IMMUNOLOGY COURSE

The Immune System: New Developments in Research - Part 1

University of Osaka, Japan
Introduction to Immunology
Inside our body, we have an amazing protection mechanism called the immune
system, which is designed to defend us against millions of bacteria, viruses,
fungi, toxins and parasites. However, this system is not perfect, and sometimes it
fails. For instance, a germ can sometimes invade successfully, making us sick. Or,
instead of getting rid of pathogens, our immune system may sometimes attack
our own body components by mistake, if this happens in your joints, you may
develop rheumatoid arthritis. Alternatively, our immune system may overreact to
certain antigens such as pollens. If this happens in your respiratory tract, you
may develop hay fever, which is one of the most common type of allergy in
developed countries. One important thing to remember concerning the immune
system is that it is a system, but not a single entity. As you may realize by
listening to my talk, our immune system is actually a collection of many different
organs, special cells, and substances, which collectively help protect you from
infections and other diseases. So, in order for the immune system to function
well, it requires balance and harmony among the various cell types and humoral
factors that comprise the immune system. First of all, let me remind you what
"immunity" means. In other words, immunity is defined as resistance to disease,
specifically infectious disease. Now, let us look at the importance of the immune
system. First of all, the immune system is important because it defends us
against infections. This point becomes clearer if you look at the people who have
been born with problems with their immune system, a condition called
"immunodeficiency". These immunodeficient people can become infected very
easily and often die of such infections. So, this tells us how important it is to have
an intact immune system. Second, the immune system is important, because it
works as defense against newly arising transformed cells, such as malignant
tumor cells. The fact that the immunodeficient patients are also prone to develop
various types of tumors underlines the importance of the immune system. Third,
elimination of non-self, such as newly introduced substances or molecules, or
tissue grafts is another important task of the immune system. Tissue grafts are
normally rejected, because the recipients' immune system attacks them and
finally eliminates them. However, if the recipient is given immunosuppressive
drugs to blunt his/her immune response, tissue graft rejection is delayed or
sometimes completely inhibited, indicating the importance of the immune
system. Finally, if the immune system works excessively, it leads to tissue injury
or inflammation, as you will see in the case of allergy and autoimmune disease.
By properly stimulating the immune system using part of pathogens or vaccines,
we have been able to dramatically reduce the incidence of certain infectious
diseases in developed countries. This is particularly the case with childhood
diseases, such as measles, rubella, mumps and polio, which were highly

prevalent among children some decades ago. However, subsequent to the

development of specific vaccines against them, these diseases are declining very
rapidly, and you hardly see them among properly vaccinated children. So, this
clearly shows the strength of the immune system.

Recognizing Invaders Part 1

So, our immune system is very effective in getting rid of pathogens or other
types of invaders. Then, how do we recognize such invaders? What kind of
strategies does our immune system exploit to recognize them? First of all, an
antigen refers to any substance that provokes an immune response. This word
originally came from ANTIbody GENerator, ANTIGEN, meaning that antigen is the
molecule that can generate antibodies or the molecule that can induce antibody
responses in the body. Pathogens normally have a number of such antigens on
their surface; they are proteins, sugars or lipids. In addition, these molecules
have a number of determinants or epitopes; an epitope is the part of an antigen
that is recognized by the immune system. Against these antigenic determinants
or epitopes, immune cells have structures necessary for sensing, and these
structures are collectively called sensors. Basically any type of immune cells has
such sensors, and hence, they are able to recognize molecules or cells invading
into our body. Functionally speaking, our immune system can be subdivided into
two. One is the innate immune system that provides non-specific host defense
against invading pathogens, whereas the other is the adaptive immune system
that provides specific and long-lasting immunity. The former, the innate system is
natural or "innate" to the host, and is ready to respond to invasion and does not
require a period of time for induction. Once pathogens invade our body by
breaching the skin or mucous membrane barriers, the innate immune system is
immediately activated. As I will describe later, the major cellular components of
this system include macrophages, neutrophils, dendritic cells and natural killer
cells. If the innate immunity could not eliminate pathogens, another immune
system, which is called the adaptive or acquired immune system, is activated,
and specific immune responses occur. This system is highly specific and acquired
in nature. Most importantly, the major players of the adaptive immune system
are lymphocytes such as T cells and B cells, T lymphocytes and B lymphocytes
and their products. The reason why activation of the innate immune system
promotes induction of the adaptive immune system is because activated cellular
components of the innate immune system such as dendritic cells and also
cytokines produced by the activated cells act on lymphocytes to stimulate their
proliferation, differentiation, and survival. Following microbial invasion, the innate
immune system, consisting of epithelial barriers, macrophages, neutrophils,
dendritic cells, natural killer cells and so on, and also complement components
that I will explain more later, plays a major role in pathogen elimination. Innate
immunity starts to act immediately after pathogen invasion and continues to act
for many hours. When the invading pathogen is not satisfactorily eliminated,
adaptive immunity ensues. B lymphocytes proliferate recognizing the pathogen
antigenic determinants and finally produce antibodies, which block infection and
eliminates the pathogen. When the pathogen invades the cytoplasm, T cells

