Immunology Test 2

3 ways the antigen binding site is generated during B-cell development in the bone marrow

• somatic recombination

• junctional diversity

• random association of heavy and light chains

Somatic hypermutation

• further increases the affinity of the antigen binding site for its antigen

• introduces random point mutations in the variable regions after B-cell activation

• results in affinity maturation

  • antibody affinity for antigen increases over time

• requires AID (activation induced cytidine deaminase)

  • made only in activated B cells

  • converts C to U

  • U removed and replaced with another base (may not be C)

Somatic Recombination

variable region is formed from _________ ______________

• brings together a single V and J (light chain) or single V,D, and J (heavy chain)

Constant Region

is not formed from somatic recombination

• not composed of gene segments

• no rearrangement necessary; exons are ready to be transcribed

• the C region is not part of the antigen binding site

Two types of recombination signal sequences

• sequences with a 12bp spacer

• sequences with a 23bp spacer

12/23 Rule

• 12bp RSS can only associate with a 23bp RSS

• ensures that segments are joined in the correct order

V(D)J recombinase complex performs somatic recombination

• RAG-1 and RAG-2: lymphocyte specific components

• ubiquitous DNA repair proteins

Overview of sequence of events to join gene segments

1. RAG complex aligns recombination signal sequences

2. RAG complex cleaves DNA

3. broken ends are joined together in such a way that introduces random DNA sequence at the joining regions

Junctional Diversity

diversity at the junction (joining region) between gene segments

Sequence of events to join gene segments

• DNA cleavage by RAG complex leaves hairpin ends

• Hairpins cleaved in a random location (generates P nucleotides)

• TdT randomly adds nucleotides

• opposite strands pair

• gaps filled by adding complementary nucleotides

After somatic recombination of the variable region, DNA is transcribed to mRNA

for the heavy chain, Cμ (IgM) or Cδ (IgD) constant region exons are used

• determined by alternative splicing/alternative use of polyA signal sequence

Naïve (unactivated) B cells express IgM and IgD receptors on the surface

• antigen binding site is identical-only heavy chain constant region differs

Alternative splicing produces a B-cell receptor or an antibody

• a B-cell has a B-cell receptor on the surface

• a plasma cell secretes the immunoglobulin molecule as soluble antibody

• membrane-bound versus secreted immunoglobulin molecules are produced by alternative splicing to include either the MC sequence (receptor) or SC sequence (antibody)

Low affinity IgM

the first antibody to be secreted

Class Switching

is required to produce antibodies of a different class

• determined by cytokines

• antibody class determines function; changing class does not affect the antigen binding site

Sequence of events for class switching

1. transcription is induced upstream of the Cμ/Cδ switch region and the switch region of the desired class

2. AID converts Cs in switch regions to Us

3. Us are removed leaving a DNA nick in switch regions

4. DNA repair at switch regions brings desired V region next to new C region

T-cells

there are 2 broad groups of T-cells: CD4 T cells (several subsets) and CD8 T cells

T-cell receptor structure

• One α chain

• One β chain

• Each chain has a variable region (V α and V β ) and a constant region (Cα and Cβ)

One antigen binding site of T cells

• formed from variable region of ⍺ chain and variable region of β chain

• formed form 6 hypervariable regions (3 per chain) (same as B-cell receptor)

T-cell receptor ⍺ chain

• composed of V and J gene segments and constant region exons (C)

• variable region formed through somatic recombination to join VJ

T-cell receptor β chain

• composed of V,D, and J gene segments and constant region exons (C)

• variable region formed through somatic recombination to join VDJ

T cell antigen binding site is generated by:

• somatic recombination

• junctional diversity

• random association of ⍺ and β chains

• NO somatic hypermutation

T cells have either α:β receptors or γ:δ receptors, but not both

• The two types of T-cells are functionally different

• α:β T cells account for approximately 95% of the total T-cell population and are the T-cells that participate in the adaptive immune response.

• γ:δ T-cells participate in more ‘innate-like’ functions

Protein antigens

are degraded by dendritic cells and peptide fragments are presented on MHC molecules to α:β T-cells

MHC

stands for major histocompatibility complex

• presents peptide antigen on the surface of cells

MHC class I

• present antigens from intracellular pathogens present in the cytosol

• activate CD8 T cells

• all cell types except red blood cells express MHC class I to present viral antigens to CD8-T cells

MHC class II

• present antigens derived from the vesicular system

• antigens from extracellular pathogens and pathogens that replicate in vesicles

• activate CD4 T cells

• only antigen presenting cells (APCs) express MHC class II

• dendritic cells

• macrophages and B-cells can also present antigen to activated CD4 T cells in order to become activated themselves

