a normal protein or protein complex (and sometimes DNA or RNA) that
is recognized by the immune system of patients suffering from a specific autoimmune disease.
These antigens should not be, under normal conditions, the target of the immune system, but their
associated T cells are not deleted and instead attack.
Viral antigens
For virus-associated tumors, such as cervical cancer and a subset of head and neck
cancers, epitopes derived from viral open reading frames contribute to the pool of neoantigens.
Tumor antigens
Tumor antigens are those antigens that are presented by MHC class I or MHC class II molecules on
the surface of tumor cells. Antigens found only on such cells are called tumor-specific
antigens (TSAs) and generally result from a tumor-specific mutation. More common are antigens
that are presented by tumor cells and normal cells, called tumor-associated
antigens (TAAs). Cytotoxic T lymphocytes that recognize these antigens may be able to destroy
tumor cells.

Factors affecting Immunogenecity:
Immunogenicity is influenced by multiple characteristics of an antigen:
• Phylogenetic distance
• Molecular size
• Epitope density
• Chemical composition and heterogeneity
• Protein structure, aa-polymers, Glu-Lys, Tyr, Phe
• Degradability (ability to be processed & presented to T cells)
• D-amino acids
Antigenic specificity
Antigenic specificity is the ability of the host cells to recognize an antigen specifically as a unique
molecular entity and distinguish it from another with exquisite precision. Antigen specificity is
due primarily to the side-chain conformations of the antigen. It is measurable and need not be
linear or of a rate-limited step or equation.
Structure of immunoglobulins
Antibody (or immunoglobulin) molecules are glycoproteins composed of one or more units,
each containing four polypeptide chains: two identical heavy chains (H) and two identical light
chains (L). The amino terminal ends of the polypeptide chains show considerable variation in
amino acid composition and are referred to as the variable (V) regions to distinguish them from
the relatively constant (C) regions. Each L chain consists of one variable domain, VL, and one
constant domain, CL. The H chains consist of a variable domain, VH, and three constant domains

CH1, CH2 and CH3. Each heavy chain has about twice the
number of amino acids and molecular weight (~50,000) as
each light chain (~25,000), resulting in a total
immunoglobulin monomer molecular weight of
approximately 150,000.
Generalized structure of an immunoglobulin (IgG).
Heavy and light chains are held together by a combination of non-covalent interactions and
covalent interchain disulfide bonds, forming a bilaterally symmetric structure. The V regions of H
and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each Ig
monomer contains two antigen-binding sites and is said to be bivalent.
The hinge region is the area of the H chains between the first and second C region domains and is
held together by disulfide bonds. This flexible hinge (found in IgG, IgA and IgD, but not IgM or IgE)
region allows the distance between the two antigen-binding sites to vary.

Classes of immunoglobulins
The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD and IgE. These are
distinguished by the type of heavy chain found in the molecule. IgG molecules have heavy chains
known as gamma-chains; IgMs have mu-chains; IgAs have alpha-chains; IgEs have epsilon-chains;
and IgDs have delta-chains.
Differences in heavy chain polypeptides allow these immunoglobulins to function in different
types of immune responses and at particular stages of the immune response. The polypeptide
protein sequences responsible for these differences are found primarily in the Fc fragment. While
there are five different types of heavy chains, there are only two main types of light chains: kappa
(κ) and lambda (λ).
Antibody classes differ in valency as a result of different numbers of Y-like units (monomers) that
join to form the complete protein. For example, in humans, functioning IgM antibodies have five Y-
shaped units (pentamer) containing a total of 10 light chains, 10 heavy chains and 10 antigen-
binding.
IgG class
Properties of IgG:
• Molecular weight: 150,000
• H-chain type (MW): gamma (53,000)
• Serum concentration: 10 to 16 mg/mL
• Percent of total immunoglobulin: 75%
• Glycosylation (by weight): 3%
• Distribution: intra- and extravascular
• Function: secondary response

IgM class
Properties of IgM:
• H-chain type (MW): mu (65,000)
• Serum concentration: 0.5 to 2 mg/mL
• Distribution: mostly intravascular
• Function: primary response
IgA class
Properties of IgA:
• Molecular weight: 320,000 (secretory)
• H-chain type (MW): alpha (55,000)
• Serum concentration: 1 to 4 mg/mL
▪ Glycosylation (by weight): 10%
▪ Distribution: intravascular and secretions
▪ Function: protect mucus membranes
IgD and IgE class
Properties of IgD:
• H-chain type (MW): delta (70,000)
• Serum concentration: 0 to 0.4 mg/mL
• Percent of total immunoglobulin: 0.2%
• Distribution: lymphocyte surface
• Function: unknown

