Immunology Exam 2

TCR

antigen-binding molecule expressed on the surface of T cells and associated with the CD3 molecule - are heterodimeric, consisting of either and alpha beta (glycosylated) or gamma delta (not glycosylated) chain

mature, naive B cell

mature B cell that has not encountered antigen

non-templated (N) nucleotide addition

mediated by TdT activity, adding in random nucleotides between joints

peptide requirements for MHC class II

1. 13-18 aa in length but up to 30 2. binding is mediated by hydrogen binding between the peptide backbone and the side chains of certain MHC aas (hang over ends like hot dog in bun)

peptide requirements for MHC class I

1. 8-10 aa long - 9 mer (nonamer) is most common 2. conserved anchor residues at N terminus and C terminus of the peptide 3. "closed ends", middle bulges up

affinity maturation

The increase in average antibody affinity for an antigen that occurs during the course of an immune response or in subsequent exposures to the same antigen.

At what level does V(D)J recombination occur? (DNA or RNA?)

- occurs at the DNA level

foreign peptide in MHC I will result in?

lysis by CD8+ T cells

what is LIG4 syndrome?

- loss of DNA ligase IV expression - low numbers of T and B cells

RAG 1/2 deficiency would lead to?

- loss of RAG protein expression - no T or B cells but normal NK and myeloid cells

which of the 5 mechanisms that generate antibody diversity fall under junctional diversity?

- P nucleotide addition - exonuclease trimming - N-nucleotide addition

12/23 rule

- The spacing and arrangement dictates that a 12 bp RSS must pair with a 23 bp RSS for recombination to occur - The RSS is comprised of two blocks of conserved sequences: a heptamer and a nonamer that have either a 12bp or 23bp spacer. When recombining V(D)J segments, only a segment containing a heptamer-nonomer RSS with a 12bp spacer can recombine with a heptamer-nonomer RSS with a 23bp spacer.

At what level does V exon to C exon joining occur? (DNA or RNA)

- at the RNA level - via RNA splicing

how was the gamma chain discovered?

- by accident while trying to fine the alpha chain - using subtractive hybridization - realized sequence had no glycosylation

name the different genes in the MHC loci for humans

- class 1: HLA-A,-B,-C (encode the alpha chains) - class II: HLA-DP,-DQ,-DR (encodes the alpha and beta chains) - class III: complement and TNF

name the different genes in the MHC loci for mice

- class I: H2-K, -D, -L (encode alpha chains) - class II: H2-A and H2-E (encode alpha and beta chains) - class III: complement and TNF

what are the non-classical molecules in MHC loci of mice?

- class I: H2-Q, H2-T, and H2-M -class II: O and M

what is the function of non-classical molecules in MHC loci?

- class I: expressed only on certain cell types and plays a role in self/non-self discrimination

multiple gene segments

- creates lymphocyte receptor diversity

Davis-Hendrick Group

- discovered T cell receptor genes - discovering the various chains

how is TCR recombination different/similar to BCR?

- essentially the same - D-J recombination occurs in beta-chian of the TCR first - followed by V-DJ recombination - primary transcript is synthesized that incorporates the constant domain gene - mRNA transcription splices out any intervening sequence and allows translation of the full length protein for the TCR beta-chain - the rearrangment of the alpha chain of TCR follows beta-chain rearrangment and resembles V-J recombination like the light chain - the assembly of beta and alpha chains results in the formation of the alphabeta-TCR that is expressed on most T cells

germ-line theory

- every B lymphocyte has all the genes for every type of immunoglobulin but transcribes only one - 1 gene = 1 protein

how was delta chain discovered?

- found within the same locus of the alpha chain - found that expression of alpha chain led to deletion of delta chain

differences between alpha/beta, gamma/delta T cells and NKT cells

- gamma/delta T cells arise first in fetal development (thymic) and provide protection prior to birth, are small percentage of Tcells in humans, located in mucosal tissues (join innate immune cells), limited variability, recognize lipid and glycolipid antigens - most cells are alpha beta T cells with TCR composed of two glycoprotein chains while gamma delta T cells have a TCR that is made up of one gamma and one delta chain - NKT cells are the smallest percentage of T cells. coexpress alpha/beta t cell receptor. recognize lipids and glycolipids presented by CD1d molecules rather than MHCs

similarities and differences between BCR light and heavy chain rearrangements

- heavy chains are recombined (D-J followed by V-DJ) and expressed first - unspliced RNA is generated with the newly combined VDJ segments and constant regions (mu and delta) - processing of this new RNA forms mRNA that is translated to produce IgM heavy chain proteins - signaling through the pre-BCR results in inhibition of heavy chain recombination and the onset of light chain recombination - kappa and lambda or the light chain rearrange (same as heavy but lacks D segment - meaning V and J combine to form VJ complex before adding constant chain) - translation of spliced mRNA for either the kappa or lambda chains results in formation of IgK or Iglambda - heavy chain is partially recombined in the lymphoid cell and finished in pro-B cell -light chain recombination occurs in pre-B cell - assembly of heavy and light chain results in formation of membrane bound form of IgM that is expressed on an immature B cell

Hozumi and Tonegawa

- important contributors to BCR - testing that a piece of DNA can change its position on chromosome - paradigm shifting

how was the alpha chain discovered?

