Genetics of antibodies and Tcell development

Question 1

affinity maturation

Answer 1

process of decreases Kd

Question 2

avidity

Answer 2

measurment of the affinity of a single antigen-antibody bond

Question 3

cross-reaction

Answer 3

antibodies produced against one antigen bind to another similar antigen

Question 4

monoclonal antibodies

Answer 4

B cells that make one specific antibody

Question 5

How is it possible to have a B cell reprtoire of 10^11?

Answer 5

germ line in-rearranged Ig, in non-B cells

somatic cell- each B cell undergoes somatic hypermutation

process that occurs during pr-b cell stage

1. 12/23 rule
   
   1. RSS marks the recombination spot
2. Rag joins regions and forms the loop and cuts the loop
3. TdT adds nucleotides to fill in the gap

chains

1. light chain
   
   1. combines VJ regions
2. heavy
   
   1. combines DJ first
   2. then combines V with DJ

Question 6

label and explain regions

Answer 6

1. antibody L chain and H chain both have
   
   1. V region
      
      1. higher degree of variability in the N-terminal region
   2. C region
      
      1. limited variation
2. antigen binding site
   
   1. formed by paired V regions
   2. BCR=high affinity in Ab
      
      1. ​compared to lower affinity TCR

Question 7

list the 5 Ab, chains, regions and domains

Answer 7

heavy chain

1. C region
   
   1. alpha, delta, gamma
2. C domain-4
   
   1. mu, epsilon
   2. extra domain is an elongated hing region

light chain

1. isotypes
   
   1. kappa and lambda
   2. either is present in any individual antibody molecule
   3. no difference between the two

Question 8

define the following

1. isotype
2. allotypes
3. idiotypes

Answer 8

1. isotypes
   
   1. antibody classes, five majore
      
      1. MDGEA
   2. difference lies in the heavy chain
      
      1. Mu, delta, gamma, epsilon or alpha
   3. light chain
      
      1. kappa or lamba
2. allotypes
   
   1. represent the genetically determined differences in antibodies between people
   2. example
      
      1. you and I both hav IgG, but unless we’re closely related, my IgG’s are very slightly different than yours
      2. used in paternity testing
3. idiotypes
   
   1. antibodies of different specificity found within the same individual due to the diversity of the Ig V region

Question 9

describe multivalence, with respect to the antigen, and its role in antibody attachment

Answer 9

Question 10

what are two ways the Ab recognize epitopes?

Answer 10

l

Question 11

What confers epitope recognition and binding diversity?

Answer 11

Gentically hypervariable regions of antibodies

1. framework regions
   
   1. regions that flank the hypervariable regions, these are much less variable
2. hyper variability
   
   1. the differences in the aa sequence of the V domains between different antibody molecules are concentrated within particular locations in the gene.
      
      1. this is the V domain
3. complementarity-determining regions (CDRs)
   
   1. another name for the hypervariable loops, since they provide the binding surface that is complementary to that of the antigen

Question 12

describe the loci associated with Ig heavy vs light

Answer 12

light-two loci

1. lambda loccus-c’some22
2. kappa loccus-c’some 2

heavy

1. one chain locus-c’some 14

Question 13

start with a germ line DNA of heavy and light, and draw lines the final product after VDJ recombination. leave out lengthy mech parts

Answer 13

Question 14

several steps are involved in rearrangment/recombination. Describe the recognition site

Answer 14

RSS- recombination signal sequences

1. specal DNA motifs direct the siter of recombination in germ-line immunoglobulin DNA
2. 12/23 rule
   
   1. RSS with 12bp spacer interacts only with RSS with 23bp spacer to ensure that gene segments are joined in the correct order

Question 15

several steps are involved in rearrangment/recombination.

describe the joining step of the 12/23

Answer 15

RAG 1 and 2 are the cleaving enzyme

1. only expressed in lymphocytes (B and T cells )
2. bring the 12/23 spacers between D and J region
3. cleave the DNA

Question 16

What is the difference between combinational diversity and junctional diversity.

describe the junction components and resolving the junciton

Answer 16

combinational

1. VDJ recombinations + light/heavy chain comombinations

junctdional diversity

1. provides third CDR
2. junctional flexibility-imprecision in the site of the join between gene segments
3. P-nucleotides additions
   
