Regulation of the Immune System

Question 1

How does the adaptive immune response start?

Answer 1

antigen presenting cell presents antigen to naive t-cell

Question 2

what is the initial event in the adaptive immune response?

Answer 2

naive t-cell activation

Question 3

what does naive t-cell activation lead to?

Answer 3

b-cell activation or cytotoxic t-cell response or both

Question 4

when do a lot of regulatory mechanisms apply”

Answer 4

to naive t-cell activation

Question 5

antibodies do the same as what?

Answer 5

t-cell receptors

Question 6

how were antibodies discovered?

Answer 6

100 yrs ago, people immunized animals w/bacteria antigen. In serum from animals there was activity - if they put bacterial toxin in serum it formed precipitation. If they then injected this serum into naive mice, they were protected from bacterial infection (how antibody came from) - neutralizing activity that neutralizes bacterial toxin. nobody knew nature of the antibody; protein, DNA or what?

Question 7

How did they discover what antibodies actually were?

Answer 7

electrophoeisis - you can see immunoglobins between 150 and 900 kd. But they still didn’t know structure of antibody (huge molecular weight)

Question 8

How did they figure out structure of antibody

Answer 8

disassembled them & tried to figure out the pieces - 1950’s Porter and Edelman won nobel prize figuring out structure

Question 9

Porter’s lab - what did it do?

Answer 9

used papain to digest antibody protein - found 3 fragments, two were identical - Fab (fragment of antigen binding), the other one was Fc - fragment of crystallizable - also the constant region of the antibody

Question 10

what is the function of the two identical parts of the antibody?

Answer 10

antigen binding part of the antibody - 2 of them -means antibody can bind to 2 antigens - divalent (antibodies are divalent)

Question 11

What did Edelman’s lab do?

Answer 11

did similar experiment to Porters, but used stronger reducing agent & broke disulfide bonds between light chains & heavy chains, so they got 4 fragments - two light chains & 2 heavy chains

Question 12

Where is the constant region of an antibody?

Answer 12

in both light & heavy chains

Question 13

what is the most important part of the antibody?

Answer 13

FAB, the variable region that recognizes the antigen (both heavy & light chains have variable regions)

Question 14

Is the variability of an antibody homogeneous?

Answer 14

Nope. - there are areas within variable region that are hyper-variable - complementary determining regions

Question 15

What is CDR?

Answer 15

Complementary determining regions of the variable region on the antibody - most important part of the antibody - this is where the antigen binds

Question 16

what does complementary mean?

Answer 16

the amino acids in the complementary determining region are complimentary to the amino acids of the antigen (the epitope)

Question 17

Where does the antigen bind?

Answer 17

only to the CDR region of the antibody (between the light chain and the heavy chain of the variable region)

Question 18

Why is there a precipitate in the serum of an immunized animal that has the bacterial toxin introduced?

Answer 18

the antibodies bind to two antigens each - makes huge complex which is not soluble - precipitates out

Question 19

What does specificity mean

Answer 19

Goodness of fit - fit between ligand and receptor - measured by affinity between antigen determinant (epitope - the binding part of the antigen) with the antibody

Question 20

how do you quantify the specificity of ligand and antigen?

Answer 20

association constant of a good antibody that binds to its counterpart antigen is 10to the 15th L/mole - if you dilute antigen to 10 to the -15th molar, still half of the antibodies are bound to the antigen - very very diluted
doesn’t disassociate very easily

Question 21

How many antibodies can one species make?

Answer 21

100,000 million antibodies

Question 22

How many antibodies does one individual make?

Answer 22

100 million

Question 23

Do we have to have 100 million genes to encode for each antibody we have?

Answer 23

Nope

Question 24

How many total genes do we have?

Answer 24

30 million

Question 25

How do we have the capacity to make so many different antibodies? What are the 2 theories?

Answer 25

2 theories: 1. instruction theory - we don't need to have anything ready, we just wait until antigen comes along & depending on the structure of the antigen, we make the corresponding antibody or t-cells 2. clonal selection theory - means we have all of these antibodies & t-cells & b-cells already there, we don't wait until antigen comes along

Question 26

Which theory makes more sense for antibody capacity?

