Immuno 3

Question 1

What is a B cell (B lymphocyte)?

Answer 1

a lymphocyte that is dedicated to making immunoglobulins and antibodies

the primary effector function of B cells is to produce antigen-specific antibodies

Question 2

Antibodies are aka?

Answer 2

immunoglobulins

Question 3

B cells (and T cells) are derived from what cells?

Answer 3

common lymphoid progenitor cells

Question 4

Where are B cells produced? What happens after they are produced there?

Answer 4

B cells are produced in the bone marrow. They mature in the bone marrow, and then B cells emerge from the bone marrow as naïve B cells; these cells bear receptors on their surface (membrane bound antibody molecules; B cell receptor) that can potentially bind to a specific antigen. It is important to note that each of the immunoglobulins produced by any one B cell clonal line all have the same antigen-specificity.

Question 5

What happens if B cell receptors bind to an appropriate antigen?

Answer 5

the B cell can be activated to proliferate (giving rise to many B cells that bear and produce immunoglobulins that are specific for the same antigen) and differentiate into either plasma cells or memory cells

Question 6

What is the primary function of plasma cells?

Answer 6

a terminally differentiated B cell whose primary function is to produce antibodies.

Question 7

Where do IgG producing plasma cells typically go?

Answer 7

migrate to bone marrow

Question 8

Where do IgA producing plasma cells typically go?

Answer 8

IgA-producers migrate primarily to the lamina propria of mucosal surfaces

Question 9

What do memory cells do?

Answer 9

a long-lived antigen-specific B cell that results from antigenic stimulation of a naïve B cell during the primary immune response. Upon subsequent exposure to specific antigen, these cells are reactivated to differentiate into plasma cells as a component of a secondary immune response

Question 10

How many antigen specificities exist in the body?

Answer 10

there can be as many as 10^11 different antibody specificities generated by somatic recombination of immunoglobulin genes in
any individual (perhaps more than 10^11)

Question 11

Note on rearrangement of light and heavy chains.

Answer 11

This is an antigen-INdependent process.

A tremendous number of different Ab molecules (10^11), that differ only in their variable regions, are produced by each
individual. This diversity is needed for recognition of the countless antigens that are associated with potential pathogens. The tremendous diversity of possible antibody specificities results from a “cassette” system of gene segments that are somewhat randomly spliced together to form the genes that encode the variable regions of light and heavy chains of antibody molecules.

NOTE: there are two sets of gene segments that can be recombined to generate the coding regions for light chains (the kappa and lambda genes); only one of these will be successfully rearranged in any B cell.

Question 12

What are the component of light chain genes?

Answer 12

variable (V) and joining (J) gene segments which are combined to create the variable region which is associated with a constant (C) region gene segment to form the complete light chain gene

Question 13

What are the component of heavy chain genes?

How much variety of each of these components exist?

Answer 13

consist of V, D (diversity), and J gene segments which combine with the C region gene to form a complete heavy chain gene

there are approx. 38-46 V gene segments, 23 D genes, 6 J genes, and 9 C genes (one for each isotype (and subtype) of Ig)

Question 14

Describe a lambda light chain gene organization.

Answer 14

there are approx. 29-33 Vλ gene segments (each preceded by a leader sequence),
followed 4-5 Jλgene segments; each of the Jλ gene segments is associated with a Cλ gene (codes for the constant
domain).

Can you draw this out? If not, refer to note set for diagram

Question 15

Describe a kappa light chain gene organization.

Answer 15

there are approx. 31-36 Vκ gene segments (each preceded by a leader sequence),
followed by up to 5 Jκ gene segments in a row which are associated with a single Cκ gene segment (i.e. 1 Ck gene segment for the total 5 Jk segments, ratio 1:5)

Can you draw this out? If not, refer to note set for diagram

Question 16

Describe a heavy chain gene organization.

Answer 16

there are approx. 38-46 VH gene segments (each preceded by a leader sequence),
followed by 23 DH gene segments, then 6 JH gene segments, and finally 9 CH segments

Question 17

What are leader sequences used for?

Answer 17

it directs the proteins into the cells’ secretory pathway following translation

Question 18

Overview of B cell proliferation.