   1. palindromes from loop cleavage and repair
   2. another enzyme cuts near the end of the RSS allowing nucleotides from the bottom (non-coding) strand to become part of the coding strand
4. N-nucleotide additions
   
   1. terminal deoxy-nucleotidyl transferase (TDT) adds a random number of random nucleotides

resolving the junction

1. b/c the number of disordered processes occurring, ther is a chance of generating “stop” codons orintroducing framshifts in the J region
2. some “Joins” will be non-productive
   
   1. non-coding
3. productive joins will generate increased diversity in the thir hypervariable region
   
   1. (CDR3)

Question 17

describe the 3 CDRs

Answer 17

Complementarity-determining regions/hypervariable regions

1. germ line contains varying sequences on
   
   1. CDR1 and 2
2. junctional diversity is responsible for the increase in diversity at
   
   1. CDR3

Question 18

allelic exclusion

Answer 18

1. allelic exclusion
   
   1. process of shutting off the other allele after rearrangment of 1H and L chain. IF the first recombination works
   2. principle idea of specificity
      
      1. one H + one L =one specific

Question 19

what is clonal selection?

what does it allow for?

Answer 19

clonal selection

1. B cells are monospecific and for a given antigen only a subset of V cells wil produce antibodies to that antigen
2. antigen specificity
   
   1. each b cell is specific for only one epitope of an antigen
3. antibody repertoire/B cell reperetorre
   
   1. the numbe rof different antibodies/B cells recognizeing

Question 20

describe the structures of the human Ig.

Answer 20

1. variation in the C region of heavy chain determines the Ig Isotype (anitbody classes
   
   1. IgM-mu
   2. G-gamma
   3. D-delta
   4. A-alpha
   5. E-epsioln
2. light chains have kappa or lambda, no difference in known

Question 21

each b cell is specific for only one epitope, is an example of ?

Answer 21

antigen specificity

Question 22

somatic recombination

Answer 22

aka: VDJ recombination

the process of DNA recombination by whic hthe functional genes encoding the variable regions of antigen receptors are formed during lymphocyte development.

1. germline
   
   1. limited
2. somatic recombination
   
   1. process occurs in developing B and T cells
   2. mediated by
      
      1. RAg 1 and RAG-2
         2.

Question 23

describe the ways B cells generate such diversity in the antibodies

Answer 23

1. germ line
   
   1. hyper variable regions (CDR1&2)
2. somatic recombination-gene rearrangment
   
   1. VDJ recombination
      
      1. 1.6x10^6 combinations
   2. junctional diversity (CDR3)
3. mRNA splicing-isotype
4. after Ag exposure
   
   1. somatic hypermutation
   2. AID assisted isotype switching

Question 24

Which type of Ig do naive B cells express? How?

Answer 24

naive B cells express both suface IgM and IgD

expression of two antibody isotypes simultaneously is acomplished by alternative mRNA splincing

Question 25

discuss the process of changing between secretory and membrane bound Ig

Answer 25

Alternative splicing produces surface Ig and secreted Ig

1. the sIgM and the secreted IgM have the same specificity and differ only in that one is membane bound and another is secreted.

Question 26

What happens to the antiboy change in the primary vs secondary immune response. What is the process for this event?

Answer 26

the IgG in late primary and secondary responses against an Ag has the same Ag specificity as the IgM in the primary response (and the same heavy chain VDJ and light chain VJ gene segment)

recombination event only at the constant regions.

Question 27

after 2 weeks the Ig in the body increase their affinity and diversity. What is the cause of this? why and how?

Answer 27

Somatic hypermutation

Affinity maturation

1. selection occurs for V regions with higher affinity
   
   1. introduction of point mutation in the V region alters Ab affinity for Ag
   2. ONLY V REGION is targeted, not the C region or other B cell genes.
2. The CDR regions (1-3)will show higher diversity

Question 28

talk about the changes in a B cells during its life

1. V region assembly from gene fragments
2. generation of junctional diversity
3. assembly of transcriptional controlling elements
4. trasciption activated with coexpression of surface IgM and IgD
5. synthesis changes from membrane Ig to sIg
6. somatic hypermutation
7. isotype switch

Answer 28

Question 29

compare TCR to BCR

1. bound types
2. receptor and effector?
3. changes in Ig?

