Lecture Notes

B Cell Development & Activation (3/4) - Wk 5

Case Study

• Complement system and kinins

• Reading up to the case was harder-

B Cell Development and Activation

• Immunoglobulin gene rearrangements

• Negative selection and central tolerance

• somatic hypermutation

• antibody isotypes and class switching

• development of plasma and memory B cell

• T-B Cell interactions

• B cell migration

• Intracellular B Cell signaling

• B Cell (AKA B Lymphocytes)

  • Develop and mature inside the bone marrow

  • Responsible for Humoral Immunity

  • Have B-Cell receptors (BCR) (antibodies stuck to the surface of a cell) that are specific for antigens

  • Make and secrete antibodies - ONLY CELL THAT DOES THIS

  • Name from bursa from chicken

Genetic Recombination

• How do you get such a large diversity of receptors on the surface of lymphocytes?

  • A human can generate trillions of different B cell receptors (which are proteins)

  • A human only has ~21,000 protein-coding genes

• Genetic recombination is responsible for the large repertoire of B cell receptors an individual person has

  • Repertoire - all the different lymphocyte receptors a single individual has in their body

• Why so many BCRs?

  • Recognize as many antigens as possible

B-Cell Development

• The process if B cell development occurs continuously throughout your life

• Stem cell = hematopoietic stem cell (HSC, found in Bone Marrow)

• Development of B Cells from HSC to Mature B cell occurs in the Bone Marrow

• Pro B Cell

  • earliest recognizable

  • No longer a HSC

  • also called B progenitor cells

  • Initial genetic recombination occurs at this stage

Genetic Recombination in B Cells

• B cells experience genetic recombination in order to produce a unique B Cell receptor, or antibody = more BCRs = more antibodies; THEY ARE THE SAME THING

• BCR (antibody) structure

  • Composed of 4 proteins

    • 2 identical heavy chains - bottom portion

    • 2 identical light chains - "top arm portion”

  • Chains are held together with disulfide bonds

• FC = constant fragment - “main body”

  • This region is the same for all BCRs of a particular isotype

• Fab = antigen binding fragment - the “arms”

  • F = fragment ab = antigen binding

    • This is what is different on each newly developed B cell

• All 4 chains have a unique region

  • These are proteins which are encoded by genes

B Cell Receptor Structure

• One region encodes for heavy chains in humans

  • H chain locus (specific region) on chromosome 14

• Two different gene regions encode for light chains in humans

  • kappa locus on Chromosome 2

  • lambda locus on Chromosome 22

B Cell Development

• During Pro B Cell

  • during the Pro B cell stage, the H chain locus begins to undergo recombination

Genetic Recombination

• Top portion - Germline

  • Each H chain locus is made up of

    • ~50 versions of V_H gene (V = variable)

    • ~20 versions of D_H gene (D = diversity)

    • ^ versions of J_H gene (J = joining)

    • 9 options for a C region (C = constant)

  • The VDJ genes will recombine to make the variable region of the heavy chain (composing Fab)

  • One C region gene will be selected to make the constant region of the heavy chain (composing the Fc) determining the isotype

• In a pro B cell, one D and J region are first cut out and recombined

• Important enzymes

  • VDJ recombinase

  • RAG 1

  • RAG 2

• In a pre B cell, a second rearrangement occurs in H chain Locus

• One V gene is recombined with DJ recombination made in the pro B stage

• VDJ unit is transcribed along with the first C genes: mu and delta

• Notice that there are two J gene options and 2 C gene options

• The primary RNA transcript is alternatively spliced so that mature mRNA ends up with

  • 1 V, 1 D, 1 J, 1 C

• These mature mRNA are translated to produce heavy chain proteins with:

  • A unique combination of VDJ to make up the variable region

  • Either C(mu) or C(delta) to make up the constant region

  • BCRs with C(mu) are known as IgM isotype and C (delta) are IgD isotype

• This is an error prone process!

  • If the H chain rearrangement does not work (does not produce a viable protein product)

    • The cell will try again using H chain locus on the matching homologous chromosome

  • If the H chain rearrangement try 2 does not work:

    • The pre B cell will undergo apoptosis (programmed cell death)

• If the H chain rearrangement is productive

  • The pre B cell sends the heavy chains to the cell surface forming a pre B cell receptor

• Once the pre BCR is on the surface of the cell, intracellular signaling occurs from the pre-BCR and tells the cell to rearrange its light chain genes

• Light chain recombination is very similar to heavy chain recombination

• Only consist of V and J (no D)

• Only one option for C region

• Cell tries to rearrange kappa locus first

  • if rearrangement is productive, stops there

• Cell tries. to rearrange kappa locus first

  • if second kappa locus try is not productive, tries to rearragne the genes on the lambda loci

• If no productive rearrangements are made from any kappa loci or lambda loci, the cell will die

• If productive rearrangements are made

  • light chains are sent to the surface

  • from. afunctional BCR

  • leads to the Immature B cell stage

B Cell Activation II (3/6-3/10) - Wk 5 & 6

Immunoglobulin Gene Rearrangements

• Takeaway: The genome of developing B cell is rearranged to form novel combinations of DNA to produce heavy and light protein chains to form antibodies/BCRs

Negative Selection and Central Tolerance

• The immunoglobulin gene rearrangements are random

  • There is nothing that stops the production of a BCR that can recognize something belonging to the self

    • How does the immune system solve the problem?

