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Immunology > Lecture 5: Adaptive Antigen Rexognition > Flashcards

Lecture 5: Adaptive Antigen Rexognition Flashcards

1
Q

Activation of B cells

A

B-cells do not need an APC for them to become activated. Instead, they can bind soluble or cell-surface assx forms of antigens.

When a B-cell is activated, they can become a plasma cell (which secretes antibodies) or a memory cell.

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2
Q

Where are plasma cells found?

A

NOT IN PLASMA!

In lymphoid organs.

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3
Q

Activation of T cells

A

In order to activate T-cells, the antigen must be presented to them by a APC within a MHC.

Remember; the antigen must be a linear peptide fragment

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4
Q

B-cell receptors: Tell me about them

Similar or different to TCR?

What is their NH terminus like?

A

B-cell receptors are structurally similar TCR.

-The -NH-terminus of heavy and ligt chains is highly variable, as is the Va and Vb of T-cells.

In the constant region, there is limited variabled.

In a single B cell or T cell, BCR and TCT are identical.

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5
Q

BCR

  • What is the antigen?
  • Diversity?
  • Signaling?
  • Effector functions?
A

Anntigens- macromolecules

Diversity: Each clone has unique specificity

Signaling is inited by [Igalpha and Igbeta proteins]

The effector functions are mediated by the constant regions of the antibodies.

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6
Q

T Cell Receptor

  1. What forms of antigens does it recognize?
  2. Diversity?
  3. Signaling functions are mediated by?
  4. Effector functions?
A
  1. Linear, short sequences of AA presented on a APC.
  2. Each clone has unique specicity
  3. CD3 and ς
  4. No effector functions
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7
Q

What is another word for antibodies?

A

Immunoglobins.

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8
Q

Most antibodies are found in the _____ slowest migrating group of globulins, named _____ globulins.

A

Third

gamma

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9
Q

What effects do antibodies have?

A

Antibodies can:

  1. Organize T-cells
  2. Opsonization
  3. Activate compliment system
  4. Neutralize toxins
  5. Have direct anti-bacterial activity
  6. Immunodulation
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10
Q

What is the structure of antibodies?

A

Anitbodies have 4 polypeptide chains: two light chains and 2 heavy chains.

The chains are bound together by disulfide bridges and non-covalent interactions

H and L chains are divided into: variable (V) and constant (C) regions.

  • Light chain has 1 variable and 1 constant regions
  • Heavy chain has 1 variable region and 3 constant regions

Antigens bind on the variable regions.

C region determines the fate of the Ag.

Anitbodies can be cleaved to make 2 Fab fragments and 1 Fc fragment

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11
Q

Antbodies can be cleaved to create, what?

A

2 Fab fragments, where the antigen binds

1 Fc fragment, responsible for effector functions

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12
Q

We mentioned that antibodies can be cleaved. What happens they are cleaved by Papain?

A

Papain will cleave Ab into

2 Fab fragments

Fc fragment

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13
Q

We mentioned that antibodies can be cleaved. What happens when they are cleaved by Pepsin?

A

Pepsin will not seperate the two Fab fragments (F(ab)2. Thus, it will create a single bivalent antigen binding fragment.

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14
Q

How do we create different types of antibodies?

A

There are 5 different types of heavy chains (___, __, __, ___, alpha), which determine the class of the antibody.

There are also two kinds of L chains (k and __).

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15
Q

1 AB can only have one ____ or ___, but not both.

A

k or __-L chain

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16
Q

How do we connect L and H chains?

A

Disulfide bridges will connect them every 90 AA, creating a polypeptide loop.

These loops are referred to as VH, VL, CH1, CH2

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17
Q

What are the Ig superfamily proteins?

A

Cells surface and soluble protein involved in the [recognition, binding or adhesion] processes of cells.

