Immuno 10

Question 1

What must T cells supply to a B cell to become fully activated?

Answer 1

The 2nd signal of activation other than MHC binding.

Once that has occurred, the B cell proliferates and differentiates, and the final effector cells that are produced are plasma cells (antibody “factories”) and memory cells (a clonally expanded population of daughter B cells that occupy secondary lymphoid tissues of the body for long periods of time).

Question 2

What is the first signal to B cell for activation?

Answer 2

The 1st signal of B cell activation is crosslinking of the B cell receptor by binding of the immunoglobulin component of that BCR with its cognate antigen. Sufficient signaling will not occur unless multiple copies of the BCR are crosslinked with antigen

Question 3

How do B cells signal to the nucleus of the cell so that the cell will know that the BCR has encountered its cognate antigenic determinant?.

Answer 3

The signal transduction unit of the BCR is known as the Ig-alpha/Ig-beta complex. Once this signal has been transmitted, the B cell has now received its first signal of activation.

Question 4

Besides the signals that are transmitted via the Ig-alpha/Ig-beta complex, that are some other signals that can increase the intensity of the 1st activation signal. What are they?

Answer 4

The B cell co-receptor, complement receptor 2, or CR2, has affinity for complement component C3b, and when it binds to C3b, additional signals are transmitted as part of the 1st signal of activation. This should seem logical to you because if an antigen that is recognized by a B cell has been opsonized by labeling with C3b, that is a pretty good sign that the antigen is not a self determinant and is likely to be pathogen-derived. This indicates to the immune system that an immune response is needed to clear an infection.

There are also CD19 and CD81

Even all of these mechanisms together isn’t usually enough to cause B cell activation and require T cell help

Question 5

What do CD19 and CD81 do?

Answer 5

surface markers of B cells that are also part of the B cell receptor complex. CD19 is an important component of the signaling apparatus of the B cell receptor because it amplifies the signals that are transmitted via the Igα/Igβ complex. The function of CD81 has not been clearly established.

Question 6

Some non-protein antigens can elicit B cell activation in the absence of T cell help (albeit, a partial activation, really). How?

Answer 6

T-independent antigens

Some non- protein antigens can also activate B cells in the absence of T cell help. In this case, the antigen crosslinks multiple BCRs on the surface of the cell, in addition to another determinant of the antigen that binds to an unlabeled receptor on the cell surface. This receptor is what is known as a pattern recognition receptor, or PRR. PRRs are receptors that recognize structures that are common to large numbers of bacteria. These structures are known as pathogen-associated molecular patterns (PAMPs). A good example would be the LPS receptor that binds to bacterial LPS, which is found on all gram-negative bacteria. The combination of BCR and PRR engagement is sufficient to partially activate the B cell.

NOTE: that B cells that are activated by T- independent antigens do not initiate germinal center reactions, and they do not result in immunological memory (no memory cells are generated).

Question 7

T or F. T-independnent antigens can activate B cells in the absence of T cell help

Answer 7

T

Question 8

There are two types of T-independent antigens: What are they?

Answer 8

TI-1 and TI-2 antigens

Question 9

What are TI-1 antigens?

Answer 9

T-independent 1 (or TI-1 antigens): this type of TI antigen has PAMPs, and can crosslink BCRs, and therefore has an intrinsic ability to initiate partial activation of B cells.

These types of antigens are known as mitogens. Interestingly, if these types of antigens are supplied in high enough concentrations (much higher than would ever be seen naturally in a host), they can activate B cells in a non-specific fashion.

Question 10

There are multiple ways that TI-1 antigens can activate B cells. Name them.

Answer 10

This graphic shows how mitogens can activate B cells. In the top panel a high concentration of mitogen results in polyclonal (meaning that multiple antibody types are produced) activation of B cells that results from engagement of large numbers of PRRs on the B cells by the PAMP component of the mitogen.

The bottom panel shows what happens when the mitogen is supplied in low concentrations. In this case, the activation of a specific B cell occurs due to crosslinking of BCRs that bind specifically to some component of the mitogen, with simultaneous engagement of the PRR by the PAMP component of the mitogen.

Both of these result in production of antibodies that are almost all of the IgM isotype. There is some programmed class switching to IgG, but because there is no germinal center reaction, no other class switching will occur. In addition, there will be no immunological memory generated.