- isolation of genomic sequence expressed on T cells only and encoded protein glycosylation sites and molecular weight

J segment

- joining - part of rearranged immunoglobulin or T-cell receptor gene that joins the variable region to the constant region

what is SCIDA?

- lack Artemis expression - lack T and B cells

MHC Class I

- on all nucleated cells - present cytosolic antigens to CD8+ T cells - alpha chain: 3 external domains, transmembrane domain, and cytoplasmic tail - beta2-microglobulin: non-covalently bound to MHC class I - peptide binding cleft between alpha1 and alpha2

What is Omenn syndrome?

- partially active RAG1 and RAG2 -leads to lack of B cells - T cells are autoreactive (attack own body)

peptide bind groove of MHC I vs II

- peptides that can bind to class I are different than peptides than can bind to class II - both exhibit promiscuous binding or broad specificity - a given MHC can bind numerous different peptides and some peptides can bind to more than 1 MHC

polyA (adenylation) site

- polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. - poly(A) tail to a messenger RNA

MHC class II

- present on antigen presenting cells (APCs) - present extracellular antigens to CD4+ T cells - alpha chain: two external domains, transmembrane domain and cytoplasmic tail - beta chain: two external domains, transmembrane domain and cytoplasmic tail - peptide binding cleft between alpha1 and beta1

class I MHC peptide interactions

- present peptides to CD8+ T cells - peptides derived from endogenous intracellular proteins - some amino acids anchor the peptide into the groove - other amino acids are available to interact with a TCR - anchor residues at both ends of peptide, generally hydrophobic at carboxyl-terminus

somatic hypermutation theory

- programmed process of mutation affecting the variable regions of immunoglobulin genes - limited number of genes encode V region, but there is some unknown mutational mechanism

SCID

- severe combined immunodeficiency - a genetic defect in which adaptive immune responses do not occur, due to lack of T cells and possible B and NK cells

what is SID?

- severe combined immunodeficiency (primary) - X-linked SCID express IL2RG gene which encodes the interleukin receptor common gamma chain - lack cytokine signaling so produce very few T cells which leads to loss of B cell and NK cell function - Ig replacement therapy is a passive immunity to help

TdT

- terminal deoxynucleotidyl transferase - enzyme that adds untemplated nucleotides at the V-D and D-J junctions of the B and T cells receptor genes

how was the beta chain gene discovered?

- using subtractive hybridization

Class II MHC-peptide interactions

- usually derived from exogenous extracellular processed antigens - present antigen peptides to CD4+ T cells (appear to lack conserved anchor amino acid residues) - conserved residues distributed along the length of the peptide anchor

what are the non-classical molecules in MHC loci of humans?

-class I: HLA-E, HLA-F, HLA-G -class II: DM and DO

what are the 5 mechanisms that generate antibody diversity?

1. Multiple gene segments 2. P nucleotide addition 3. exonuclease trimming 4. N-nucleotide addition 5. Combinatorial diversity

what are the 3 mechanisms for junctional diversity?

1. P nucleotide addition = palindromic sequences added by DNA repair enzymes 2. exonuclease trimming = occurs mainly in heavy chain 3. N nucleotide addition = non-template encoded nucleotides added by TdT occurs only in heavy chain

what are the mains steps of V(D)J recombination?

1. binding of RAG1/2 to RSS 2. synapsis between gene segments 3. cleavage of signal and coding joints (RAG1/2 and HMG proteins) 4. generation of functional variable region gene (NHEJ proteins, DNA-dependent protein kinase, Artemiz, DNA ligase IV, and TdT)

extra V(D)J recombination steps on heavy chains

1. exonuclease - cleavage of coding nucleotides at coding joint 2. Tdt- terminal deoxynucleotidyl transferase adds non-templated nucleotides 3. ligation

what are the 5 differences between TCR and BCR expression?