  1. TCR
  2. MHC molecules
  3. CD4 receptor of T cells
  4. CD28
  5. costimulatory receptor on T-cells
  6. adhesion molecule ICAM-1
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18
Q

How are secreted IgG different from membrane bound IgM?

A

Secreted IgG: the heavy chain C-regions end in TAIL PEICES.

Fc receptor/compliment binding sites are approcimated.

Membrane bound IgG: has one more CH4 domain and is anchored into the cytoplasm.

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19
Q

How are antibodies so flexible?

A

Antibodies have hinges in between CH1 and CH2 domains.

How are the constant chains are from one another is determined by these hinges.

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20
Q

What are the 5 major classes of antibodies?

A
  1. IgA
  2. IgD
  3. IgE
  4. IgG
  5. IgM
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21
Q

IgA

Types?

Heavy chains?

Secreted forms?

Functions?

A

Remember; antibodies are classified based on their heavy chain and each may only have a k or delta light chain.

There are two types: IgA1 IgA2

Heavy chains are: alpha1 and alpha2

Secreted mainly as dimers, but also monomers and trimers.

Functions in [mucosal immunity].

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22
Q

IgD

Main function

A

Naive B cell antigen receptor

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23
Q

IgE main function

A

Defend against parasites and immediate hypersensitivity

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24
Q

IgG main function

A
  1. Opsonization
  2. Compliment activation
  3. Antibody dependent cell-mediated cytoxicity
  4. Neonatal immunity
  5. Feedback inhibition of B cells
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25
Q

IgM function

A

Naive B cell antigen R

Compliment activation

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26
Q

Which antibody is released as a pentamer?

A

IgM

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27
Q

The tightness of antibody-antigen binding is called affinty.

Do antibodies formed in the primary response, soon after infection of an antigen, have higher or lower affinity?

A

Lower.

Antibodies produced by a memory response have HIGH affintiy.

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28
Q

When is the affinity of the ab and antigen CRITICAL?

A

When the antigen is a toxin and needs to be neutrilized quickly at low titers.

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29
Q

What is antibody valence?

A

Antibody valence is the number of antigens a antibody can bind.

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30
Q

What is the valence of IgG?

A

IgG has 2 Fab regions; thus it can bind 2 molecules of an antigen OR 2 identical sites on 1 particle

Thus; valence= 2

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31
Q

How is valence related to binding affinity?

A

Increase valence; increase binding affinity

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32
Q

What is avidity?

A

Avidity is the overall strength of the Ab-Ag complex. It depends on

  1. Affinity
  2. Valence of both Ab and Ag
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33
Q

What is the affinity of IgM?

A

IgM has low affinity.

This is useful when we microogranism with a large number of binding sites.

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34
Q

Ben got infected with a microorganism with a LARGE number of binding spots. Which antibody is the most effective in killing dat shit.

A

IgM, which has low affinity.

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35
Q

Which has the highest avidity?

Monovalent antibodies

Bivalent antibodies (IgG)

Polyvalent antibodies (IgM)

A

IgM> IgG> monovalent antibodies.

Although IgM has low affinity, it has a high valence; meaning that its avidity will be HIGH.

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36
Q

How do we make monoclonal antibodies?

A
  1. Spleen cells from a mouse that have been immunized are isolated.
  2. Polyethylene glycol is then used to combine [isolated spleen cells] and [immortizalized myeloma cells that do not secrete antibodies].
  3. Cells are placed on a selection medium (HAT) that only allows immortilized cells to survive. HAT includes (hypoxanthine, aminopterin, and thymidine)
  4. Screen for anti-X anitbody and expand the + clones
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37
Q

Why do only hybrid cells survive in a HAT medium?

A

Because myeloma cells do not have HGPRTransferase that is used to purine salvage.

The aminopterin will block purine synthesis.

THUS; PURINES (DNA) CANNOT BE MADE :)

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38
Q

What is the basic structure of TCR?

A

T-cell receptors are heterdimeric (1 alpha and 1 beta chain).