Question 11

How do TI-2 antigens work?

Answer 11

T-independent type-2 (TI-2) antigens can cause B cell activation by heavily crosslinking BCRs on the surface of the B cell. These antigens are typically highly repetitive structures that have many copies of the antigenic determinant that is recognized by a B cell. A good example of such an antigen is a cell wall polysaccharide.

There is a subset of B cells that have a restricted BCR repertoire that account for most of this type of immune response. These cells are known as B1 B cells

Question 12

Which type of antigens: T-dependent, TI-1, or TI-2- will result in: Antibody response in the absence of cognate T cells or in the absence of a thymus (no T cells)?

Answer 12

There will be no antibody response made in response to a T-dependent antigen, but because no T cell help is required for antibody responses against T-independent antigen, both TI-1 and TI-2 antigens will elicit a response.

Question 13

Which type of antigens: T-dependent, TI-1, or TI-2- will result in antibody response in infants?

Answer 13

infants can produce effector T cells, and therefore can make antibody responses to T-dependent antigens. Infants can also make antibodies in response to TI-1 antigens. However, because the B1 B cell population is not formed until about 5 years of age, infants cannot respond to TI-2 antigens.

Question 14

Which type of antigens: T-dependent, TI-1, or TI-2 will activate T cells?

Answer 14

this only occurs in response to T-dependent antigens because (at least for the purposes of this course), T cells can only recognize protein determinants.

Question 15

Which type of antigens: T-dependent, TI-1, or TI-2- will cause induction of immunological memory?

Answer 15

T-dependent antigens can elicit memory B cells, but TI-1 and TI-2 antigens cannot.

Question 16

Which type of antigens: T-dependent, TI-1, or TI-2- will cause activation of B cell in an antigen-independent fashion?

Answer 16

this only occurs in the case of mitogens (TI-1 antigens).

Question 17

Which type of antigens: T-dependent, TI-1, or TI-2- requires repeated epitopes?

Answer 17

TI-2 only

Question 18

What are 3 examples of TD antigens?

Answer 18

1) Diphtheria toxin
2) Viral hemagglutinin
3) Purified protein derivative (PPD) of Mycobacterium tuberculosis

Question 19

What are 2 examples of TI-1 antigens?

Answer 19

1) Bacterial LPS
2) Brucella abortus- I have no idea why Brucella abortus (a bacterium) is listed under TI-1 antigen, and I take some issue with that because all bacteria make a large array of protein determinants that would all be listed in the T-dependent column. It is probably listed because it is a gram negative pathogen (so it has LPS).

Question 20

What are 2 examples of TI-2 antigens?

Answer 20

1) Pneumococcalpolysaccharide
2) Polymerized flagellin (Salmonella)

Capsular polysaccharides and flagellin are both highly repetitive structures that are produced by an array of different bacterial pathogens, and both are good examples of TI-2 antigens.

Question 21

Where do germinal centers form?

Answer 21

form in the lymphoid follicles within the B cell zone of secondary lymphoid tissues.

Question 22

What occurs in a germinal center reaction?

Answer 22

The reaction is formed by the daughter cells of rapidly dividing B cells (all derived from a single B cell that was activated) and T cells (all descendent from the helper T cell that supplied the secondary activation stimuli to that B cell).

As the proliferating B cells mature, they undergo somatic hypermutation (which results in affinity maturation) and isotype switching (driven by the cytokines produced by the helper T cells.

The positively selected high-affinity isotype-switched B cells differentiate into either plasma cells or memory cells.

Question 23

B cell activation (mostly) requires T cell help. How does this work?

Answer 23

Once a naïve B cell receives its 1st activation signal (binding to its cognate antigen), it will move into the T cell zone of the secondary lymphoid tissue. In the T cell zone, the B cell will interact with many T cells. If an effector CD4 T cell recognizes its cognate determinant bound to an MHC class II molecule on the B cell, it will supply the second signal of activation. The B cell and the T cell will then begin to proliferate rapidly, forming a primary focus in the T cell zone.

Please note that either a TH1 or TH2 cell can supply the second signal of activation to any B cell.

As the B and T cell proliferate, some of the B cells will differentiate into plasma cells (and will produce IgM antibodies because they have not yet class switched).