1. secretion of product. This only occurs in BCR (Ab) 2. constant regions. C region of Ab determines its function while C region of TCR anchors receptor to membrane and connects to signal transduction 3. multiple D segments in recombined chains. TCR delta chains 4. allelic exclusion. absolute in BCR but not in TCR alpha chain 5. somatic hypermutation. occurs in B cells after antigenic stimulation and does not occur in T cells

DNA ligase

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.

allelic exclusion

A process that permits expression of only one of the allelic forms of a gene. For example, a B cell expresses only one allele for an antibody heavy chain and one allele for a light chain

(See screen shot image) Where does RAG-1/2 cut the dsDNA depicted in the figure? A. Between VL and the 7 bp regions B. Between the 7 bp and 12 bp regions C. Between the 12 bp and 9 bp regions D. Within the 12 bp region E. RAG-1/2 does not cut dsDNA

A. Between VL and the 7 bp regions

Regions of TCRs and BCRs that are particularly variable in their amino acid sequences are known for: A. Binding to antigen B. Containing the ITAM domains C. Spanning the plasma membrane D. Binding to adaptor proteins in the cytoplasm E. Becoming phosphorylated

A. Binding to antigen

Which of the following contributes to antibody diversity in B cells after antigenic stimulation? A. Somatic hypermutation B. Random pairing of heavy-light immunoglobulin chains C. P & N nucleotide addition D. Exonuclease trimming E. Multiple V, D & J segments in the germline

A. Somatic hypermutation

N nucleotide addition requires which of the following enzymes: A. Terminal dideoxytransferase (TdT) B. DNAligase C. DNA repair enzyme D. DNA-PK E. RAG-1 & RAG-2

A. Terminal dideoxytransferase (TdT)

the organization of the alpha locus and delta locus helps to ensure that each T cell cannot express both types of T cell receptors. The mechanism involved is: A. the rearrangement of T cell receptor alpha gene deletes the delta locus on that allele B. the rearrangement of T cell receptor delta gene deletes the alpha locus on that allele C. The RAG recombinase enzymes are down-regulated immediately after the first T cell receptor genes rearrange D. The alphabeta T cell receptor signals the T cell to delete the delta locus E. The gamma delta T cell receptor signals the T cell to delete the alpha locus

A. the rearrangement of T cell receptor alpha gene deletes the delta locus on that allele

variable region

Amino-terminal portions of immunoglobulin and T-cell receptor chains that are highly variable and responsible for the antigenic specificity of these molecules.

Nonhomologous end joining (NHEJ)

An error-prone mechanism of double-stranded DNA break repair in eukaryotic genomes in which damaged nucleotides are removed and blunt ends of strands are joined.

What cleaves interstrand hairpin in V(D)J coding regions? A. TdT B. Artemis C. RAG-1 & RAG-2 D. DNA ligase IV E. Ku 70 and Ku 80

B. Artemis

If an animal were deficient in RAG-1 and RAG-2 which of the following processes would be affected? A. Defects would occur in addition of nucleotides during V(D)J gene rearrangement leading to a decrease in diversity of antibody molecule B. Defects would occur in V(D)J gene rearrangement leading to no (or negligible) numbers of T or B cells C. Defects would occur in isotype switchingD. Defects would occur in production of secreted vs. membrane immunoglobulin E. None of the above

B. Defects would occur in V(D)J gene rearrangement leading to no (or negligible) numbers of T or B cells

5. Which of the following is capable of undergoing successive rearrangements to prevent the formation of autoreactive B lymphocyte receptors in a process called receptor editing? A. IgHδ B. IgLλ C. IgHμ D. Both A and B E. all the above

B. IgLλ

(See screenshot image) The indicated items are found in an Ig light chain (IgL) gene. Which of the labeled elements serves as a spacer? A. VL B. 7 bp C. 12 bp D. 9 bp E. None of the above

C. 12 bp

combinatorial diversity

BCRs and TCRs generate this type of diversity from the rearrangement of one heavy chain with different light chains or one light chain with different heavy chains.

haplotype

Because the loci for MHC alleles are located so close in proximity to each other, they are linked and typically inherited together and the set of alleles is known as a

What introduces a transient hairpin that links the two strands in the V(D)J coding regions? A. TdT B. Artemis C. RAG-1 & RAG-2 D. DNA ligase IV E. Ku 70 and Ku 80

C. RAG-1 & RAG-2

Which of the following does NOT contribute to the generation of diversity (i.e. changes in the antigen binding region) of B cell antigen receptors? A. Multiple V genes in the germline B. Combinatorial diversity generated from light and heavy chains C. P & N nucleotide addition D. Multiple C-region genes E. Exonuclease trimming

D. Multiple C-region genes

Which of the following TCR gene variable regions possesses a "D" segment? A. β B. δ C. γ D. α E. Both A and B

E. Both A and B

(See screen shot image) What length must the RSS spacer be to link the above V element to its appropriate D segment? A. 7 bp B. 23 bp C. 9 bp D. 12 bp E. None of the above

E. None of the above

light chain

Immunoglobulin polypeptides of the lambda or kappa type that join with heavy-chain polypeptides to form the antibody heterodimer.

codominance of mIgM and mIgD

Mature naive B express both mIgM and mIgD.