Each chain has a variable (V) region and a constant (C) region (same as in antibodies).

The v region has 3 complemtarity-determining regions (CDR) that each form a loop.

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39
Q

Describe the TCR Complex

A

TCR is a heterodimeric protein with an alpha and beta subunit.

Alpha and beta subunits both have variable and constant regions.

The variable regions have 3 complimentarity determining regions (CDRs)

TCR will form a complex with CD3 and _ proteins due to charged areas in the transmembrane region.

The alpha and beta subunits of the TCR have tails that go into the cytoplasm that are 5-12 aa long. However, they cannot transduce signals. SO. CD3 and __ serve to transduce the signals for the TCR complex.

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40
Q

Structure of CD4 co-receptor

A
  • Has 4 extracellular-antibody like domains.
  • hydrophobic transmembrane region
  • Basic tail with 38AA
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41
Q

Structure of CD8 receptor

A

2 chains:

CD8alpha

CD8beta

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42
Q

CD8 is made up of 2 related chains: CD8a and CD8b. What are characteristics of them?

A
  • they have a single extracellular Ab domain
  • hydrophobic transmembrane region
  • basic cytoplasmic tail that is about 25 AA long

CD8 binds to class I MHC, which interact with B2 microglobin

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43
Q

Describe the antigen binding regions on immunoglobulin (Ig)

A

VH has 3 CDRs

VL has 3 CDRs

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44
Q

Describe the changes in the constant regions of Ig

A

There is heavy chain class switching and change from membrane–> secretory Ig

45
Q

Affinity of antigen binding for Ig

A

Kd= 10-7–> 10-11M

This is increases during immune responses.

46
Q

What is the on and off-rate for Igs?

A

On-rate is rapid

Off-rate is variable

47
Q

What is the antigen binding area of T-cell receptors

A

Valpha is made up of 3 CDRs

Vbeta is made up of 3 CDRs

48
Q

Changes in constant regions of TCRs?

A

The constant region does not change

49
Q

Affinity of antigen binding for TCR

A

Kd= 10-5 to 10-7M

Affinity does not change during a immune response.

50
Q

On and off rate of TCRs

A

Slow on-rate

Slow-off rate

51
Q

What antibodies are expressed on B-cells as they develop?

  1. Pre-B cell
  2. Immature B-cell
  3. Mature B-cell
  4. Activated B-cell
  5. Antibody secreting cell
A
  1. Pre-B cell has a cytoplasmic u heavy chain and pre-B receptor
  2. Immature B-cell- IgM
  3. Mature B cell- IgM and IgD
  4. Activated B cell- low rate of antibody secreteion, heacy chain isotype switching and affinity maturation
  5. Antibody secreting cell; high Ab secretion and reduced membrane Ig
52
Q

List the steps in maturation of lymphocytes (B-cells)

A
  1. Hematopoietic progenitors must commit to the B or T cell lineage.
  2. Pre-BT cells express 1 chain of the antigen receptor
  3. Progenitors must proliferate
  4. Immature B/T cells express a complete antigen receptor

If the cell has weak antigen recognition, it becomes a mature B/T cell.

53
Q

How do we generate diverse BCRs and TCRs?

A

Somatic recombination of gene segments.

Each immunoglobulin (Ig) heavy-chain constant (C) region and T cell receptor (TCR) C region has multiple exons that encode C regions.

The basic organization is the same in all species, but the order and number of gene segments may vary.

54
Q

Somatic recombination creates diverse BCRs. What is this process like?

A

2 recombination events occur:

D (diversity segment) and J (joining segment) combine

Then a V (variable segment) joins the DJ complex.