At least one pair of the daughter B and T cells will eventually migrate into the lyphoid follicle of the B cell zone and continue proliferating. This will become a germinal center.

Question 24

Describe a germinal center.

Answer 24

The helper T cells are found around the periphery of the germinal center. There is a region that is filled with large B cell precursors that are at the early stage of development, known as centroblasts. These cells are rapidly dividing and are beginning to undergo somatic hypermutation. As they develop and become smaller (now called centrocytes) they move into an area that is filled with follicular dendritic cells.

Question 25

How are follicular dendritic cells distinct from the dendritic cells?

Answer 25

FDCs do not present peptide antigens to naïve T cells. Instead, these cells express a high density of Fc receptors and complement receptors on their surface. They use these receptors to trap immune complexes (antigens that have been bound by C3b, antibody, or both) on their surface. The developing centrocytes compete for binding to immune complexes that are trapped on the FDCs. Those that have the highest affinity for antigen will receive survival signals from the FDCs, and they will process the antigen and present it to the effector CD4 T cells that will, in turn, produce cytokines that drive isotype switching in the developing B cell.

B cells that cannot complete effectively for binding to antigen trapped on the FDCs will not receive survival signals and will become anergic and die. Therefore, B cells bearing receptors that received somatic mutations that resulted in lower or similar affinity for the antigen will typically die, while those bearing receptors that received mutations that increased the affinity of the BCR for antigen will be positively selected to continue developing. Over time, this results in what is called affinity maturation.

Question 26

In summary, during a germinal center reaction, the following important things occur:

Answer 26

1) B and helper T cell proliferation
2) Somatic hypermutation and affinity maturation
3) Isotype switching (driven by the helper T cell cytokine array)
4) B cell differentiation into plasma cells and memory cells.

Question 27

IL-4 induces antibody switching to?

Answer 27

most importantly, it induces class switching to IgE. It also promotes switching to IgG isotypes that are weakly opsonizing (IgG2 and IgG4 in humans).

Question 28

IL-5 induces antibody switching to?

Answer 28

augments class switching to IgA (both subtypes in humans)

Question 29

IFN-gamma induces antibody switching to?

Answer 29

Promotes class switching to strongly opsonizing isotypes of IgG (IgG1 and
IgG3 in humans)

Question 30

TGF-beta induces antibody switching to?

Answer 30

to weakly opsonizing subtypes of IgG (2 and 4)

Note: each of these cytokines inhibit class switching to their opposing isotypes (TH1 cytokines inhibit class switching to weakly opsonizing IgGs (2 and 4), IgA, and IgE, while TH2 cytokines inhibit class switching to strongly opsonizing IgG (1 and 3) subtypes.

Question 31

THI cytokines would induce class switching to which antibody types?

Answer 31

IgG1 and IgG3

Question 32

TH2 cytokines would induce class switching to which antibody types?

Answer 32

IgGs (2 and 4), IgA, and IgE

Question 33

Plasma cells have a large ___ organelle.

Answer 33

ER/golgi apparatus. This reflects the fact that a plasma cell really has a single purpose: to produce and secrete antibodies.

Question 34

What are the intrinsic features of naive B cells?

Answer 34

A resting B cell has surface immunoglobulin and MHC class II molecules (which it needs to present antigen to naïve or effector T cells), but it does not secrete very much antibody

Question 35

What are the intrinsic features of plasma B cells?

Answer 35

A plasma cell, on the other hand, produces large quantities of its antibodies, but it no longer displays surface immunoglobulin or MHC class II. This reflects that fact that plasma cells are terminally differentiated effector B cells that no longer have the ability or need to present antigens to T cells or to recognize cognate antigen.

Question 36

What are the inducible features of naive B cells?

Answer 36

a resting or naïve B cell can be induced to proliferate (or grow), and undergo both somatic hypermutation and isotype switching. However, there is no need for a plasma cell to do any of these things.

Question 37

T or F. IgG is found in the circulation, but not in extravascular or extracellular spaces of the body

Answer 37

F. IgG is found in the circulation as well as in the extravascular and extracellular spaces of the body,

Question 38

Where is IgA found in the body?

Answer 38

found primarilly in the lumen of the gut and respiratory tract, in the secretions, and even in the circulation (in low levels)

Question 39

Where is IgM found in the body?