recognize the pathogen with the aid of dendritic cells. Full-blown adaptive
immune responses thus require several days. Here I show you the major cellular
components involved in innate immunity. Some of them engulf pathogens;
Macrophages, neutrophils, dendritic cells. They are called phagocytes and others
act as effector cells, releasing bactericidal proteins or proteins that can kill
microbes, and some act as cytokine producing cells. Cytokines, such as G-CSF, IL
means interleukin, IL-1, IL-6. They mediate inflammatory and immune reactions,
and are principal mediators of communication between cells of the immune
system. A recent addition to the innate immune system is a group of cells, here,
innate lymphoid cells or ILCs. We know now that there are, at least, three types of
ILCs. They all secrete cytokines that can activate immune cells including T and B
lymphocytes. So, in this system, you have many different kinds of cells and they
secrete bactericidal proteins and cytokines. Most ... Quite few of them are called
interleukins.
Recognizing Invaders Part 2
The complement system is made up of over 25 proteins and protein fragments,
which assist the ability of antibodies and phagocytic cells to clear pathogens from
an organism. They are normally produced by the liver cells and circulate as
inactive precursors in the blood. When infection occurs, microbial products may
activate proteases in the system, which cleave specific proteins to release
cytokines, and this amplifies a cascade of further cleavages. The end-result of
this activation cascade is massive amplification of the response and activation of
the membrane attack complex on the surface of the pathogen, which leads to
killing of microbes. Complement proteins are inactive until they are cleaved by a
protease, which in turn, converts the proteins into a protease. For instance,
binding of an antibody to a pathogen triggers the complement activation through
the so-called classical pathway. And the antibody bound to the pathogen can
activate C1, which activates C2 and C4, which activates C3 and C5, which leads
to the sequential activation of C6, C7, C8 and C9, which finally results in the
formation of the membrane-attack complex or MAC on the surface of the
pathogen. The MAC in turn functions as a channel, allowing the passage of ions
and small molecules, which causes osmotic swelling and killing of the pathogen
or infected cells. Alternatively, when a complement component C3 binds to the
microbial cell surface, this activates a number of other complement proteins in
sequence. This is called an alternative pathway. Some of the products induce
blood vessel dilatation, histamine release from mast cells, recruitment of
phagocytes. Some of them induce promotional microbial phagocytosis and killing
locally. Now, in addition to the classical pathway and the alternative pathway,
there is another complements activation pathway - the lectin pathway. Upon
activation of complement proteins through these pathways, now, inflammatory
leukocytes are recruited to the site of infection, phagocytosis by the activated
leukocytes is promoted, and pathogens are killed at the site of infection. So, you
can see now how important the complement system is as a component of the
innate immune system.