  1. D+J
  2. DJ+V
55
Q

FIGURE 4-13 Mechanisms of diversity in antigen receptors. Diversity in immunoglobulins and T cell receptors is produced by random combinations of V, D, and J gene segments, which is limited by the numbers of these segments and by removal and addition of nucleotides at the V-J or V-D-J junctions, which is almost unlimited. The numbers of gene segments refer to the average numbers of functional genes (which are known to be expressed as RNA or protein) in humans. Junctional diversity maximizes the variations in the CDR3 regions of the antigen receptor proteins, because CDR3 includes the junctions at the site of V-J and V-D-J recombination. The estimated contributions of these mechanisms to the potential size of the mature B and T cell repertoires are shown.

Also, diversity is increased by the ability of different Ig heavy and light chains, or different T cell receptor (TCR) α and β chains, to associate in different cells, forming different receptors (not shown). Although the upper limit on the number of immunoglobulin (Ig) and TCR proteins that may be expressed is extremely large, each individual contains on the order of only 107 clones of B cells and T cells with distinct speci cities and receptors; in other words, only a fraction of the potential repertoire may actually be expressed.

A
56
Q

Chromosome 14 has a gene for which antibody?

A

H-chain

57
Q

What chomosome has a gene for k-chain?

A

2

58
Q

What antibody does chromosome 22 encode?

A

___-chain

59
Q

What is allelic exclusion?

A

Allelic exlcusion is one reason why we have a diverse group of BCR and TCRs.

We inherit codominant alleles for both H and L chains from our mom and dad. However, only one of the light chain alleles and one of the heavy chain alleles is expressed on 1 B cell.

However, because we have so many B and T cells, both alleles are expressed equally.

60
Q

Do TCRs exhibit allelic exclusion?

A

Yes; both TCRs and BCRs

61
Q

Mechanism of VDJ Recombination

A

somatic recombination of VDJ gene segments is mediated by VDJ recombinase (made up of RAG1 and RAG2 proteins), which the causes dsDNA breaks.

These breaks are resolved via nonhomologous end joining.

62
Q

What is the major mechanism for epitope-speific diversity of BCR and TCR?

A

DNA chromosomal rearragement. Doing so involves deletion of DNA/RNA nucleotides and reannealing.

This is completed via Rag1 and Rag2 recombination enzymes.

63
Q

How is BCR diversity acheived?

A

VDJ recombination

-Chromosome 14 has the genes for heavy chains.

Heavy chains have 4 different parts: V (variable), D (diversity), J (joining), C (constant).

Each of those segments will have multiple copies of that segment.

B cells create different variations of V-D-J, creating diversity.

Recombination begins with the heavy chain. If the heavy chain is functional, the light chain undergoes recombination.

While in the developmental stages, B and T cells undergo VDJ recombination, initiated by RAG1 and RAG2.

  1. D + J; DNA in between is deleted
  2. DJ + V; DNA in between is deleted

Productive rearrangements only occur about 10% of the time. If they are productive, they will undergo transcription and translation (a test run).

When a productive arrangement is confirmed, mom and dad H chain genes stop competing and other segments stop undergoing recombination.

Light chain

If the VH recombination is production, B cell proliferated and cares for VL. VL undergoes a similar process, but does not have D segments.

THIS CREATES UNIQUE VH AND VL REGIONS.

64
Q

TCR Rearragement by Somatic Recombination

A

When TCR undergo recombination, the B-chain goes first; then the A-chain.

65
Q

Does VDJ introduce enough diversity so that we can respond to all antigens?

A

No. It is limited by the number of V, D and J segments that we have.

66
Q

If VDJ diversity is not enough, how can we increase it?

A

Junctional diversity

67
Q

What is junctional diversity?

A

Junctional diversity is the changes in nucleotide sequences at the junctions where V, D and segments recombine. This occurs via terminal deoxynucleotidyl transferase (TdT).

68
Q

What enzyme mediated the process of junctional diversity?

A

TdT

Terminal deoxynucleotidyl transferase

69
Q

What are the three mechanisms junctional diversity occurs?