Answer 18

Through a process known as somatic recombination, our immune system produces a B cell repertoire that has more than 10^11 distinct B cell receptor/antibody specificities. Each B cell expresses many identical copies of the B cell receptor on its surface. When one of the millions of B cells becomes activated during an infection, it divides rapidly (proliferates), resulting in clonal expansion and differentiation of that B cell specificity line. The antibodies produced by effector B cells, or plasma cells from that B cell line, then participate in inactivation/removal of the pathogen.

Question 19

T or F. Somatic recombination is a random process that occurs continuously throughout the life of the host.

Answer 19

T. It does not occur in response to infection, and it is completely antigen- independent.

Question 20

Describe somatic recombination of light chains.

Answer 20

occurs in germline DNA

light chain gene construction: the first step is the random rearrangement of a single V gene segment with a single J gene segment to form a continuous piece of DNA that encodes the entire variable region of the light chain (catalyzed by a RAG gene); During this, all of the DNA separating the two segments is removed from the germline DNA; Once the germline DNA has been rearranged successfully, transcription of the rearranged DNA results in the production of a primary RNA transcript. since a C gene segment adjacent to the J gene segment is separated from the VJ junction by non-coding (intronic) DNA sequence, RNA processing following transcription of the rearranged DNA brings the VJ junction together with the C gene segment to create the complete coding region for the light chain. The primary RNA transcript then undergoes splicing to generate the final mRNA. Once the mRNA has been produced, it serves as a template for translation to produce the light chain protein.

Question 21

Describe somatic recombination of heavy chains.

Answer 21

occurs in germline DNA

heavy chain gene construction: the variable region of heavy chains are constructed from 3 gene segments (VH, DH, and JH); the first step is the random rearrangement of a single DH gene segment with a single JH gene segment to form a DJH junction, removing all DNA operating them; the second step is a random rearrangement of a single VH gene segment with the previously joined DJH sequence to form a complete DNA sequence that encodes the variable domain of the heavy chain (VDJH); as in the light chain, the C gene segment (always Cμ) adjacent to the J gene segment is separated from the VDJH junction by non- coding (intronic) DNA sequence; RNA processing following transcription of the rearranged DNA brings the VDJ junction together with the Cμ gene segment to create the complete coding region for the heavy chain.

The mRNA then serves as a template for translation to produce the heavy chain protein. Each heavy chain protein becomes disulfide-bridged to a single copy of the light chain that is produced by that B cell, and two copies of the heavy-light chain combination are disulfide bridged together to create a complete monomeric copy of the antibody that is produced by this B cell. In all cases, this antibody is of the IgM isotype.

Question 22

Somatic recombination is mediated by which genes?

Answer 22

two recombination activation genes, or RAG genes: RAG-1 and RAG-2. People that have a deficiency that affects the function of one or both of these gene products experience extreme clinically susceptibility to virtually all pathogens.

Question 23

T or F. The sequences that flank the V, D, and J gene sequences all have highly conserved motifs

Answer 23

T

Question 24

The flanking sequences of the V regions of heavy and light chain gene segments are composed of what?

Answer 24

A conserved block of 7 nucleotides (the heptamer 5’-CACAGTG-3’) followed by a
spacer that is either 12 or 23 nucleotides long (no conservation of sequence, just length; corresponds to either 1 or two turns of the DNA double helix), and followed by a second conserved block of 9 nucleotides (the nonamer 5’- ACAAAAACC-3’)

Question 25

What is the significance of the length of the spacer between the gene segments of the V region of light and heavy chains?

Answer 25

the length of the spacers ensure that the heptameter and nonomer are on the same side of the DNA double helix, where they can be bound by the protein complex that catalyzes recombination

VDJ recombination only occurs between gene segments on the same chromosome

VDJ recombination only occurs between a gene segment that is flanked by a 12-mer RSS and a gene segment that is flanked
by a 23-mer RSS (the 12/23 rule)

Question 26

the heptamer-spacer, nonuser motifs are called?

Answer 26

RSSs, recombination signal sequences

Question 27

What is the basis of junctional diversity on heavy chain recombination?