Innate Immune System Activation

Next, I would like to explain how the innate immune system senses microbes
invading our body. As I told you a while ago, innate immune cells have sensors
that detect pathogens, which are collectively called innate immunity receptors.
These receptors are activated by mainly two types of molecular patterns. One of
them is called pathogen-associated molecular patterns or PAMPs, which are
structures produced by microorganisms but not mammalian cells. Typical
examples are microbial DNAs, RNAs, and cell wall components such as bacterial
lipopolysaccharides. The other is called damage or danger-associated molecular
patterns. They are endogenous molecules produced by or released from
damaged or dying cells. Examples include high-mobility group box1 (HMGB1)
proteins, S100 proteins, uric acid crystals, and purine metabolites such as
extracellular ATP (Adenosine Triphosphate). Because the innate immunity
receptors recognize these structures conserved or shared among pathogens or
damaged cells, they are often called pattern recognition receptors. So, sensors of
the innate system are pattern recognition receptors. And, these patternrecognition receptors are expressed ubiquitously. They include cell surfaceassociated Toll-like receptors (TLRs) and lectin receptors that recognize microbial
cell surface components, cytoplasmic NOD-like receptors and RIG-like receptors,
and endosome-associated Toll-like receptors that recognize nucleic acids of
ingested microbes. So, innate immunity receptors are expressed ubiquitously.
Among the pattern recognition receptors, Toll-like receptors or TLRs have been
the most studied so far. There are at least nine different Toll-like receptors,
depending on their localization, and Toll-like receptors respond to various PAMPs
and DAMPs. For instance, Toll-like receptor-1, -2, -4, -5, and -6 recognize microbial
cell wall components, whereas Toll-like receptor-3, -7, -8, and -9, which are all
expressed on the endosomal membrane, recognize nucleic acids. These Toll-like
receptors all contain a ligand-binding domain composed of leucine-rich motifs
and a cytoplasmic signaling domain or TIR domain. When microbial components
bind to Toll-like receptors via the Toll-like receptor's leucine-rich like domain, the
signals are transmitted intracellularly via the TIR domain, and as a result, various
adaptor proteins are recruited locally, and cytoplasmic transcription factors
including NF-B and interferon-regulatory factors or IRFs are subsequently
activated. IRF activation induces production of type-1 interferons such as
interferon alpha and beta, thus inducing anti-viral states in the cell. NF-B
activation induces expression of certain proinflammatory cytokines and adhesion
molecules, both of which help promote acute inflammation. NF-B activation also
induces expression of costimulatory molecules that are required for induction of
adaptive immunity. I will tell you more about costimulatory molecules later in this
talk. Among the innate immune receptors, NOD-like receptors or NLRs sense
PAMPs and DAMPs in the cytoplasm. When NLRs are engaged, it stimulates the
formation of the inflammasome complex, which is a multiprotein complex that
mediates activation of caspase-1, and activated caspase-1 proteolytically
generates the active form of interleukin-1 or IL-1 from an inactive precursor. IL-1
is one of the strongest cytokines that can induce inflammation. So, because IL-1
is, so called, a typical proinflammatory cytokine, this process helps promote
inflammatory responses. In this slide, one of the NLRs, NLRP-3, is activated by

PAMPs or DAMPs, and forms oligomers with an adaptor protein and an inactive
form of caspase-1, which leads to activation of caspase-1, which in turn activates
IL-1. IL-1 is constitutively produced in inflamed cells as inactive form. But
activated caspase-1 activates IL-1 to induce acute inflammation. And Toll-like
receptor signaling also enhances this process. So, upon sensing of PAMPs and
DAMPs, NLRP-3 oligomerizes with an adaptor protein and inactive form of
caspase-1. Once recruited, caspase-1 is activated, and cleaves pro-IL-1 (inactive
from of IL-1), to generate biologically active IL-1. And IL-1 induces acute
inflammation and causes fever. And among these proinflammatory cytokines, you
have IL-1, IL-18, TNF (tumor necrosis factor), and Interleukin-6 and so on.