A

3 mechanisms:

  1. Exonucleases remove nucleotides
  2. TdT adds nucleotides between [V and D] and [D and J] segments, forming N regions.
  3. RAG cleaves hairpin loops, creating overhanging DNA sequences. They are then filled in with P nucleotides, forming palondromic sequences.
70
Q

RAG removes hairpin loops to create single stranded overhangs. What happens to them?

A
  1. They can be removed
  2. Filled in with P nucleotides.
71
Q

Junctional diversity increases diversity by how much?

A

Ig: 10^6–> 10^11

TCR: 3*10^6–> ~10^16

72
Q

Where does the maturation of B cells occur?

A

Bone marrow.

73
Q

Stages from

Stem cell–> mature B cell

A
  1. Stem cell
  2. Pro-B
  3. Pre-B
  4. Immature B
  5. Mature B
74
Q

Progenitors committed to the B-cell lineage proliferate and create what?

A

Pro-B cells

75
Q

How do we go from pro-B–> pre-B cell?

A

Pro-B will become a Pre-B if it can make a

1. [Ig u heavy-chain protein], located in the cytoplasm.

  1. Some of this protein will be on the cell surface and associate with [surrogate light chains].
  2. [u-chain + surrogate light chain] + Igalpha and Igbeta–> pre-BCR complex
76
Q

During the development of T and B cells, cells go through checkpoints to see if they can procede with the process. What are the 1st and 2nd checkpoints in lymphocyte development?

A

1st- production of the 1st polypeptide chain of the two-chained antigen receptor

2nd- production of the 2nd polypeptide chain.

This process makes sure we dont have self-reactive lymphocytes.

77
Q

In the development of B and T cell, pre-antigen receptors and antigen receptors send signals so they can proliferate and mature. As we said, this having this is a checkpoint. What are the pre-antigen receptors called in B cells and T-cells?

Do they have both polypeptide chains?

A

B cells- pre-BCRs (have one Ig-u heavy chain)

T cells- pre- TCRs (have one TCR-B chain)

These receptors only have 1 polypeptide chain and surrogate receptor chain.

78
Q

what is the first antigen receptor gene to be completely rearranged in B cells? What happens if successful rearrangement occurs?

A

Ig heavy chain.

B cells that successfully rearrange express u-heavy chain and create the pre-BCR.

79
Q

what is the first antigen receptor gene to be completely rearranged in B cells? What happens if successful rearrangement occurs?

A

TCR-B chain gene.

Doing this productively created the pre-TCR.

80
Q

Developing B and T cells rearrange Ig heavy chain and TCR-B chain, respectively, to form pre-BCR/pre-TCRs.

What re-arrangements need to be made: in-frame rearrangments or out-of-frame rearrangements?

A

In-frame rearrangements.

If they make out of frame rearrangments, the cells do not receive SURVIVAL signals and undergo apoptosis.

81
Q

Now that pre-BCRs and pre-TCRs are made; what do they do?

A

They send signals for [survival, proliferation and development] of B and T cells and begin to form the secnond chain.

82
Q

Second checkpoint:

Describe how we determine which cells are undergo “positive selection”.

A

If a cell expresses a functional second chain of the antigen receptor (completing it). they are positively selected. Positive selection makes sure these cells attack MHC molecules, not self.

*When the full receptor is displayed, the cell is still immature.

83
Q

Second checkpoint:

Describe what happens to cells who undergo “negative selection”.

A

Being sure we “negative select” immature B and T cells is important in maintaing tolderance to agonists. It ELIMINATES harmful T-cells and ALTERS harmful B-cells.

Clonal deletion- the apoptosis of harmful T-cells with a high affinity for self

Receptor editing- harmful B cells are given a second try to rearrange their Ig gene. If they fail, clonal deletion occurs.

84
Q

Clonal deletion

A

Clonal deletion is the process whereby immature T- cells under apoptosis because they do not have a functional two-chain antigen receptor. This gives them the opportunity to be harmful to self.