Answer 27

the joining of the heavy chain gene segments is not precise, and consequently generates added diversity of the recombined V region coding sequence

1. Rag complex cleaves the heptameter RSSs from the D and J gene segments to yield DNA hairpins
2. RAG complex opens the hairpins on the D and J by nicking one strand of the DNA, generating palindromic P-nucleotides
3. N-nucleotide additions by TdT
4. Pairing of strands, removal of unpaired bases by an exonuclease, and gaps filled in by DNA synthesis

Question 28

What chromosome are the lambda light chain genes found on?

Answer 28

22

Question 29

What chromosome are the kappa light chain genes found on?

Answer 29

2

Question 30

What chromosome are the heavy chain genes found on?

Answer 30

14

Question 31

T or F. once the V region genes have rearranged (to productively form VJ (light) or VDJ (heavy) exons), no further rearrangement of that chromosome can take place

Answer 31

T. all progeny of that B cell will express the same V region genes; the only change in the variable region genes of that B cell line arise from somatic hypermutation

Question 32

What are the 3 main processes (associated with gene segment recombination) that help to generate antibody diversity?

Answer 32

1. different pairing of the many gene segments to form the variable domains of both the light and heavy chains
2. imprecise joining of gene segments of heavy chain genes during the somatic recombination process (junctional diversity)
3. different pairing of the many possible light chains with the many possible heavy chains that can be produced

heavy chain rearrangement occurs first!!

*** each of these mechanisms of generating diversity occur during initial development of the B cell in the bone marrow

Question 33

What is allelic exclusion?

Answer 33

a process by which the protein from only one allele is expressed while other allele(s) are silenced. It has been observed most often in genes for cell surface receptors and has been extensively studied in immune cells such as B lymphocytes. In B lymphocytes, successful heavy chain gene rearrangement on one chromosome results in the shutting down of rearrangement on the second chromosome. If no successful rearrangement occurs, rearrangement takes place on the second chromosome. If no successful rearrangement occurs on either chromosome, the cell dies. As a result of allelic exclusion, all the antigen receptors on an individual lymphocyte will have the same amino acid sequence in the variable domains of the light and heavy chain proteins. The mechanism by which allelic exclusion occurs is not fully understood.

Question 34

What do RAG enzymes do?

Answer 34

These enzymes catalyze the double stranded DNA breaks and recombination events that result in V-D or V-D-J recombinations.

Question 35

People that lack one or both of these enzymes produce very few B cell or T cells and suffer from what disease?

Answer 35

severe combined immunodeficiency syndrome (or SCID)

Question 36

What is the function of the enzyme, terminal deoxynucleotidyl transferase (or TdT)?

Answer 36

This enzyme catalyzes the addition of “N” nucleotides in the junctions between rearranging gene segments during somatic recombination of B cell heavy chain genes (but not light chain genes) as well as for beta chain rearrangements in T cell. When the gene segments are spliced together during recombination events, TdT catalyzes the addition of random nucleotides between the gene segments that are joined. Therefore, there are many different possible coding regions that can be created when the same two gene segments are recombined. This results in a huge increase in the number of possible antibody specificities (and T cell receptor specificities) produced via somatic recombination.

Question 37

What is Somatic hypermutation?

Answer 37

occurs after binding of B cells to an antigen; i.e. antigen dependent

single nucleotide substitutions (point mutations) that occur at very high frequency in the rearranged variable region DNA of immunoglobulin genes (both light and heavy chain genes). This process occurs in activated B cells during a germinal center reaction and results in production of variant antibodies, some of which have higher affinity for the antigen.

enzyme: activation-induced cytidine deaminase (AID)

Question 38

What is the basis of affinity maturation?

Answer 38

B cells that produce higher affinity Ab are selectively stimulated to proliferate and differentiate. This process results in antibody responses that “mature”, resulting in Ab populations that have increasing affinity for specific antigen

Question 39

Where do most of the point mutations that occur during somatic hypermutation occur?

Answer 39

they are confined to the complementarity-determining regions (CDRs) (also known as the hypervariable regions (HVs)

these mutations occur at a rate of approx. 1 mutation per V-region sequence per cell division; this rate is about 1000000 times greater than ordinary mutation rate for a gene

NOTE: this process is an antigen-dependent process that occurs in secondary lymphoid tissues during a germinal center reaction

Question 40

What is the order of the heavy chain constant region in all people and what type of Ab do they produce?