D segment

Only present in heavy chain and TCR β chain

production of mIgD/mIgM occurs at what level? (DNA or RNA)

RNA level - alternate RNA splicing

activated B cell

Type of B cell that begins antibody production after differentiating

MHC II expressed by APCs present extracellular antigen to ___ cells

Th

constant region

The nearly invariant portion of the immunoglobulin molecule that does not contain antigen-binding domains; The sequence of amino acids in the constant region determines the isotype of heavy chains and the type of light chains

RAG 1/2

The protein complex of RAG1 and RAG2 that catalyzes V(D)J recombination of B- and T-cell receptor genes. These proteins operate in association with a number of other enzymes to bring about the process of recombination, but RAG1/2, along with TdT, represent the lymphoid-specific components of the overall enzyme complex.

alternative splicing

This event takes place following the addition of polyA sites on the mRNA primary transcript of BCRs and determines whether that transcript will produce membrane or soluble IgM or IgD

subtractive hybridization

This is a fine tune search for unique genes. cDNA libraries contain many housekeeping genes (non-unique genes that carry out normal functions for all cell types) and subtractive hybridization will remove these genes and enrich unique mRNA pool. DNA from two cultures are allowed to hybridize and the unique cDNA will pass through the filter while the housekeeping genes will not.

exonuclease

enzyme that cleaves off nucleotides, one at a time, from the end of the RNA

MHC II activation to kill extracellular pathogens occurs via?

activation of B cells

artemis

an enzyme that is a member of the non-homologous end joining (NHEJ) DNA repair pathway. During V(D)J recombination, it opens the hairpin loops formed after RAG1/2-mediate cleavage of the immunoglobulin genes

difference between germ-line and somatic hypermutation theories

both proposed theories for immunoglobulin gene structure diversity - germ-line: every B lymphocyte has all the genes for every type of immunoglobulin but transcribes only one. so 1 gene = 1 protein - programmed process of mutation affecting the variable regions of immunoglobulin genes.

BCR

complex comprising a membrane-bound immunoglobulin molecule and two associate signal-transducing Igalpha/Igbeta molecules

MHC I present antigens from ______ locations

intracellular

glycosylated

descriptive of molecules to which sugar groups have been added

Junctional diversity

diversity in antibody and T-cell receptor genes created by the imprecise joining of coding sequences during the assembly of the rearranged genes

immature B cell

express a fully-formed IgM receptor on their cell surface. Contact with antigen at this stage of B cell development results in tolerance induction rather than activation. - express lower levels of IgD and higher levels of IgM than do mature B cells - they also have lower levels of anti-apoptotic molecules and higher levels of Fas than mature B cells, reflective of their short-half lives

MHC II present antigens from _______ spaces

extraellular

syngeneic

genetically identical at all loci

congeneic

genetically identical except at a single genetic region

presence of self peptides in MHC I indicates?

healthy cell

recombination sequence signals (RSS)

highly conserved heptamer and nonamer nucleotide sequences that serve as signals for the gene rearrangement precess and flank each germ-line V, D, and J segment

MHC loci

in humans = HLA complex in mice = H-2 complex

where do gene rearrangements occur?

primary lymphoid organs (bone marrow for B cells and thymus for T cells)

V segment

the 5' coding portion of rearranged immunoglobulin and T-cell receptor genes - there are multiple V gene segments in germ-line DNA, but gene rearrangment leaves only one segment in each functional gene

C terminal

the end amino acid in a peptide chain with a free carboxyl group

N terminal

the end amino acid in a peptide with a free amino group.

heavy chain

the larger polypeptide of an antibody molecule - comprised of one variable domain (V) and three or four constant domains. there are five major classes in humans which determine isotype of an antibody

surrogate light chain

the polypeptides Vpre-B and lambda5 that associated with u heavy chains during the pre-B-cell stage of B-cell development to form the pre-B-cell receptor

absence of self peptides in MHC I or lack of MHC I indicates?

virus infected or tumor cell killing by NK cells

what is non-productive gene rearrangement?

when VJ or VDJ creates a stop codon or frameshift mutation resulting in a non-functional protein