B-cells will also undergo clonal deletion if receptor editing fails.

85
Q

What is receptor editing?

A

If B-cell do not exhibit a functioning two chain antigen-receptor, they are given a second try. If they fail, the self-reactive B cells will undergo clonal deletion.

86
Q

In v immature B cells, there is a strong affinity of IgM by self-antigen. This is why we pay attention to self-reactivity. As we develop our antigen receptor, what do we want to do?

A

Prevent the cell from reacting with self.

87
Q

B-cells have B1 and B2 subsets.

Most B cells that develop from the fetal liver SC will differentiate to ______.

B-cells that arise from the bone marrow will AFTER birth will give rise to _______.

A
  1. B-1 lineage
  2. B-2 lineage
88
Q

B-1 cells are derived from fetal liver. Are they diverse?

A

B1 cells are not diverse because the fetal liver does not have TdT; which causes junctional diversity.

Thus, these cells do not undergo junctional diversity

89
Q

B-1 cell antibody secretion

A

B-1 cells will spontaneous secrete IgM.

These antibodies are sometimes called natural antibodies because they live in our without immunization.

90
Q

B-_ cells cause most of the serum IgM during the early phases of infection.

A

B-1.

B-1 cells will spontaneously secrete IgM that are called our natural antibodies; because they are made without immunizations.

91
Q

Negative and positive selection removes B-cells via what mechanism?

A

Central tolerance

92
Q

Immature B-2 cells that have passed both checkpoints and were positively selected now do what?

A

The move to the spleen.

93
Q

Immature B2 cells in the spleen will differentiate into what?

A
  1. Marginal zone B-cells (MZ-B2-cells)
  2. Mature follicular B-cell (FO-B2-cells)
94
Q

Where do B2 cells in the marginal zone live?

A

Marginal zone of the spleen AND lymph nodes.

95
Q

MZ B2 Cells respond to what types of antigens?

A

Blood borne.

96
Q

Features of MZ B2 Cells (marginal zone B2 cells)

A
  1. Self-renewing
  2. Control T-cell INDEPENDENT and DEPENDENT responses.
  3. Have limited diversity and make natural antibodies
  4. Develop into SHORT-LIVING: Ig-M secreteing plasma cells
97
Q

Are mature follicular B2 Cells (FO-B2 cells) constantly in stock?

A

No; we constantly need to make the from bone marrow.

98
Q

What kind of antigens to mature follicilar B2 cells respond to?

A

Antigens presented to them by T-cells.

Thus; they develop into LONG-LIVING plasma and memory b cells.

99
Q

Antigent-independent maturation of T-cells occurs where?

A

Thymus

100
Q

Thymus cells types

A
  1. Cortical thymus epithelial cells
  2. Medullary thymus epithelial cells
101
Q

Thymocytes will evolve into mature T-cells that have different antigens on them. These antigens will differentiate them into what stages?1

A
  1. Double negative (DN)
  2. Double positive
  3. Single positive (express with CD4 or CD8)
102
Q

Differentiation of T-Cells occurs where?

A

In the stroma (medulla) of the thymus.

mTECHs here have a group of genes that code for organ specific proteins

103
Q

promiscuous gene expression

A

the expression of SP antigens on T cells;

playing a big role in t-cell tolerance

104
Q

The faith of DP cells depend on what?

A

the ability of the TCR to interact with a self-peptide MHC complex

105
Q

Positive selection

A

DP thymocytes are programmed to die unless they get a signal from a MHC complex that they recognize them. 95% of them do not but 5% will bind with mid avidility.

This causes DP–> SP

106
Q

+ selection occurs where?

A

thymus cortex

107
Q

Negative selection

A

occurs in the medulla of the thymus.

If both TCR and CD28 bind to CD80 and CD86

108
Q

getting rid of self reactive thymocytes via clonal deletion requires what?

A

TSA- tissue specific antigens

Immunology (12 decks)

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