Answer 39

only in circulation

Question 40

Where is IgE found in the body?

Answer 40

IgE is found in the epithelium underlying the skin and in the linings of the gut and respiratory tracts. One fact to remember is that IgE is not found there in free form. Almost all IgE that is produced very rapidly binds to the high affinity IgE receptors that are expressed on the surface of mast cells as well as eosinophils. This is why there is so little IgE found in the serum, despite the fact that relatively large quantities are produced.

Question 41

What is transcytosis?

Answer 41

The process of actively transporting antibodies into secretions or into extravascular spaces.

Question 42

How does transcytosis work?

Answer 42

Accomplished by binding of the Ab to a specific receptor that is then endocytosed and transported from the basolateral surface of the cell to the apical surface, and then released from the cell.

Question 43

What is the basis of transcytosis of IgA work?

Answer 43

Multimeric antibodies (IgA and IgM) bind to a receptor known as the poly-Ig receptor for transport across the epithelial layers that line the gut and respiratory tracts as well as into secretions such as saliva and breast milk.

IgM is so large that, even though it can bind to the poly-Ig receptor, its transport is very inefficient (why it is only found in circulation).

Question 44

Where do most plasma cells that make IgA migrate to?

Answer 44

the secondary lymphoid tissue of the gut and the respiratory tract (the GALT and the MALT, respectively).

Question 45

How does transcytosis of IgA work?

Answer 45

As IgA is secreted, it binds to the poly Ig receptor that is displayed on the basolateral surface of epithelial cells that line the lumen of the gut or respiratory tract or glands that produce secretions. Once it binds to the poly Ig receptor, the complex is endocytosed and transported in a vesicle to the apical side of the cell. When it arrives there, the poly Ig receptor is cleaved, releasing the IgA dimer.

Please note that a piece of the poly Ig receptor is left bound to the antibody. This is known as the secretory component.

Question 46

What is the purpose of the secretory component of IgA?

Answer 46

The secretory component helps to stabilize IgA multimers, increasing their half-life in mucosal secretions.

Question 47

How does movement of IgG into extravascular spaces work?

Answer 47

A different receptor, known as the Brambell receptor (or FcRB or FcRn) is responsible for active transport of IgG across vascular endothelium and into extravascular spaces. It is also responsible for transporting IgG across the placental barrier into the fetal circulation during pregnancy.

Question 48

How does a Brambell receptor work?

Answer 48

Bramble receptors are expressed on vascular endothelial cells within the lumen of capillaries. When the Fc region of an IgG molecule binds to FcRB, the complex is endocytosed and transported within an acidified vesicle to the other side of the cell. When the complex reaches the basolateral surface, the basic pH of the extravascular fluid causes the IgG molecule to be released.

It is important for you to know that FcRB transports IgG into extravascular spaces as well across the placental barrier during pregnancy, providing passive immunization of the child for the first 6-9 months after birth.

Another interesting tidbit relates to the role of this receptor in protecting IgG during its transport through the liver. It is believed that binding to FcRB during its time in the lever protects IgG from degradation, extending its serum half-life over that of other immunoglobulins.

Question 49

What is Passive immunization (or passive transfer of immunity)?

Answer 49

the transfer of immunity to a non-immune person by introduction of specific antibody, immune serum, or T cells from an immune individual. Antibodies or T cells can be obtained from immunized people or animals, and antibodies can also be obtained from hybridoma cell lines. Passive immunization is used to protect against pathogens or toxins.

Question 50

What is Natural passive immunity?

Answer 50

The transfer of antibodies from a mother to her fetus via placental transfer of IgG (by FcRB), or transfer of IgA antibodies from the mother to her child via colostrum or breast milk.

Question 51

IgG is transferred into fetal circulation via active transport by the Brambell receptor (or FcRB). You should know that when the child is born, it will have essentially the same IgG levels (and array of IgG antigen specificities) in its body that the mother does. Because the half-life of IgG is approximately 20 days, the maternal IgG will provide protection to the child for between 6 and 9 months.

IgA antibodies can be passively transferred to a child via nursing because IgA is actively transported by the poly Ig receptor into the mother’s secretions, including breast milk.