Innate Immunity vs. Adaptive Immunity Part 1

Here I summarize some important properties of the innate immune system,
compared with those of the adaptive immune system. First, innate immunity
receptors recognize PAMPs and DAMPs, and thus their specificity is broad. This is
also underlined by the fact that the innate immunity receptors are encoded in the
germline and do not mutate as frequently as B-cell receptors and T-cell receptors,
both of which are encoded by genes that undergo gene rearrangements and
somatic mutations. So, basically, all cells have more or less the same innate
immunity receptors, whereas, as I will explain to you later, each lymphocyte
expresses a unique receptor, because lymphocytes respond to antigens on the
principle of clonal selection. Therefore, innate immunity receptors have limited
diversity compared with antigen-specific receptors of the adaptive immune
system, while both types of receptors can discriminate self from non-self
molecules. OK, so much for the innate immune system. I will now focus on the
adaptive immune system that is more specific, more adaptable, and hence, more
potent than the innate immune system. As you see here, the major cellular
components of this system are T and B lymphocytes. T cells express specific
antigen receptors called T-cell receptor, and upon activation, they can
differentiate into several subsets, such as Th1, Th2, Th17, Treg and Tc. These
subsets are diverse in terms of how they are activated and what kind of cytokines
they produce. On the other hand, B cells express antigen specific receptors called
B cell receptors or BCRs and consist of two major subsets, B1 and B2. Both
subsets have the potential to produce antibodies but of different properties. Here
I show you antigen receptors of T cells and B cells. They are similar in a sense
that they are polypeptides recognizing a specific antigen. However, they are very
different in structure and different in the nature of the antigen they recognize. For
instance, a T-cell receptor is alpha-beta heterodimer and recognizes only peptides
with the aid of dendritic cells. As I will explain to you later, a T-cell receptor
recognizes so-called peptide-MHC complexes presented by dendritic cells. In
contrast, a B-cell receptor is tetramer consisting of four polypeptides, namely two
heavy chains and two light chains. A B-cell receptor can recognize such antigens
as proteins and sugars in the absence of dendritic cells. This is an important
point. In the adaptive immune system, T cells and B cells have their individual
function. B cells mediate so-called humoral immunity where B cells respond to
extracellular microbes by producing antibodies that can neutralize and eliminate

these molecules such as microbes and microbial toxins. In contrast, T cells

mediate cell-mediated immunity in response to microbes or parasites that are
internalized into or synthesized in cells. Helper T cells eliminate phagocytosed
microbes by secreting cytokines and activating macrophages, whereas cytotoxic
T cells kill infected cells and eliminate reservoirs of infection. Here I summarize
important properties of the adaptive immune responses. The responses are
highly specific, because the responses are mediated by lymphocyte clones
expressing specific antigen receptors. I will tell you more about clones a little
later. The adaptive immune responses are also highly diverse, which makes the
adaptive immune system to respond to a large variety of antigens successfully.
The adaptive immune responses can also generate memory. When the adaptive
immune system is exposed to the same antigen it has encountered before, it
remembers this and shows a more rapid and more effective response, and this is
why vaccines can confer long-lasting protection against infections. The adaptive
immune responses are self-limited and decline as the infection is eliminated. The
last not least important property of the adaptive immune system is that, although
it can respond to a whole variety of microbes and other foreign antigens, it
normally does not react with self antigens. As you will hear later in this lecture
series, self-reactivity is normally down-regulated so that no concomitant tissue
injury occurs when this system responds to foreign antigens.
Innate Immunity vs. Adaptive Immunity Part 2
Now, I will tell you about lymphocyte clones. We have within our body many
clones of lymphocytes that are specific for different antigens. What I mean by
clone here is a population of lymphocytes expressing identical antigen receptors
and hence identical specificities; here I show you three different clones for
example. These clones are derived from a single precursor cell. They have the
ability to respond to a specific antigen and proliferate subsequently. For instance,
when a B cell clone specific for antigen X encounters this the antigen X, it gets
activated and differentiates into the cells that can produce a specific antibody
against X, that is, anti-X antibody. Similarly, when a B cell clone specific for
antigen Y encounters the antigen Y, it gets activated and differentiates into the
cells that can produce specific antibody against Y, that is, anti-Y antibody.
However, B cell clones with irrelevant specificities do not get activated and
produce no antibody. Basically, T cells also respond to antigen in an identical
manner, except that they require antigen presentation by dendritic cells, as I will
describe later. So, lymphocytes respond to antigens at the clonal level. A
particular antigen selects the clone to be activated. So, this concept is called the
clonal selection theory, which was initially put forward in the 1950s and
eventually yielded several Nobel Prize winners. In the case of B cells, after some
rounds of cell division, the antigen-responding cells, which are called plasma
cells, produce antibodies and antibodies produce membrane-bound antigen
receptors they are secreted as antibody molecules from plasma cells. And this
concept: a specific clone is selected for proliferation by antigen, is called clonal
selection theory. Here I show you the way how the adaptive immune system
responds to antigens. The lymphocytes that are seeing antigens for the first time
are called nave cells. They are immunologically inexperienced, but upon
encounter with a specific antigen, for instance, antigen X, they mount a so-called