Answer 40

1. C(mu)= IgM
2. C(delta)=IgD
3. C(gamma3)= IgG3
4. C(gamma1))=IgG1
5. C(alpha1)=IgA1
6. C(gamma2)=IgG2
7. C(gamma4)=IgG4
8. C(epsilon)=IgE
9. C(alpha2)=IgA2

My dad got 3 gifts 1 ano1, gave 2 great forks, eggs, And 2thpaste

The first one in line is the only one that will be expressed. Therefore, all B cells initially produce IgM antibodies.

Question 41

the V-region exons produced by any given B cell are determined during the pro- and pre-B cell stage of maturation; except for somatic hypermutation, a given B cell and all of its progeny will express the same VL and VH genes. By contrast, the CH- region genes expressed by a B cell line can change as the response matures

Answer 41

the V-region exons produced by any given B cell are determined during the pro- and pre-B cell stage of maturation; except for somatic hypermutation, a given B cell and all of its progeny will express the same VL and VH genes. By contrast, the CH- region genes expressed by a B cell line can change as the response matures

Question 42

Class switching (or isotype switching) only changes the ____ region of the heavy chain

Answer 42

constant. It does not have any effect on the variable region of the heavy chain.

Question 43

Each B cell initially produces which Ab?

Answer 43

IgM

Question 44

What is the basis of isotype switching?

Answer 44

This is an antigen-dependent process.

each B cell initially produces IgM. Later in the response, the same V-region exons can be expressed with a different CH gene resulting in either IgG, IgE, or IgA antibodies with the same specificity as the initial IgM antibody

isotype switching to IgD does not occur, as IgD, and IgM are simultaneously expressed on mature naive B cells

Question 45

T or F. isotype switching only occurs after B cells have been stimulated by antigen, and have received T cell “help” in the form of
cytokine signals

Answer 45

T. the cytokine signals produced by T helper cells (TH1 or TH2) promote class switching to a particular antibody isotype; the cytokines communicate to the B cell what type of immune response is needed to most efficiently eliminate the particular pathogen, and the B cell switches to the most appropriate heavy chain

Question 46

How does isotype switching occur?

Answer 46

Each of the heavy chain constant domain coding regions, except for IgD, is immediately preceded by what is known as a switch region. Because there is no switch region preceding Cdelta, no class switching to IgD is ever possible.

Ex.
An enzyme known as activation-induced cytidine deaminase catalyzes a recombination event between the switch regions that precede the IgM and IgG3 gene segments. This causes the intervening germline DNA to be circularized.

The intervening DNA is then released, leaving the IgG3 heavy chain constant domain coding region as the first in line, and therefore the one that will be expressed. This B cell now will express IgG3, and it will never be able to class switch back to IgM because that coding region is permanently removed.

Be aware that class switching to one of the remaining constant domain coding regions (IgG1, IgA1, IgG2, IgG4, IgE, and IgA2) is still possible in this particular B cell because the germline DNA encoding those heavy chain constant regions has been left undisturbed.

Question 47

How does isotype switching add functional diversity to the Ab reptiroire?

Answer 47

Although class switching does not affect the antigen-combining region of the Ab molecule, it does lead to functional diversity of the antibody repertoire by producing different isotypes of antibody that have the same specificity as the parental B cell.

Question 48

What is the enzyme needed for both somatic hypermutation and isotype switching?

Answer 48

activation-induced cytidine deaminase

Question 49

a genetic deficiency of AID results in what is known commonly as ____.

Answer 49

hyper IgM syndrome. These patients
can activate a B cell response, but their B cells cannot undergo class- switching. These patients are left with heightened susceptibility to extracellular infections (bacteria, fungi, and parasites) as well as some viral pathogens (such as influenza, because neutralizing antibodies are the most effective immune response to flu).

These patient’s B cells are also unable to undergo somatic hypermutation/ affinity maturation. This problem alone would probably be subclinical.

Question 50

What are the phases of B cell development?