      • Negative Selection

• Negative selection - The elimination of self-reactive B cells during development

• Before leaving the bone marrow, the immature B cell is exposed to self-antigens from the stroma of the bone marrow

• If an immature B cell BCR binds to a self-antigen in the bone marrow

  • Receptor editing OR

  • Apoptosis

• Receptor Editing

  • BCR + self-antigen interaction in the bone marrow reactivates VDJ recombinase

  • V and J genes that were not deleted in the first rearrangement can. be edited out so that the cell can try again to make a different receptor

  • Originally in this example, V1 and J5 were not edited out of the genome

  • They either were not transcribe (V1) or alternatively spliced out (J5)

  • They reactivated VDJ recombinase can edit V2 and/or J4 out of the genomes to try make a different light chain

  • If receptor editing does not work OR

  • receptor editing produces another , different self-reactive BCR

  • then the cell undergoes apoptosis

• Negative selection leads to deletion of self-reactive immature B cells in the bone marrow

• This contributes to the development of central tolerance

  • The self tolerance that is developed in the primary lymphoid organs

  • peripheral tolerance later!

• Immature B cells that survive negative selection leave the bone marrow and travel to the spleen

• Once in the spleen, they become fully mature, naive B cells (hasn’t seen antigen yet)

B Cell Activation and Somatic Hypermutation

• A B cells job is to become activated when it sees its appropriate antigen

• Once activated it should

  • Clonally expand (make a bunch of copies of itself)

  • Differentiate into either

    • A plasma cell (to pump out antibodies for a short amount of time)

    • A memory cell (long-lived, non-proliferating cells that live in tissues and wait for future antigen exposure)

• A mature B cell migrates btw secondary lymphoid organs

  • spleen, lymph nodes, tonsils,

• Antigens arrive in secondary lymphoid organs

  • lymph nodes drain all body tissues, including foreign antigens

  • APCs bring processed antigen from anywhere in the body to secondary lymphoid organs

• Some antigens require T-cell help to fully activate their B cells

  • These are called Thymus Dependent (TD) antigens

    • If someone doesn’t have a thymus, they won’t respond to TD antigens bc they won’t have mature T cells to provide help to B cells

• B Cell Activation by TD antigens

  • B cells and CD4+ T cell that both recognize the same antigen are required

    • They can recognize different epitopes (parts) of the same antigen

  • These are cognate B and T cells

    • They recognize the same antigen

  • B cells spend time in the follicle area of secondary lymphoid organs

  • If a B cell recognizes a TD antigen in the follicle, its BCR signaling will cause it to move toward the T cell region of that organ

  • Simultaneously, a T cell in the same organ has recognized the same antigen (multiple copies of the antigen are present simultaneously) and its TCR signaling tells it to move toward the follicle

B Cell Activation

• B Cell activation by TD antigens - THE B CELL IS NOT ALWAYS PRODUCING ANTIBODIES

  • The B cell presents the antigen T_helper Cell

  • The cognate B and T cell interact for several hours

  • The B cell presents the antigen to the T_helper Cell

  • The cognate B and T cell interact

  • This interaction causes some proliferation of B cells

  • Some of these cells are short-lived plasma cells that start making antibody of the IgM isotype

  • The cognate T and B cells develop a germinal center in the follicle ~48 hours after they see each other

  • In the Germinal Center

    • Somatic Hypermutation

    • Isotype Switching

B Cell Activation and Somatic Hypermutation

• B Cell Activation by TD antigens

  • In the Germinal Center, B cells begin proliferating in what is called the ‘dark zone’

  • These B cells turn on expression of any enzyme called AID

  • AID is important in Somatic Hypermutation

• Somatic Hypermutation

  • AID = Activation Induced Cytidine Deaminase

  • AID deaminates (removes an amine group from) Cytosine, converting it to Uracil, specifically in the immunoglobulin gene loci

  • When the DNA replicates next, the Uracil base is complementary base paired with A instead of the G that would have been present if AID hadn’t acted

  • This generates mutations at 100,000 times the expected rate in immunoglobulin gene loci

  • The mutant offspring cells have BCRs that are altered by a few random amino acids bc of these mutations

  • These altered BCRs may be better, worse, or the same at recognizing their antigen

  • The pool of B cells with slightly altered BCRs migrate to the light zone of the germinal center

  • They have a competition to try to recognize their antigen, which is presented by T_helper and follicular DCs