Answer 26

problem with instruction theory, is that when we are born it means we don't have antibodies for anything. When we are infected with something, we then make a gene out of nowhere & that gene encodes for antibody - means that the outside world will determine our genome (that's what instruction theory entails) - that hasn't happened in biology. Scientist from CU & another scientist from Australia put forth clonal selection theory - now that is the accepted hypothesis - each b-cell & t-cell in our immune system is programmed to make one antibody or one t-cell or b-cell receptor. Choice of which antibody the cell can make is random. Entire b-cell population is already there when we are born. Antigen causes activation of one clone of t-cell or b-cell, so we get immunity against that antigen

Question 27

What is the N-terminal?

Answer 27

End of the antibody where light chain and heavy chain has different sequences between other antibodies of different specifications - the variable domain (V)

Question 28

Describe the clonal selection theory

Answer 28

(1950's posed by David Talmage & MacFarlane Burnet) - proposed that each Bcell of the immune system is programmed to make only one antibody; that the choice of which antibody the cell will make is random, not dependent on outside information; and that the entire population preexists in a normal individual, even before contact with any antigens. When a new antigen is introduced into the body, it comes into contact with a huge number of B lymphocytes and when it encounters one to whose receptors it binds with sufficient affinity, it activates that cell, resulting in expansion of that clone of b cells and production of antibody. The best fitting clones are selected by the antigen

Question 29

How do we get so much antibody diversity with fewer genes?

Answer 29

1. Multiple genes that create the v domain 2. genes are organized into segments that can be rearranged to give diversity 3. combinatorial diversity - creates more diversity & flexibility & randomness 4. somatic mutation 5. pairing of heavy and light chains (each antibody has 2 heavy & 2 light chains) 1000 heavy chains + 1000 light chains gives you 1,000,000 different antibodies with just 2000 genes

Question 30

What are the gene segments that code for heavy chains?

Answer 30

V, D and J

Question 31

What are the gene segments that code for light chains?

Answer 31

V and J

Question 32

How many genes encode for each segment on an antibody?

Answer 32

``` For heavy chains: 65 - V 27 - D 6 - J =10,500 different antibodies by using only 98 genes for light chain 35 v-segment 5-J =175 antibodies by only 40 genes ``` only one gene encodes for constant region for each antibody

Question 33

How many genes for constant region?

Answer 33

``` 5 (5 flavors) - each one encodes for a different kind IgG gamma IgM Mu IgA alpha IgD delta IgE epsilon ```

Question 34

What accounts for the majority of the complexity of the variable region of antibodies?

Answer 34

recombination in developing B cell making antibody genes in D region come closer to genes in J regin - intervening DNA in between is removed & D & J region are joined same thing happens for V - genes in V region then come into the D and J and the DNA's in between are removed, then we have VDJ recombined variable region

Question 35

What enzyme does the V-D-J recombination?

Answer 35

RAG 1 & RAG 2 recombinase | without these two enzymes, we wouldn't have any b-cells or t-cells

Question 36

How does RAG recombinase work?

Answer 36

recognizes splicing signal to right of D segment, and splicing signal to left of J segment, brings the two together, cut everything in between & then join the two segments then recognizes splicing signal to right of V segment, brings it to the D & cuts the intervening DNA & joins the V and D - how we get VDJ recombination - then transcribed to mRNA & we get the antibody

Question 37

What mechanism adds unpredictability or randomness to the antibodies we make?

Answer 37

recombinational inaccuracy: during joining, two ends of each segment, there are nucleotides that can be removed or added before the two segments are joined

Question 38

What enzyme removes or adds nucleotides to the ends of the V,J & D segments before they are joined to each other?

Answer 38

terminal deoxynucleotide (TDT) transferase - doesn't need a template - that's why there's randomness - can't predict what's going to happen based on the genome

Question 39

Which kind of cell recognizes antigen on its own?

Answer 39

B-cells recognize antigen on its own. T-cells do NOT.