Answer 50

1) expression of surface receptor molecules (immunoglobulins); rearrangement of light and heavy chain genes must occur
2) negative selection in the bone marrow to remove B cells bearing receptors that bind to “self” antigens
3) positive selection of developing B cells that will continue to develop into mature B cells
4) searching for infection: mature B cells recirculate between lymphoid tissues until they become activated

5) activation and proliferation of B cells upon encounter with cognate antigen (must receive 2nd signal of activation unless T-
independent antigen)

6) proliferating B cells differentiate giving rise to plasma cells and memory cells

Question 51

What is one of the major functions of bone marrow stromal cells in relation to B cell development?

Answer 51

interleukin-7 (or IL-7) is a critical B cell growth/developmental factor that is produced by the bone marrow stromal cells. IL-7 is required for B cells to continue developing properly. Patients that cannot produce functional IL-7 (or its receptor—the IL-7 receptor) typically have normal numbers of B cells, but those B cells are non-functional.

NOTE: Lymphoid progenitor cells interact with bone marrow stromal cells via adhesion molecule interactions

Question 52

What are the two stages of B cell maturation?

Answer 52

1) antigen independent, and

2) antigen-dependent

Question 53

What are the four broad phases of antigen-independent development of B cells?

Answer 53

1. Pro-B cell
2. Pre-B cell
3. Immature B cell
4. Mature B cell

Question 54

What happens during the Pro-B cell phase of B cell development?

The early pro-B cell?
The late pro-B cell?

Answer 54

rearrangement of heavy-chain Ig genes takes place in these cells

Early: D-J rearrangement occurs
Late: V-DJ rearrangement occurs

Question 55

What happens during the Pre-B cell phase of B cell development?

The large pre-B cell?
The small pre-B cell?

Answer 55

productive VDJ is complete and the μ chain is expressed on the B cell surface; light chain rearrangement begins

• large pre-B cell: μ-chain (all of heavy chain) is expressed on B cell surface
• small pre-B cell: Initially, rearrangement of the kappa light chain genes occurs. If this is successful, the B cell will produce a kappa light chain. If it is not successful, the lambda light chain genes begin to rearrange. If this rearrangement is successful, the B cell will produce a lambda light chain.

Question 56

What happens during the immature B cell phase of B cell development?

Answer 56

light chain rearrangement is complete and IgM only is expressed on the cell surface; to this point, all maturation stages have occurred in the bone marrow; immature B cells migrate to peripheral lymphoid tissues and undergo self tolerance selection.

Question 57

What is Clonal deletion?

What is Clonal ignorance?

Answer 57

immature B cells that specifically interact STRONGLY with multivalent “self”-antigens in the bone marrow undergo apoptosis (programmed cell death), and are thus depleted from the B cell population (negative selection)

B cells that bind with weak affinity to self determinants in the bone marrow will continue to develop and will migrate to peripheral lymphoid tissues; the term “clonal ignorance” is, in my opinion, somewhat meaningless. These cells are essentially the same as a B cell that has no recognition of self determinants. However, peripheral tolerance mechanisms that will be discussed in a later lecture are more important for these types of B cells.

It is critical that you understand B cell (and T cell) tolerance. You must remember that somatic recombination is a random process that generates many trillions of lymphocyte receptor specificities and that many of them will bind tightly to determinants of the host. This can result in autoimmune disease.

Question 58

What is Anergy?

Answer 58

immature B cells that specifically interact with soluble antigens in the bone marrow are signaled to down-regulate cell surface expression of IgM and are left anergic (or unable to respond to specific antigen). These cells usually do not survive more than a few days after leaving the bone marrow (negative selection)

Question 59

What are mature B cells?

Answer 59

immature B cells that survive self-tolerance selection further differentiate to become mature B cells. This process is complete when the B cell expresses both IgM and IgD on its surface. These cells (also called naïve B cells) recirculate through peripheral lymphoid tissues where they may encounter their specific foreign antigen and become activated.

Question 60

B cell development in the bone marrow is dependent on _________, which provide specialized microenvironments for B cells at various stages of development

Answer 60

non-lymphoidal stromal cells

Question 61

What are the functions of non-lymphoidal stromal cells?