Answer 51

IgG is transferred into fetal circulation via active transport by the Brambell receptor (or FcRB). You should know that when the child is born, it will have essentially the same IgG levels (and array of IgG antigen specificities) in its body that the mother does. Because the half-life of IgG is approximately 20 days, the maternal IgG will provide protection to the child for between 6 and 9 months.

IgA antibodies can be passively transferred to a child via nursing because IgA is actively transported by the poly Ig receptor into the mother’s secretions, including breast milk.

Question 52

Describe the typical kinetics of antibody levels in the circulation of a child, starting with fetal development and ending at adulthood.

Answer 52

The levels of maternal IgG antibodies is the highest at the time of birth, and the levels of these maternal antibodies decrease at a constant rate after birth until it is completely gone at about 9 months of age (begin to rise constantly from 3 months after conception). This reflects the half- life of IgG. It is also important to note that the IgG levels in extravascular spaces of the child’s body will decrease at essentially the same rate.

The child begins making it own IgM during fetal development (starts at about 3 months after conception), and the rate of IgM production increases dramatically after birth because the fetus is removed from its sterile in utero environment and thrust into the heavily contaminated ex utero world where he/she encounters many foreign materials and makes antibody responses against them. IgM levels reach their peak at about 1 year of age and remain constant for life.

The child begins making its own IgG and IgA shortly after birth, and the levels of IgG really do not peak until adulthood. IgA levels rise but remain lower than IgG and IgM levels in adulthood

Question 53

Describe toxin neutralization.

Answer 53

Many toxins are produced in an inactive form. They have affinity for some receptor molecule that is expressed on the surface of host cells, and when they bind to that receptor, the receptor:toxin complex is internalized. Once inside the cell, the toxin undergoes a change that results in it becoming active, and it now does its damage to the host cell.

Now, it you make an antibody that has specificity for that toxin, the antibody can bind to the toxin and sterically hinder the binding of the toxin to its cellular receptor. This prevents the toxin from gaining access to the interior of host cells and from damaging the host.

Question 54

Which antibody types can neutralize toxins?

Answer 54

Please remember that not all antibody isotypes have neutralizing activity. Each of the IgG and IgA subtypes have strong neutralizing capacity.

Question 55

Describe virus neutralization.

Answer 55

Many (if not most) viruses gain access to cells via binding to a specific surface molecule (or receptor) on host cells. Once bound to the receptor, the virus is endocytosed. Once endocytosed, the virus begins its replicative cycle, and it takes over and eventually kills the host cell.

Antibody responses directed at envelope proteins of the virus can interfere with the virus’s ability to bind to its host cell receptor, preventing it from gaining access to the cell.

Bacterial neutralization is very similar

Question 56

What is the preferred antibody type for the FcεRI receptor?

Answer 56

This is the (Very) high affinity IgE receptor.

It has binding strength that is orders of magnitude higher than the other Fc receptors.

It is the only receptor that binds to its antibody ligand when that antibody is not bound to its cognate antigen.

Question 57

What cell types express FcεRI receptors?

Answer 57

Mast cells, eosinophils, basophils, FDCs

Question 58

What is the preferred antibody type for the FcγRIII (CD16) receptor?

Answer 58

IgG1

Question 59

What cell types express FcγRIII (CD16) receptors?

Answer 59

**NK cells, Eosinophils, macrophages, neutrophils, mast cells, FDCs

FcγRIII is especially important because of its role in antibody-dependent cell-mediated cytotoxicity (or ADCC) via NK cells.

Question 60

T or F. One of the most important roles of humoral immune responses is opsonization of pathogens that encourages uptake and destruction by macrophages or neutrophils.

Answer 60

T. In this example, antibodies that have specificity for some surface structure of a bacterium have bound to that bacterium. A macrophage can now recognize that bacterium as foreign because its Fc receptors bind to the Fc region of the opsonizing antibodies. This encourages the macrophage to phagocytose and destroy the bacterium.

This is of particular importance for bacteria that express a polysaccharide capsule. Phagocytes have no pattern recognition receptors that recognize capsular material, so thay have no way of seeing such pathogens as foreign.

Question 61

T or F. Antibody binding is a reversible reaction. Binding of complement component C3b is a covalent binding interaction, and is therefore permanent.