Question 40

How do t-cells recognize antigen?

Answer 40

MHC restricted recognition of antigen discovered with experiment took cytotoxic t-cells from inbred strain A mice infected with virus V - found t-cells kill v virus infected cells, but don't kill e-virus infected cell (makes sense -antigen specificity) cytotoxic t-cells taken from strain a mice did not recognize infected v cells from strain b mice, even though they were infected with the same virus. that suggests that t-cells don't just recognize antigen - it recognizes something else that has to be self (recognize antigen plus something identical that is self-protein)

Question 41

What can t-cells "see"?

Answer 41

only antigen presented to them on the surface of a genetically identical cell to themselves

Question 42

What is the self molecule important for t-cell antigen recognition?

Answer 42

major histocompatibility complex | or human leukocyte antigen (MHC, HLC)

Question 43

how many classes of MHC are there?

Answer 43

``` 2 class 1 - ABC class 2 - DP, DQ and DR ```

Question 44

Where do you find MHC's?

Answer 44

``` Class one - all nucleated cells in our body class 2 - only on antigen presenting cells macrophages &dendritic cells & b-cells ```

Question 45

What kind of t-cells recognize what kind of MHCs?

Answer 45

CD8 T- cells (cytotoxic T-cells) recognize MHC class 1, CD4 T-cells recognize MHC class 2

Question 46

What are cytotoxic t-cells?

Answer 46

CD-8 t-cells, killer t-cells

Question 47

What do CD-4 cells become?

Answer 47

t-helper cells - after they recognize antigens presented by dendritic cells, macrophages & sometimes b-cells - they then release cytokines to help b-cells or cytotoxic t-cells

Question 48

What is the structure of an MHC receptor?

Answer 48

alpha beta chains, antigen sits in the groove (beta barrel forms groove, & sides are alpha helices), t-cell receptor recognizes antigen but also binds to regions on the sides of MHC molecule - that's why it is MHC restricted recognition

Question 49

What is meant by MHC restricted recognition?

Answer 49

The t-cell recognizes the antigen that sits in the groove of the MHC molecule - so the t-cell also has to recognize the side of the MHC molecule where the antigen binds

Question 50

What are the requirements in the thymus for a successful t-cell?

Answer 50

it should not recognize self peptide (derived from self protein) or the self MHC - they do need to recognize self MHC molecule when bound to antigen, but they cannot respond to MHC too strongly (MHC alone, or MHC loaded with self peptide) should recognize antigenic peptide plus self MHC should not recognize free antigen

Question 51

How many selections to t-cells go through in the thymus?

Answer 51

2 - negative selection, and positive selection

Question 52

what is negative selection?

Answer 52

cells that respond to self are removed - go through apoptosis If t-cell responds to self-peptide loaded onto MHC with high affinity, they apoptose if the t-cell receptor doesn't recognize anything - not even MHC with low affinity - those cells are also destroyed by apoptosis - only those that recognize self-MHC or self-MHC + self peptide with low affinity The AFFINITY is what makes the difference - high affinity makes them self-reactive. If they don't respond at all, they won't recognize pathogens. have to recognize with low affinity - not enough to cause t-cell activation, but when self-peptide is exchanged with pathogenic peptide, the hope is it will have high affinity

Question 53

What is positive selection?

Answer 53

If they recognize MHC class II, they become CD4 cells, if they recognize MHC class I cells, they become CD8 cells

Question 54

What is central tolerance?

Answer 54

education process of t-cells in the thymus - the most important part of central tolerance is negative selection positive selection determines what kind of t-cells they will become

Question 55

How do you define low affinity?

Answer 55

ambiguous - in the thymus, even though we do our best & we get the right t-cells, but when they get out we need to have more regulations to make sure we respond to infection & we don't respond to self

Question 56

What are the three regulatory mechanisms in the periphery for t-cells?

Answer 56

1. anergy 2. activation-induced cell death 3. suppression by regulatory t-cells

Question 57

How is a naive t-cell activated?