Intracellular pathogens are present in the cytosol

• pathogen peptides are delivered to the ER

• peptides are loaded onto MHC class I in the ER

Extracellular pathogens are brought into the cell in the endosomes/vesicles

peptides are loaded onto MHC class II in vesicular

CD4

• expressed on helper T cells and regulatory T cells

• binds to MHC II

CD8

• expressed on cytotoxic T cells

• binds to MHC I

Cross presentation

• antigens taken up from outside the cell are presented on MHC class I molecules

• allows a dendritic cell to activate cytotoxic T cells when the dendritic cell is not itself infected

Dendritic cells

are professional antigen presenting cells that activate T-cells

MHC molecules

• are also referred to as human leukocyte antigens (HLA)

• are encoded by conventional genes that do not rearrange

MHC genes are located on chromosome 6

• 3 MHC class I molecules involved in antigen presentation (only A,B, and C)

• 3 MHC class II molecules involved in antigen presentation (DP, DQ, and DR)

• Each person has a total of 6 MHC class I and at least 6 MHC class II molecules involved in antigen presentation (3 of each type from mother and 3 of each type from father)

MHC class I molecule structure

• variant ⍺ chain

• invariant β2-microglobulin

MHC class II molecules structure

variant ⍺ and β chain

Diversity of MHC molecules in the human population

• most MHC (HLA) genes are polymorphic (have multiple different alleles)

• most individuals are heterozygous for the highly polymorphic and polymorphic MHC genes

  • i.e., the genes that encode the MHC molecules involved in antigen presentation

• Heterozygosity increases the range of antigens that an individual can present to the immune system during an infection

• multiple alleles in the population reduces the probability that the population will succumb to a particular pathogen

Determination of MHC Antigen Binding

• MHC polymorphism arises in sequences that bind peptide and T-cell receptor

• MHC molecules have promiscuous binding specificity

• Anchor residues determine the antigens that bind to an MHC

The T-cell receptor

is specific for both peptide and MHC molecules

B-cell development in the bone marrow

• production of immature naïve B-cells with a functional B-cell receptor

• negative selection— removes B-cells that are moderately/strongly reactive to “self” antigens, i.e., antigens that are a part of the body (central tolerance)

B-cell development in peripheral circulation (blood & secondary lymphoid tissues)

• negative selection— removes B-cells that are moderately/strongly reactive to “self” antigens, i.e., antigens that are a part of the body (peripheral tolerance)

• B-cell maturation to mature naïve B-cells

• B-cell activation if B-cell receptor binds antigen

B-cell development: production of heavy chain

• everyone has 2 copies of the heavy chain gene (one maternal and paternal)

  • DhJh joining takes place in both gene copies

  • Vh-DhJh rearrangement takes place only one chromosome at a time

• each heavy chain gene has a 1/3 chance of a productive somatic recombination

  • random addition of nucleotides at the junction during production of junctional diversity can alter the reading frame

Rearranged Heavy chains

are tested for the ability to form a pre-B-cell receptor

• is tested to see if it can associate with surrogate light chain

• a functional pre-B-cell receptor sends survival signals, and the cell moves to the next stage

• if the heavy chain from the first allele is non-functional, the second allele completes Vh to DhJh somatic recombination

• if the heavy chain from the second allele is non-functional, the cell undergoes apoptosis

Allelic Exclusion

• a cell expresses only one allele of a gene pair

• joining of Vh to DhJh takes place one chromosome at a time

  • if the first heavy chain allele produces a functional pre-B-cell receptor, then the second heavy chain is not produced

  • ensures only one type of heavy chain is expressed by a B-cell

  • ensures each B-cell makes only one receptor

Light Chain Rearrangement

is more efficient

• everyone has 4 light chain gene copies (2 kappa and 2 lambda)

• several attempts can be made at a successful somatic recombination

• only one attempt can be made on each heavy chain gene because there are no remaining D segments

Production of light chain

• somatic recombination on each k gene is attempted first

• somatic recombination on each λ chain is tried if there are no productive k chain rearrangements

• rearranged light chains are tested for ability to associate with heavy chain to form a functional IgM B-cell receptor

  • if receptor is functional, cell moves to the next stage

Negative selection of B-cells

• many immature B-cells have affinity for self-antigens

• B-cells cannot leave the bone marrow with receptors that have a high affinity for self antigens

• removes B-cells moderately/strongly reactive to self antigens— results in “self tolerance”

Self-reactive B-cells can

• try somatic recombination again using different gene segments (receptor editing)

• die by apoptosis

Receptor Editing

B-cells with a receptor that binds self antigen continue somatic recombination of the light chain gene to produce a different receptor