Properties of IgE:
• H-chain type (MW): epsilon (73,000)
• Serum concentration: 10 to 400 ng/mL
• Percent of total immunoglobulin: 0.002%
• Distribution: basophils and mast cells in saliva and nasal secretions
• Function: protect against parasites
Subclasses of immunoglobulins
In addition to the major immunoglobulin classes, several Ig subclasses exist in all members of a
particular animal species. Antibodies are classified into subclasses based on minor differences in
the heavy chain type of each Ig class. In humans there are four subclasses of IgG: IgG1, IgG2, IgG3
and IgG4 (numbered in order of decreasing concentration in serum).
Variance among different subclasses is less than the variance among different classes. For
example, IgG1 is more closely related to IgG2, IgG3 and IgG4 than to IgA, IgM, IgD or IgE.
Consequently, antibody-binding proteins (e.g., Protein A or Protein G) and most secondary
antibodies used in immunodetection methods cross-react with multiple subclasses but usually not
multiple classes of Ig.
Function
The main categories of antibody action include the following:
• Neutralisation, in which neutralizing antibodies block parts of the surface of a bacterial
cell or virion to render its attack ineffective
• Agglutination, in which antibodies "glue together" foreign cells into clumps that are
attractive targets for phagocytosis
• Precipitation, in which antibodies "glue together" serum-soluble antigens, forcing them
to precipitate out of solution in clumps that are attractive targets for phagocytosis

• Complement activation (fixation), in which antibodies that are latched onto a foreign
cell encourage complement to attack it with a membrane attack complex, which leads
to the following:
✓ Lysis of the foreign cell
✓ Encouragement of inflammation by chemotactically attracting inflammatory cells
Activated B cells differentiate into either antibody-producing cells called plasma cells that secrete
soluble antibody or memory cells that survive in the body for years afterward in order to allow the
immune system to remember an antigen and respond faster upon future exposures.
Monoclonal antibodies:
Monoclonal antibodies (mAb or moAb) are antibodies that are made by identical immune
cells that are all clones of a unique parent cell. Monoclonal antibodies can have monovalent
affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the
antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are usually made by
several different plasma cell (antibody secreting immune cell) lineages. Bispecific monoclonal
antibodies can also be engineered, by increasing the therapeutic targets of one single monoclonal
antibody to two epitopes.
History
The idea of a "magic bullet" was first proposed by Paul Ehrlich, who, at the beginning of the 20th
century, postulated that, if a compound could be made that selectively targeted a disease-causing
organism, then a toxin for that organism could be delivered along with the agent of selectivity. He
and Élie Metchnikoff received the 1908 Nobel Prize for Physiology or Medicine for this work,
which led to an effective syphilis treatment by 1910.
In the 1970s, the B-cell cancer multiple myeloma was known. It was understood that these
cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the
structure of antibodies, but it was not yet possible to produce identical antibodies specific to a
given antigen.
In 1975, Georges Köhler and César Milstein succeeded in making fusions of myeloma cell lines
with B cells to create hybridomas that could produce antibodies, specific to known antigens and

that were immortalized. They shared the Nobel Prize in Physiology or Medicine in 1984 for the
discovery.
In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal
antibodies, eliminating the reactions that many monoclonal antibodies caused in some patients.
Production
Hybridoma technology:
Monoclonal antibodies are typically made by cell culture that involves fusing myeloma cells with
mouse spleen cells immunized with the desired antigen. Rabbit B-cells can be used to form
a rabbit hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes, but the
success rate is low, so a selective medium in which only fused cells can grow is used. This is
possible because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic
acids. The absence of HGPRT is not a problem for these cells unless the de novo purine
synthesis pathway is also disrupted. Exposing cells to aminopterin (a folic acid analogue, which
inhibits dihydrofolate reductase, DHFR), makes them unable to use the de novo pathway and
become fully auxotrophic for nucleic acids, thus requiring supplementation to survive.
The selective culture medium is called HAT medium because it contains hypoxanthine,
aminopterin and thymidine. This medium is selective for fused (hybridoma) cells. Unfused
myeloma cells cannot grow because they lack HGPRT and thus cannot replicate their DNA.
Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid
cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell
partner supplies HGPRT and the myeloma partner has traits that make it immortal.
This mixture of cells is then diluted and clones are grown from single parent cells on microtitre
wells. The antibodies secreted by the different clones are then assayed for their ability to bind to
the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most
productive and stable clone is then selected for future use.

The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be
injected into mice (in the peritoneal cavity, surrounding the gut). There, they produce tumors
secreting an antibody-rich fluid called ascites fluid.
The medium must be enriched during in vitro selection to further favour hybridoma growth. This
can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as
briclone. Culture-media conditioned by macrophages can be used. Production in cell culture is
usually preferred as the ascites technique is painful to the animal. Where alternate techniques
exist, ascites is considered unethical.
Purification
After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the
desired antibodies must be extracted. Cell culture sample contaminants consist primarily of media
components such as growth factors, hormones and transferrins. In contrast, the in vivo sample is
likely to have host antibodies, proteases, nucleases, nucleic acids and viruses. In both cases, other
secretions by the hybridomas such as cytokines may be present. There may also be bacterial
contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the
complexity of the media required in cell culture and thus the contaminants, one or the other
method (in vivo or in vitro) may be preferable.
The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids and clotted
material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter.
These large particles can cause a phenomenon called membrane fouling in later purification steps.
In addition, the concentration of product in the sample may not be sufficient, especially in cases
where the desired antibody is produced by a low-secreting cell line. The sample is therefore
concentrated by ultrafiltration or dialysis.
Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These can
be separated by ion exchange chromatography. Either cation exchange chromatography is used at
a low enough pH that the desired antibody binds to the column while anions flow through,
or anion exchange chromatography is used at a high enough pH that the desired antibody flows
through the column while anions bind to it. Various proteins can also be separated along with the
anions based on their isoelectric point (pI). In proteins, the isoelectric point (pI) is defined as the

pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge and
when the pH < pI, a protein has a net positive charge. For example, albumin has a pI of 4.8, which
is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. Thus, at a pH
between 4.8 and 6.1, the average charge of albumin molecules is likely to be more negative, while
mAbs molecules are positively charged and hence it is possible to separate them. Transferrin, on
the other hand, has a pI of 5.9, so it cannot be easily separated by this method. A difference in pI of
at least 1 is necessary for a good separation.
Transferrin can instead be removed by size exclusion chromatography. This method is one of the
more reliable chromatography techniques. Since we are dealing with proteins, properties such as
charge and affinity are not consistent and vary with pH as molecules are protonated and
deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low
resolution, low capacity and low elution times.
A much quicker, single-step method of separation is protein A/G affinity chromatography. The
antibody selectively binds to protein A/G, so a high level of purity (generally >80%) is obtained.
However, this method may be problematic for antibodies that are easily damaged, as harsh
conditions are generally used. A low pH can break the bonds to remove the antibody from the
column. In addition to possibly affecting the product, low pH can cause protein A/G itself to leak
off the column and appear in the eluted sample. Gentle elution buffer systems that employ high
salt concentrations are available to avoid exposing sensitive antibodies to low pH. Cost is also an
important consideration with this method because immobilized protein A/G is a more expensive
resin.
To achieve maximum purity in a single step, affinity purification can be performed, using the
antigen to provide specificity for the antibody. In this method, the antigen used to generate the
antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly
synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such
as KLH during development and to support purification. The antibody-containing media is then
incubated with the immobilized antigen, either in batch or as the antibody is passed through a
column, where it selectively binds and can be retained while impurities are washed away. An
elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover
purified antibody from the support.

Applications
Diagnostic tests
Once monoclonal antibodies for a given substance have been produced, they can be used to detect
the presence of this substance. The Western blot test and immuno dot blot tests detect the protein
on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed
tissue sections and immunofluorescence test, which detect the substance in a frozen tissue
section or in live cells.
Analytic and chemical uses
Antibodies can also be used to purify their target compounds from mixtures, using the method
of immunoprecipitation.
Therapeutic treatment
Therapeutic monoclonal antibodies act through multiple mechanisms, such as blocking of targeted
molecule functions, inducing apoptosis in cells which express the target, or by modulating
signalling pathways.
Cancer treatment
One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-
specific antigens and induce an immune response against the target cancer cell. Such mAbs can be
modified for delivery of a toxin, radioisotope, cytokine or other active conjugate or to
design bispecific antibodies that can bind with their Fab regions both to target antigen and to a
conjugate or effector cell. Every intact antibody can bind to cell receptors or other proteins with
its Fc region.
Autoimmune diseases
Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which
are effective in rheumatoid arthritis, Crohn's disease, ulcerative Colitis and ankylosing spondylitis
by their ability to bind to and inhibit TNF-α. Basiliximab and daclizumab inhibit IL-2 on
activated T cells and thereby help prevent acute rejection of kidney transplants. Omalizumab
inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.

Abzymes:
An abzyme (from antibody and enzyme)also called catmab (from catalytic monoclonal antibody),
and most often called catalytic antibody, is a monoclonal antibody with catalytic activity.
Enzymes function by lowering the activation energy of the transition state of a chemical reaction,
thereby enabling the formation of an otherwise less-favorable molecular intermediate between
the reactant(s) and the product(s). If an antibody is developed to bind to a molecule that is
structurally and electronically similar to the transition state of a given chemical reaction, the
developed antibody will bind to, and stabilize, the transition state, just like a natural enzyme,
lowering the activation energy of the reaction, and thus catalyzing the reaction. By raising an
antibody to bind to a stable transition-state analog, a new and unique type of enzyme is produced.
Some abzymes have been engineered to use metal ions and other cofactors to improve their
catalytic activity.
Abzymes however do occur naturally in the human body.
Uses in Medicine
The abzymes could target a specific site on the HIV infected cells that do not mutate and then
make the virus inert. These abzymes are chosen from monoclonal antibodies which are created by
immunizing mice with haptens which mimic the transition states of enzyme-catalyzed reactions.
The rate of this reaction is promoted by enzyme catalysts that stabilize the transition state of this
reaction, thereby decreasing the activation energy and allowing for more rapid conversions of
substrate product . To successfully create abzymes that are complementary in structure to this
transition state, mice were immunized with an aminophosphonic acid hapten.

Cells and organs of the immune system

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