Answer 61

T

Question 62

Antibody-dependent cell-mediated cytotoxicity (ADCC) is driven by what kind of cell?

Answer 62

dependent on the effector function of NK cells, a purely innate immune cell type.
NK cells are very important players in the innate immune response to viral infection, and they are also involved in suppression of tumor formation (we will talk about both of these roles later in the course.
During the replicative cycle of many (if not most) intracellular pathogens, some pathogen-derived proteins are transported to, and anchored into the cytoplasmic membrane of the infected host cell. If antibodies that have specificity for one or more of those proteins are produced, they can bind to their cognate determinant on the surface of the host cell.
A critical effector function of NK cells is to recognize host cells that have been opsonized with IgG1 or IgG3 antibodies (via their FcγRIII receptors) and to kill the host cells by initiating programmed cell death (using the same mechanism that is used by CTLs); this effector function is termed ADCC.

Question 63

NK cells are derived from which cell?
What cell are they similar to? Differences?
Are they considered innate or acquired? Why?

Answer 63

NK cells are derived from lymphoid precursor cells in the bone marrow, and are virtually identical to CD8 T cells, except that they do not express CD8 and they do not have a T cell receptor.

NK cells are a purely innate cell type because they have no receptors that recognize specific determinants of pathogens.

NK cells are very important players in the innate immune response to viral infection, and they are also involved in suppression of tumor formation

Question 64

What is the critical effector function of NK cells? Which antibodies do they bind to? Via what receptor?

Answer 64

To recognize host cells that have been opsonized with IgG1 or IgG3 antibodies (via their FcγRIII receptors) and to kill the host cells by initiating programmed cell death (using the same mechanism that is used by CTLs); this effector function is termed ADCC.

Question 65

Can Fc receptors bind to the Fc region of antibodies when they are not bound to their cognate determinant?

Answer 65

No. When the antibody is bound to its antigen, its conformation is altered, opening up the determinant on the antibody that binds to the Fc receptor.

The one exception is IgE, which binds to the high affinity FcεRI receptor in the absence of binding to its cognate antigen.

Question 66

Why are mast cells considered a purely innate cell?

Answer 66

because they do not make any antigen specific receptors. Mast cells use their FcεRI receptors to essentially “steal” IgE molecules so that they can use them as antigen-specific receptors.

There are a very large number of FcεRI receptors on each mast cell. Therefore, each mast cell effectively has thousands of specificities of antigen-specific receptors displayed on its surface.

When IgE molecules become crosslinked by antigen binding (antigen is recognized by multiple IgE molecules that are bound to the surface of the mast cell), the mast cell is triggered to degranulate, and it releases its powerful inflammatory mediators and anti-parasite compounds.

Question 67

Eosinophils are another innate cell type that can be recruited into an acquired response by antibody responses. Like mast cells, eosinophils express the high affinity IgE receptor (FcεRI) on their surface and steal IgE as soon as it is secreted by plasma cells. Their most important role in immune responses is to ______.

Answer 67

kill multicellular parasites.

An interesting fact: Parasite pathogens tend to elicit very IgE-centric antibody responses. These pathogens make a large number of proteases that enable them to remodel tissue as part of their life cycle. For unknown reasons, antibody responses to antigens that have enzymatic activity are very likely to elicit IgE responses.

Question 68

What does the term hybridoma mean?

Answer 68

Refers to a cloned B cell line that has been fused with a tumor cell. B cells will survive in culture for no more than a week
unless very rigorous steps are taken to keep them alive and growing. Tumor cells, on the other hand, have abnormal growth and replication properties that make them easy to maintain and expand in culture. When a B cell is fused to a myeloma cell, the resulting cell grows and expands easily in culture, and it produces and secretes the antibody that the parent B cell expressed. Thus, the antibodies that are secreted by this culture of cells are all essentially identical.

Question 69

What are monoclonal antibodies?

Answer 69

Mono means single: in the case of monoclonal antibodies, mono refers to a single antibody specificity.

A monoclonal antibody preparation is produced by a cloned hybridoma cell line and consist of antibodies that all have an identical specificity for antigen and are of the same isotype.

Question 70

What are polyclonal antibodies?