  • There is a limited amount of antigen

  • Only the best BCRs with the highest affinity will get it

  • B cells with BCRs that don’t manage to compete for antigen will die

  • Macrophages will eat up and dispose of dead B cells

  • The B cells that leave this process are better at binding their antigen than the original B cells

  • This is called affinity maturation and is due to somatic hypermutation

B Cell Activation and

• B cell activation by TD antigens

  • In the Germinal Center

    • Somatic Hypermutation

    • Isotype switching

Antibody Isotypes

• there are 5 different classes of heavy chains

• Each class is known as an isotype

  • IgG, IgM, IgA, IgD, IgE

  • IgM and IgD are the most common

• There are 5 different classes of heavy chains

• each class is known as an isotype

• Each isotype has different properties

• IgG: Most prevalent isotype in serum, critical roles in pathogen defense

• IgM: First isotype produced after B cell activation

• IgA: isotype especially important in secretions (including breastmilk)

• IgD: Mystery isotype - what is its function?

• IgE: Isotype especially important in responses to parasites, worms, and during allergies

Isotype Switching

• Recombination of heavy chain initially does not recombine the constant region genes that determine isotype

• IgM and IgD are produced initially bc they are the first TWO constant region genes next to the VDJ region

• Therefore, mature naive B cells produce IgM and/or IgD BCRs and antibodies

• In the Germinal Center after a B cell has interacted with…:

  • its antigen AND

  • its cognate T cell

• …it will undergo istoype switching

• Isotype switching rearranges the H chain DNA to choose a different C gene for transcription and translation with VDJ unit

• The VDJ unit does not change!

• Allows plasma cells to make different isotypes of antibody with the same specificity

• Which C gene is ‘switched’ to will depend o the cytokines in the environment

B Cell Development & Activation III (3/11) - Wk 6

Effector Function

• In the Germinal Center after a B cell has undergone (found in 2ndary lymphoid organs)

  • Somatic Hypermutation

  • Isotype Switching

• it is ready to become either a Plasma Cell or a memory cell

• The plasma cells that are produced from a germinal center

  • Are much longer lived than the ones produced before the germinal center

  • Migrate to lymphoid organs (especially bone marrow) to produce antibody

• The memory B cells that are produced from germinal center

  • are very long lived and non proliferating

  • Migrate into various body tissues

  • Reactivate to produce more plasma cells upon second antigen exposure without germinal center formation

B Cell Migration

• How do all these B cells get to where they are going

  • They use the leukocyte homing system

• Important molecules in leukocyte homing

  • chemokine receptors

  • integrins

  • selectins

• All produced on the surface of the B cell

• Chemokine receptors

  • Allow B cells to sense and respond to chemical gradients in the body

    • Respiratory burst, histamines, etc.

• Integrins & Selectins

  • These molecules adhere to other molecules expressed in various tissue types

  • B cells alter which types of these are expressed based on development, and this helps them stick to different places

Intracellular B-Cell Signaling

• Intracellular B cell signaling: What happens inside a cell when its BCR binds its antigen?

• BCRs do not function alone

• BCRs are associated with two molecules called Ig(alpha) and Igß (called together CD79)

  • sends signal to nucleus to tell what is happening on the outside of the cell

  • w/o CD79 singaling will not work and receptors will not react to antigen binding

• When BCR binds its antigen more than one BCR complex gets brought together in the membrane

• IgAlpha /Igß become phosphorylated, starting a signaling cascade on the inside of the cell

• Co-Receptors also enhance B cell signaling

• BCR co-receptors are a complex of CD19/21/81/225

  • both bind to the antigen

• When complement activated (C3dg) and the BCR is signaling, the signal is enhanced through the co-receptor

T Cell Development & Maturation (3/13) - Wk 6

BE ABLE TO COMPARE AND CONTRAST B & T CELLS FOR EXAM (Both, Similar, Diff)

T Cell Receptors

• T Cell Receptors (TCRs) are specific for a particular antigen

• TCRs are found on the surface of all T cells

• TCRs are generated using genetic recombination

  • This process is very similar to that for BCRs/Antibodies

• TCRs are made of two protein chains

  • Most T cells have 1 alpha and 1 beta chain

• Like BCRs, there is a variable region (Valpha & Vbeta) and a constant region (Calpha & Cbeta) for each chain

• BCRs could bind to lots of different kinds of biological molecules

• TCRs mostly bind to peptides - bind btw alpha & beta chains

• BCRs bind to antigens free in solution - body fluids, blood

• TCRs bind of antigens that are presented on molecules called MHCs on the surface of another cell

• TCRs DO NOT undergo processes that are analogous to somatic hypermutation or isotype switching

• REFER TO COMPARE AND CONTRAST SLIDE

Molecules on the T Cell Surface

• All T cells express CD3

  • This marker can be used in Flow Cytometry assays to identify T Cells