Answer 61

1) they make specific cell surface contact with the B cells through interactions between their adhesion molecules; and
2) they produce growth factors that are essential survival/developmental signals for B cells (stem cell factor and IL-7)

Question 62

What are the odds that each B cell precursor will productively rearrange their immunoglobulin genes?

Answer 62

approximately a 40% chance. If a B cell precursor does not productively rearrange both its heavy and light chains and express IgM on its surface, it will die.

Question 63

What are the RAG enzymes responsible for doing?

Answer 63

somatic recombination is dependent on the activity of two enzymes that are encoded by the recombination activation genes (RAG-1 and RAG-2); these proteins are responsible for making double-stranded breaks in DNA during somatic recombination

Question 64

What does Bruton’s tyrosine kinase (Btk) do?

Answer 64

nvolved in the transduction of signals from cell-surface receptors during B cell development; individuals that do not have a functional Btk gene have little or no circulating antibody because B cell development halts at the pre-B cell stage; this gene is encoded on the X-chromosome

Question 65

A deficiency in Btk leads to what disease?

Answer 65

X- linked agammaglobulinemia

Question 66

When does negative selection occur?

Answer 66

in the bone marrow, once heavy and light chains are expressed and IgM is displayed on the surface of cells

Question 67

What are possible destinies of B cells that are negatively selected upon?

Answer 67

 B cells whose B cell receptor molecules bind with high affinity to self cell surface molecules are induced to
undergo apoptosis (programmed cell death)

 B cells whose B cell receptor molecules bind with high affinity to soluble self antigens are rendered
unresponsive (or anergic), and they migrate to the periphery where they will die within a couple of days

 B cells whose receptors do not bind to self proteins migrate to the periphery where they continue the
maturation process and become mature naive B cells

Question 68

Mature naive B cells express what on their surface?

Answer 68

IgM and IgD

Question 69

What do mature naive B cells do?

Answer 69

recirculate through the peripheral lymphoid tissues until they encounter their specific antigen (or until they die). Once the B cell encounters specific antigen and receives the appropriate T cell-derived signals in the T cell areas of the lymphoid tissues, the B cell is activated to begin proliferating.

Question 70

What happens when B cells begin to proliferate?

Answer 70

1. some of the progeny b cells form a germinal center in a nearby follicle
2. Germinal center B cells proliferate vigorously, and their variable region genes undergo somatic mutations (somatic hypermutation, resulting in affinity maturation)
3. actiavted B cells are stimulated to undergo class switching
4. selected B cells then differentiate into plasma or memory cells

Question 71

What do plasma cells do?

Answer 71

a terminally differentiated B cell whose primary function is to produce antibodies. Many IgG- producing plasma cells migrate to the bone marrow, while IgA-producers migrate primarily to the lamina propria of mucosal surfaces; the half-life of the cells is between 30-60 days

Question 72

What do memory cells do?

Answer 72

a long-lived antigen-specific B cell that results from antigenic stimulation of a naïve B cell during the primary immune response. Upon subsequent exposure to specific antigen, these cells are reactivated to differentiate into plasma cells as a component of a secondary immune response

Question 73

Memory B cells express which Igs on their surface?

Answer 73

G, E, and A following isotype switching (mature B cells express M and D)

Question 74

The surface Ig (the B cell receptor) serves what functions?

Answer 74

1) Serves as a receptor for specific antigen; delivers the 1st activation signal to the B cell following interaction with specific
antigen

2) Via receptor-mediated endocytosis, surface Ig delivers the specific antigen to the interior of the B cell where it is degraded. Peptide fragments of the antigen are then displayed on the surface of the B cell in association with MHC class II (antigen presentation). The peptide:MHC complex can then be recognized by antigen-specific T helper cells. Once the T helper cells encounter the peptide:MHC complex, they produce signals (lymphokines) and CD40 ligand (a cell surface marker, CD40L) which together trigger B cells to proliferate and to differentiate into either memory or plasma cells

Question 75

What are Lymphokines (also called cytokines)?

Answer 75

proteins made by cells that affect the behavior of other cells; these proteins bind to specific receptor molecules on their target cells. Lymphokines are cytokines produced by lymphocytes.