primary response in which activated B cells secrete antibodies specifically

reactive against antigen X. This response declines with time, but when the same
antigen X comes in for the second time, memory B cells are now activated,
developing a much stronger and much more long-lasting response, which is
called the secondary response, or secondary immune response. When a different
antigen comes in, it evokes only the primary response, showing the specificity of
the adaptive immune response. The way T cells respond to antigens is very
similar to this, but T cells produce cytokines but not antibodies. Now, let us look
at the issue of where lymphocytes are produced, where they mature, and where
they respond in the body. All lymphocytes are derived from stem cells. These
stem cells reside in the bone marrow. Subsequently, B cells are produced and
mature in the bone marrow, whereas T cells are produced and mature in the
thymus. The bone marrow and the thymus are thus the places where these cells
are produced, and hence, they are called primary lymphoid organs. From these
organs, only mature cells leave and enter the circulation, and continue to
circulate throughout the body. These are nave T and B cells you find in the blood
and lymphoid organs. They may encounter specific antigens in the secondary
lymphoid tissues, such as the lymph nodes, spleen, and other peripheral
lymphoid tissues, and they respond there. If lymphocytes see antigens in the
secondary lymphoid tissues, one may wonder how antigens get into these
tissues. Microbes enter the body usually by crossing the epithelial barriers, such
as those in the skin, the gastrointestinal tract, or the respiratory tract. Dendritic
cells are abundant in these areas and capture the invading microbes and their
products. They sense the pathogen-associated molecular patterns or PAMPs of
the microbes and become activated to produce proinflammatory cytokines. They
then lose adhesiveness to the tissue and move into the lymphatic vessels or
blood vessels. Antigens that evaded uptake by dendritic cells also move into the
lymphatic vessels and blood vessels. Thus, the microbes and their products are
transported into the peripheral lymphoid tissues and are subsequently recognized
by T cells and B cells. So, in the case of lymphoid antigens, antigens are delivered
into lymphatics, finally a lymph node captures these antigens and T cells respond
there. In the case of blood-borne antigens, the spleen captures antigen and
immune response takes place there. So, antigen that enter the lymphatic vessels,
are transported into lymph nodes. Whereas those that enter the blood vessels,
are transported to the spleen. So, microbial antigens are displayed in peripheral
lymphoid tissues for T cell recognition. Upon encounters with antigens, nave
lymphocytes differentiate into effector cells and memory cells. Here, you see
nave cells, effector cells and memory cells. B cells can recognize native antigens
in the absence of dendritic cells and differentiate into plasma cells to secrete
antibodies or into memory cells that can respond to the same antigen more
rapidly and more vigorously upon a second encounter. In contrast, T cells
recognize antigens only with the aid of dendritic cells; and this step is called
antigen presentation, because dendritic cells process the internalized antigen and
present it as antigenic peptides to T cells. T cells then proliferate and differentiate
into cytokine-secreting effector cells or memory cells. Nave cells, effector cells,
and memory cells have the following properties. Nave cells are quiescent,
meaning not cycling or dividing, and live for weeks to months. They have no

effector function and migrate mainly to lymph nodes. In contrast, effector cells
are usually cycling and live only for days. Activated B cells secrete antibodies,
whereas helper T cells secrete cytokines, and cytotoxic T cells can kill microbeinfected cells. On the other hand, memory cells are quiescent, live long, but have
no effector function, migrating through the lymph nodes and bone marrow.