Answer 70

In contrast, polyclonal antibodies are defined as “a preparation of antibodies that were secreted by B cells of different lineages within the body”. A polyclonal antibody preparation is likely to include antibodies of all isotypes (except IgD, of course), and it will also contain antibodies that have many specificities.

The term polyclonal antibody often refers to antibodies that were generated against a specific antigen, so the different specificities of the antibodies in the preparation are for different antigenic determinants (or epitopes) on that specific antigen.

However, when you collect blood from a patient, the serum will contain antibodies that have specificity for most of the foreign antigens that person has ever encountered. This is polyclonal antibody.

Question 71

How are monoclonal antibodies made?

Answer 71

(Left Panels): When a monoclonal antibody preparation with specificity for some antigen is needed, the first step is to immunize a mouse or rat (usually a mouse) with that antigen to encourage the animal to make a B cell response to that antigen.

Once it has been determined that the animal has made a response, the animal is sacrificed and B cells from the animal are collected. They are then mixed with an appropriate myeloma cell line and then treated with polyethylene glycol to induce membrane fusion (I don’t care that you remember anything about polyethylene glycol). This results in three different types of fusion products: (1) B cell-to-B cell fusions, (2) myeloma-cell to-myeloma cell fusions, and (3) B cell-myeloma cell fusions (the desired product).

(Next): Now a selection step must be used to isolate all of the B cell-myeloma cell fusions from this population of fused cells. This is done by a combination of positive and negative selection. First we will discuss the negative selection: I told you earlier that B cells will not survive long in culture without laborious work to keep them alive. When these fused cell populations are grown in culture without providing the needed care to keep B cells alive, the B cell-B cell fusions will only survive for 5-6 days. Now for the positive selection: B cells produce an enzyme, that myeloma cells do not produce and that I will not ask you about: hypoxanthine-guanine phosphoribosyltransferase), that allows the B cells to grow in culture medium that contains a specific drug that I will not ask you about (hypoxanthine-aminopterin-thymidine). Because myeloma cells do not make this enzyme, they will not survive in medium containing the drug. So, if the fused cells are grown in culture for a week or more, the only remaining cells will be products of B cell-to-myeloma cell fusions.

(Next): Now, you must select for hybridomas that make antibodies that are specific for the antigen of interest. The first step is to perform what is known as a limiting dilution. The hybridoma cells are counted and then plated in 96- well culture plates at a dilution that would give you one-half of a cell per well. Therefore, if done properly, approximately half of the wells will have one cell in them (cloned), and the other half will have no cells. The cells are then cultured for 2-3 weeks, and then the supernatants from each well are screened for antibodies that are specific for the antigen of interest. This is done using techniques (Western blotting and/or ELISA) that we will discuss in lecture 11.

(All panels): Once the hybridoma has been cloned, it can be expanded infinitely in culture, and as the cells grow, they produce the antibodies of interest and secrete them into the culture medium. It is relatively easy to purify the antibodies from the culture supernatants. Another important point is that once these hybridomas have been cloned, they can be preserved cryogenically and stored indefinitely.

Question 72

To make monoclonal antibody-based drugs safer, hybridomas can be genetically engineered to replace the coding regions for the constant domains of the heavy and light chain of a murine antibody with the corresponding coding regions of human origin. The resulting hybridoma produces chimeric antibodies that have the same specificity of the original B cell as well as heavy and light chain constant domains that will appear as “self” to a patient. Humanized antibodies will also have increased half-life in the human body and efficient recruitment of human effector functions.

Humanizing monoclonal antibodies can be accomplished with murine/rat hybridomas via recombinant DNA techniques, or the monoclonal antibodies can be generated using
transgenic mice that have been engineered to express human immunoglobulin constant domains.

Answer 72

To make monoclonal antibody-based drugs safer, hybridomas can be genetically engineered to replace the coding regions for the constant domains of the heavy and light chain of a murine antibody with the corresponding coding regions of human origin. The resulting hybridoma produces chimeric antibodies that have the same specificity of the original B cell as well as heavy and light chain constant domains that will appear as “self” to a patient. Humanized antibodies will also have increased half-life in the human body and efficient recruitment of human effector functions.

Humanizing monoclonal antibodies can be accomplished with murine/rat hybridomas via recombinant DNA techniques, or the monoclonal antibodies can be generated using
transgenic mice that have been engineered to express human immunoglobulin constant domains.