• B-cells are eliminated if they cannot produce a receptor that is not moderately/strongly reactive to a self-antigen

Production of mature naïve B-cells

• complete maturation in the primary lymphoid follicles in the spleen

• interact with follicular dendritic cells and receive survival signals, including BAFF cytokine

  • survival signals drive maturation

  • die by apoptosis if they don’t gain access to primary follicle

• continued survival requires regular recirculation through primary lymphoid follicles

Negative selection (peripheral tolerance)

• immature B-cells that encounter an antigen before they have had the chance to mature will undergo apoptosis or become anergic

• a mature B-cell that binds antigen requires activating signals from a T-cell in order to become activated otherwise it undergoes apoptosis

• no receptor editing outside the bone marrow

Activation of B-cells

• activated when the B-cell receptor binds its antigen in secondary lymphoid tissue

  • some B-cells differentiate immediately to plasma cells and secrete low affinity IgM

  • other B-cells form a germinal center and undergo somatic hypermutation and class switching before differentiating into a plasma cell that produces high-affinity, class-switched antibody (or memory B-cell)

B-1 cells

minor B cell subset that account for approximately 5% of human B cells

• they are activated by thymus independent antigens

most cells arise during fetal development and self-renew in the periphery

produce a constitutive supply of antibodies against common carbohydrate antigens in bacteria

• not as much receptor diversity as B-2 cells

• considered more of an innate-like immune cell

Bone Marrow

generation of T cell precursors

Thymus

• production of functional T-cell receptor

• positive selection

• negative selection (central tolerance)

Peripheral Circulation (blood and secondary lymphoid tissues)

• negative selection (peripheral tolerance)

• activation if receptor binds antigen

Somatic Recombination in double negative thymocytes

• somatic recombinations of the λ,δ, and β chains commence simultaneously

  • somatic recombination occurs one chromosome at a time for each gene

• if γ and δ successfully rearrange before β then cell becomes committed γ:δ T-cell

• If β chain successfully rearranges before both γ and δ then cell pauses somatic recombination of γ and δ (and will recommence later)

β chains

are tested for their ability to form a pre-T-cell receptor by seeing if they can associate with a surrogate alpha chain

• if the first gene copy does not produce a functional β chain after both attempts, the second gene copy undergoes somatic recombination

α chain rearrangement

eliminates the δ chain gene segments

• therefore, it is more likely that a functional α:β receptor will be the outcome rather than a γ:δ receptor

α-chains

are tested for their ability to associate with the β chain to form a T-cell receptor

• if a functional T-cell receptor is formed, somatic recombination ceases

• if a functional T-cell receptor is not formed, somatic recombination continues using remaining gene segments on the α chain

Positive Selection

• T-cells are positively selected for their ability to bind an MHC molecule

  • positive selection occurs simultaneously with alpha chain somatic recombination

• Self peptides are presented to T-cells on MHC class I and II molecules by cortical epithelial cells

  • T-cells that bind a self-peptide/MHC complex with moderate/strong affinity are signaled to survive

  • T-cells that bind self-peptide/MHC complex with low affinity or not at all continue somatic recombination of the alpha chain using remaining gene segments

  • T-cells that cannot bind a self-peptide/MHC complex after 3-4 days undergo apoptosis

Positive Selection determines

which co-receptor is expressed: CD4 or CD8

• the thymocyte becomes a committed CD4 T cell or a committed CD8 T cell

which MHC molecule a T-cell is “restricted” to

• it is restricted to recognizing the MHC molecule that positively selected it

MHC Restriction

a T-cell receptor has specificity for the MHC molecule that positively selected it during development

Negative Selection (central tolerance)

• dendritic cells, macrophages, and specialized medullary thymic epithelial cells participate in this

• thymocytes are again presented with self-peptide/MHC complexes

  • all T-cells will bind because all have been positively selected already

• T-cells that bind strongly are induced to undergo apoptosis( i.e., receptor has affinity for MHC AND self-peptide)

  • or some become regulatory T cells (Tregs)

  • no receptor editing during negative selection in T cells

• T-cells that bind moderately are released (i.e., receptor has affinity for MHC BUT NOT self-peptide)

Negative Selection (Peripheral Tolerance)

tolerance to self-antigens continues to develop in the periphery

2 major mechanisms:

• T-cells that bind self-antigen in the absence of inflammation become anergic

• regulatory T-cells

  • some T-cells that were negatively selected become natural Tregs instead of undergoing apoptosis

  • recognize self-antigen/MHC complex and suppress naïve T-cells that recognize an antigen presented by the same dendritic cell