How do T cells recognize antigen - Part 1

Now, let us move on to the subject of antigen recognition by Tcells. How do T
cells recognize antigens and how is it different from antigen recognition by B
cells? As you see here, T cells require dendritic cells for antigen recognition.
When microbes are coming into the tissue, they are captured by dendritic cells,
they are degraded into peptides in endocytic vesicles and transported to the cell
surface. This is antigen uptake and degradation by dendritic cells. The microbial
products enter the cleft of major histocompatibility complex or MHC molecules
and displayed as peptides to T cells. This is antigen presentation. When a T cell
has an appropriate T-cell receptor that can recognize the peptide, the T cell
makes a conjugate with the antigen-displaying dendritic cells. However, this
alone is not sufficient for T cells to recognize an antigen successfully. At the same
time, costimulatory molecules that are expressed by dendritic cells and T cells
must bind to each other. So, when both T-cell receptor-peptide-MHC interactions
and costimulatory molecule interactions take place, T cells can recognize
antigens successfully, and as a result, they become activated. Let us look at this
issue a bit more closely. The binding of MHC-peptide complex with T-cell receptor
alone provides only one of the signals required for T cell activation. When
antigen-presenting dendritic cells are unstimulated, they are lacking in
costimulatory molecule expression, although T cells always express costimulatory
molecules such as CD28. In the absence of costimulatory molecules binding
between dendritic cells and T cells, binding of MHC-peptide complex with T-cell
receptor alone can provide only signal 1, which alone is not sufficient for T cell
activation. So, no effective T cell responses occur. However, when dendritic cells
have been activated by microbes and/or by their products sufficiently, they show
increased expression of costimulatory molecules, such as B7, now B7 is
appearing on the surface of dendritic cells, which can provide strong binding
between dendritic cells and T cells. The activated dendritic cells can also
stimulate T cells by secreting cytokines, thus providing another signal, which is
called signal 2, that is essential for proper T cell activation. So, T-cell receptorMHC-peptide interactions provide only signal 1, whereas interactions among
costimulatory molecules provide signal 2 to T cells. In order for a T cell to respond
to an antigen properly, signal 1, which is antigen-specific, and signal 2, which is
not antigen-specific are both required. So, T cell activation requires not only T cell
receptor ligation with antigen, but also costimulation. Costimulation is absolutely
essential for T cell recognition.

How do T cells recognize antigen - Part 2

Next, to better understand the molecular mechanism underlying antigen
recognition by T cells, we should understand what the MHC is. Simply speaking,
MHC is the marker of self. Every cell in our body carries the same set of
distinctive surface proteins that distinguish us as self. This set of markers is
called the major histocompatibility complex or MHC. There are two major classes
of MHC proteins. One group is called MHC class I proteins, and they are expressed
on virtually all nucleated cells. The other group is called MHC class II proteins,
and they are expressed only on certain types of cells, namely, antigen presenting
cells. So, let us look at the human MHC, which is called the HLA complex. Here I
show you a schematic of the genes encoding the HLA complex. There are at least
three different genes in the class I locus, and they are called HLA-A, B, and C. So,
HLA class I and class II proteins that I express are completely identical to those of
my brother. So, I would assume that antigenic peptides presented by our MHC
molecules would also be almost identical or very similar between these two
brothers. But an interesting thing is that our immune responses are not identical;
I am allergic to pollens but my brother is not. This is probably because T-cell
receptors and B-cell receptors we express are not likely to be identical; this is
because T-cell receptor and B-cell receptor diversities depend mainly on somatic
recombination of the DNA encoded segments in individual cells. I will come back
to this issue a little later. Similarly, each Class MHC class II molecules consist of
two chains, called alpha and beta chains. In the case of Class I, the alpha 1 and
alpha 2 domains form a peptide-binding groove or cleft that can accommodate
peptides of 8 to 11 amino acids. Class II molecules also have a peptide binding
groove, which is formed by alpha 1 and beta 1 domains, and can accommodate
peptides of approximately 15, and they are actually 10 to 30 amino acid residues.
Thus, although Class I and Class II proteins are slightly different in subunit
composition, they are very similar in overall structure, and both can display
antigens for recognition by T cells. So, MHC molecules are membrane proteins
that can display peptide antigens for T cell recognition; Importantly, MHC I
proteins are expressed by all nucleated cells, whereas MHC class II proteins are
abundantly expressed in antigen-presenting cells. They are dendritic cells,
macrophages and in some cases, B cells. An important thing here is that each
MHC molecule can present only one peptide at a time, because there is only one
binding groove, but that each MHC molecule is capable of presenting many
different peptides. Then, where do these peptides come from? These peptides are
derived from protein antigens that are inside the host cells. In the case of MHC
Class I molecule, peptides derived from cytosolic proteins bind to its groove,
whereas in the case of MHC Class II molecule, peptides derived from proteins that
are taken up into intracellular vesicles bind to its groove. Now, let me tell you a
little bit more about antigen presentation. As shown here, when a microbe or
microbes enter the cell, microbial proteins are degraded into peptides and bind to
class I MHC in the endoplasmic reticulum or ER, and the MHC peptide complex is
subsequently delivered to the cell surface. In this case, a T cell subset expressing
CD8 recognizes the MHC-peptide complex. So, the first event is antigen uptake,
followed by antigen processing within antigen-presenting cells. And because MHC
proteins are constitutively produced and enter the endoplasmic reticulum, the