Answer 57

t-cell receptor recognizes antigen presented by MHC molecule is signal 1 (not enough) also needs signal 2 - provided by co-stimulatory molecules CD4 molecule plays as an anchor to make sure interaction lasts long enough adhesion molecules make sure they interact for 9-12 hours, these molecules are signal 2

Question 58

What molecules are signal 2 for naive t-cell activation?

Answer 58

t-cells express CD40Ligand and CD28, bind to CD40 and B7 on antigen presenting cells - those provide the signal 2. (costimulatory molecules)

Question 59

what does anergy mean?

Answer 59

t-cells see antigen but don't respond - they only see signal one but not signal 2 - they are anergized.

Question 60

When do our antigen presenting cells have B7 molecules?

Answer 60

when we have an infection

Question 61

Why do we get b7 when we have infection?

Answer 61

toll-like receptor recognizes PAMPs - transcription factor - NFkB, a lot of functions of innate immune cells (or antigen presenting cells) are made possible by activation of NFkB, toll-like receptor recognition of PAMPs leads to NFkB activation upregulates MHC molecule - normally in resting state there aren't enough MHC molecules to present antigen, B7 and CD40 are induced (not expressed on naive APC's)

Question 62

What is CTLA-4?

Answer 62

When t-cell gets activated, start to express cytotoxic t-lymphocyte associate molecule - 4 this molecule also binds to B7-1 and B7-2, when it binds it causes inhibition (shut down T-cell activation) part of anergy (peripheral tolerance)- a way to regulate t-cells

Question 63

What is the difference between CD28 and CTLA-4

Answer 63

When CD28 binds to b7, t-cell gets activated, when CTLA-4 binds to B7, the t-cell gets shut down

Question 64

What is the importance of CTLA-4

Answer 64

way of controlling t-cells so we don't have t-cell activation for a long time - especially when pathogen has already been irradiated. T-cells need to be inhibited. CTLA-4 molecule is unregulated when T-cells are already activated

Question 65

How is CTLA-4 used clinically?

Answer 65

in tumor immunity - used in clinic to boost immune response against cancer cells - when t-cells are activated, they express CTLA-4 which inhibits t-cells, in immunity against tumor, we want t-cells to be activated - especially cytotoxic t-cells that recognize the tumor antigen - so we block CTLA-4 - CTLA-4 blockade is used in clinics - inhibits anergy opposite - autoimmune diseases t-cells are over-activated, so we want to have CTLA-4

Question 66

What happens to the majority of t-cells once pathogen is eliminated?

Answer 66

they die, a few become memory cells

Question 67

What mechanism controls apoptosis of t-cells once they are activated?

Answer 67

They up regulate Fas and FasLigand - transduce signal to the cells & activates extrinsic apoptosis pathway either autocrine or paracrine fashion

Question 68

What is another molecule expressed on t-cells when they are activated other than Fas and FasL?

Answer 68

PD1 - programmed death

Question 69

how is PD1 related to cancer?

Answer 69

PD1 on t-cells will bind to PDL-1 on cancer cells a lot of cancer cells express PDL - will cause t-cell death cancer therapy, you want to inhibit interaction between PDL and PD1 so that t-cells don't die

Question 70

how is Fas related to autoimmune diseases?

Answer 70

If you delete gene for Fas or FasLigand in mice, they develop lupus. Demonstrates how important activation induced cell death mechanism is to make sure we don't develop auto-immune diseases

Question 71

What is Active Suppression by regulatory t-cells and how does it work?

Answer 71

regulatory t-cells suppress effector cells when they get signal 1 and signal 2, t-cells will proliferate. IL-2 is important growth factor for c-cell proliferation. Regulatory t-cells suppress effector t-cells several ways: act as IL-2 sync - neutralize IL-2 so effector cells won't have enough IL-2 to proliferate 2. regulatory t-cells produce immunosuppressive cytokines (most potent are IL-10 and TGF-beta & those will suppress effector t-cells.

Question 72

What are the most potent cytokines that regulatory t-cells produce?

Answer 72

TGF-beta and IL-10

Question 73

What happens if you don't have regulatory t-cells?

Answer 73

develop autoimmune diseases