Answer 29

TCR has 3CDRs along with gene rearrangments to generate extensive variability

Question 30

diagram and discuss the TCR gene rearrangments

Which Csomes, and what is different/same about each?

Answer 30

Question 31

What manifests from a mutation in the of the protein that cleaves and develops thae hair pin structure in junctional combination?

Answer 31

RAG1/2 mutations lead to

1. SCID
   
   1. decrease in B and T cells
2. Omen syndrome
   
   1. inflammation from auto-reactive T cells
3. treatment
   
   1. bone marrow transplant- to restore immune system

Question 32

what are the two classes of T-cell receptor?

Answer 32

ab- default and more numerous

1. yd
   
   1. 1-5% of circulating T cells
   2. locatin
      
      1. gut mucosa
      2. epithelial
      3. skin epidermis
   3. structure
      
      1. fewer v segments
      2. restricted TCR may act as a PRR
   4. function
      
      1. may be danger signal
      2. nteract with non-classical MHC1
         
         1. CD1 receptors

Question 33

what are the two major subpopulation of T lymphocytes?

Answer 33

T helper- secretes cytokines

CTL- kills virus infected cells, tumor cells

Question 34

What happens to the thymus as you age?

describe regions of the thymus

Answer 34

The thymus begins to atrophy and become substituted with fat after the age of 3.

As a youngster you need to mature many T cells which prepare you for the world.

Question 35

descreibe the T-lymphocyte development maturation and selection, with respect to anatomic site

Answer 35

Question 36

Describe the generation of TCR comple from the proT - pre T

Answer 36

1. once in the thymus
   
   1. Pro-t (double negative)
      
      1. changes CD32 to CD2
         
         1. commit to double negative T cell progenitor
      2. somatic recombination for B chain
         
         1. VDJ recombination at beta-chain locus
            
            1. uses rag1/2 along with RSS:12/23 rule
            2. P-nucleotide addition
            3. N-nucleotide addition by TdT
         2. has two attempts
         3. failure may lead to gamma/delta T cell or death
      3. B-chain is expressed on surface with pre-Talpha (invariant chain)
   2. Pre-T
      
      1. rearrangment of the alpha-Gene
         
         1. VJ segments only (no D segment)
         2. delta locus is cleaved out, rearrangment leaves the cell in a comitted process to alph.beta or death.
   3. double positive
      
      1. the sucessful somatic recombination of the beta then alpha gene leads to the TCR receptor expression
      2. the expression of CD4 and CD8 on the surface
   4. from here the Tcells go through
      
      1. positive selection-in the cortex
      2. negative selection-in the medulla

Question 37

list the steps of maturation of a stem cell to naive T cell, brefly with emphasis on the location, purpose and outcomes of the selection process.

What protects us from our own Tcells?

Answer 37

1. Bone marrow
   
   1. stem cell
2. thymus
   
   1. cortex
      
      1. pro
      2. Pre
      3. double posistive-positive selection
   2. medulla
      
      1. single positive-negative selection

selection

1. cortex
   
   1. positive selection
      
      1. immature T cell (thymocytes) express both CD4/CD8 are selected on their low avidity.
      2. receive a signal from the thymic epithelial cell to continue to the medulla.
         
         1. they need to be able to recognize the peptides on the APC in the thymus.
         2. the narrow activation threshold for low-moderate TCR affinity to thymocyte TCR allows thymocytes to escape death.
            
            1. ​the Tcells can be killed by
               
               1. apoptosis
               2. neglect
      3. converted into single-positive cell
         
         1. cells will have a”preference” of CD4 or CD8 and the opposite CD gene is repressed and the cell moves to the medulla. The CD preference is usually from whichever MHC they bind to first.
   2. medulla
      
      1. negative selection
         
         1. high affinity/avidity recognition of self peptide-MHC complexes in the medulla of the thymus.
            
            1. AIRE
               
               1. ​**a transcription factor which induces expression of ~ 400 different tissue-specific (self) genes in medullary thymic epithelial cells (**mTEC)
      2. deletion of self recognizing T cells develops
         
         1. central tolerance
         2. protects against autoimmunity
      3. T cells are killed by
         
         1. death

more than 90% of immature T lymphocytes die during maturation/selection process

Question 39

A tcells meets its cognate antigen in the thymus, what is the fate of this cell?

Answer 39

The T cell will die in positive selection or negative selection.