MHC and peptide meet and are delivered to the cell surface, and finally presented
to T cells. This is the MHC class I pathway. In contrast, when microbes are
endocytosed, they enter the endocytic vesicle and are degraded into peptides in
infected cells. Because class II molecules enter the same vesicles, they bind to
these peptides and are transported to the cell surface. In this case, a T cell
subset expressing CD4 recognizes the MHC-peptide complex. So, once again, the
first event is, here, antigen uptake, followed by antigen processing and then you
have MHC biosynthesis, and peptide-MHC association, and finally these peptides
are presented on cell surfaces to be recognized by T cells. This is the MHC class
II pathway.

How do B cells recognize antigen - Part 1

So, T cells recognize antigens complexed with MHC molecule. Then, how do B
cells recognize antigens? The way B cells respond to antigens is much simpler
than that of T cells. As you see in this slide which I showed you before,
lymphocytes respond to antigens at the clonal level. When antigens bind to a Bcell receptor appropriately, only the B cell clone expressing the B-cell receptor
starts to proliferate, and after several rounds of cell division, the proliferating B
cells differentiate into antibody-secreting plasma cells. The antibody molecules
secreted from the plasma cells have the same antigen-binding site as the initial
B-cell receptor. So, one can say that antibodies exist in two forms one as
membrane-bound antibodies on B cells, and the other as secreted antibodies.
Membrane-bound antibodies or B-cell receptors are used for antigen recognition
and also signal transduction into B cells, whereas secreted antibodies are also
specific to antigens, and can kill invading microbes and neutralize their products.
So, a specific clone is selected for proliferation by antigens, and eventually
produces antibody molecules in the case of B cells. In the case of T cells,
activated T cells produce cytokines. In this slide, I show you membrane-bound
IgM on the left and secreted IgG on the right. Both of them are immunoglobulins
and have two identical heavy chains and two identical light chains, with each
chain containing a variable region and a constant region. In this picture, V and C
mean variable and constant, respectively. And, each loop-like structure, you find
quite a few of them on the structure of immunoglobulin, this loop is called the
immunoglobulin domain or Ig domain. On the N-terminus, you see two identical
antigen-binding sites consisting of the V regions of the heavy chain and the light
chain. This is the same with IgG. The rest of the antibody molecule, this part, the
rest of the antibody molecule is required for structural integrity and effector
functions. The C-terminal end of the heavy chain may be anchored in the cell
membrane, as seen with membrane-bound IgM, or it may have no such
anchoring domain so that the antibody is produced as a secreted protein, as you
see with secreted IgG. As you see in this slide, there are at least five different
antibody or immunoglobulin isotypes, IgG, IgE, IgD, IgA and IgM. IgM found in
plasma is pentameric, five-mers. And they play an important role for early stage
protection against pathogens. IgG is the major immunoglobulin playing an
important role in mid- to late-stage protection against invading pathogens. Both
IgM and IgD are expressed on nave B cells as membrane-bound. IgA is mainly

found in the mucosa and prevents colonization by pathogens. IgE binds to

allergens or molecules that induce allergy, and triggers histamine release from
mast cells. So, each isotype has distinct biological properties and effector
functions. It is important to remember that, upon antigen stimulation, IgM is
produced first. Then, how are these different Ig isotypes produced? Upon
activation, B cells differentiate into plasma cells and usually secrete IgM.
However, when helper T cells are activated concurrently, various signals derived
from the activated T cells determine which Ig isotype is to be produced. For
instance, when Interferon-gamma is provided by helper T cells, B cells switch to
IgG subclasses, whereas when IL-4 is provided, B cells switch to IgE.In the
mucosal tissues, locally produced cytokines such as TGF beta and BAFF help
switch B cells to secrete IgA. So, it is mainly helper T cells and also
microenvironment that influence which Ig isotype to be produced at a given time
during the course of immune responses. This is isotype switching. These
immunoglobulin isotypes have different effector functions as you see in this slide.
IgM promotes complement activation in the classical pathway, IgG subclasses
play an important role in Fc receptor-dependent phagocyte responses; they also
activate complements and play an important role in neonatal immunity, because
IgG can cross the placenta. IgE is important for immune responses against
helminths by inducing degranulation in mast cells. IgE also causes an immediate
type of hypersensitivity by inducing mast cell degranulation. IgA is important in
mucosal immunity; IgA is transported through the intestinal epithelial cells and
displayed in the intestinal lumen; such IgA is important for neutralization of
microbes and microbial toxins. Therefore, immunoglobulins are essential for
protection against pathogens.

How do B cells recognize antigen - Part 2

Next, I would like to briefly explain how antigen receptors or antibody diversity
are generated. Genes for antibodies and T-cell receptors are present in pieces
that can be combined in many different ways in different lymphocytes. This is
called combinatorial diversity, which is the method by which functional antibody
genes are created. It involves the rearrangement of many gene segments that
code for the heavy and light chain proteins of immunoglobulins or alpha and beta
chains of T-cell receptors, and it only occurs in lymphocytes. So, diversity of
immunoglobulins and T-cell receptors is produced mainly by random combination
of V, D, and J gene segments. This somatic recombination occurs not only in the
heavy chain but also in the light chain, so the number of possible combinations
goes up to the order of 1 million in the case of immunoglobulin. Basically the
same principle works in the T cell receptor genes, and possible combination
arising on this recombination in this case is the order of 3 million. Each different
combination yields an antigen receptor with a different specificity. The reason
why such recombination occurs only in lymphocytes is because only lymphocytes
express the VDJ recombinase, which is an enzyme required for this
recombination. In addition, this diversity is further increased by adding or
deleting nucleotides at the junctions, which is induced by imprecision of the
joining. This mechanism is called junctional diversity and this is estimated to add

up to 10 fold to overall diversity. Thus, the total potential repertoire of

immunoglobulin is the order of 10 in the case of B cell receptors, and this is
even larger with T-cell receptors. OK, so this is the very important issue in the
generation of antigen receptor diversity. And, once again, I will repeat that
potential repertoire of immunoglobulin is the order of 10, and this is even larger
with T-cell receptors. Let me describe one more thing about antigen receptors,
which is about how they transmit signals into the cell. Upon antigen binding,
neither a T-cell receptor nor a B-cell receptor can deliver a signal on its own. It is
the associated invariant chains that can deliver intracellular signals. In the case
of B cells, Ig-alpha chain and Ig-beta chain, these are so-called signaling proteins.
And these signaling proteins are brought into proximity and close to the antigen
receptors and trigger complex signaling cascades in the case of B cells. Here in
the case of T cells, you have CD3 molecule, and together with the function of the
T-cell receptor, this antigen receptor and CD3 signaling, you have effective signal
transduction. Therefore, antigen receptors are non-covalently attached to other
invariant molecules the function of which is to deliver to the inside of the cell the
activation signals that are triggered by the antigen recognition. Finally, I
summarize my talk using two slides which I showed you before. First, following
microbial invasion, the innate immune system plays a major role in pathogen
elimination. Innate immunity starts to act immediately after pathogen invasion
and continues to act for many hours. The major cellular constituents would be
phagocytes, that is, neutrophils, dendritic cells and macrophages, you need
complements and you also need NK cells. Subsequently, adaptive immunity
follows. B lymphocytes proliferate recognizing the pathogens antigenic
determinants and finally produce antibodies, which block infection and eliminate
the pathogen. In contrast, T cells recognize the pathogen with the aid of dendritic
cells. They proliferate, and finally eradicate the intracellular microbes. Full-blown
adaptive immune responses thus require several days. So, please remember that
innate immunity provides the initial defense against infections, and adaptive
immunity follows next to provide more specific and long-lasting infections.