Immunology Module 2

Functions of B Cells

1. Antigen Presentation (phagocytosis)

2. Autoantibody production

3. Cytokine Production

Stages of B cell Development

1. HSC

2. Pro-B

3. Pre-B

4. Immature B

5. Mature B

6. Activated B

7. Blast B

8. Plasma B

What are B cell developements outlined by?

cell surface markers (CD)

Transcription factor expression

immunoglobulin DNA arrangements

Role of Stromal Cells

cells in the bone marrow that provide support, cytokines, growth factors to instruct HSC to differentiate into B cells

ex. CXC112 and IL7

Antibody/Immunoglobulin structure

2 heavy chains and 2 light chains

held together by intra and inter-sulfide binds

Variable and constant regions

Variable Region

Region near antigen binding site; the first 110 amino acids are highly variable

Constant Region

Everywhere except the first 110 amino acids; highly conserved

Germ line theory

States that one gene codes for one anitbody

later disproven, too many antibodies (10^11) compared to genes

Somatic Recombination Theory

B cells have limited germline antibody genes, but mutational process in B cells alters genes

Tonegawa Experiment

• collected DNA from B cells and Liver cells

• Cut DNA with BamHI

• labeled light chain with radioactive nucleic acid proteases to track DNA (this tags what codes for the BCR)

• measure migration/size of DNA

Found that B cell dna cut and recombines, the pieces are smaller than liver cells

Early Pro B cells

DJ recombination of the heavy chain occurs

starts VDJ of heavy chain recombination

self-renewal property is eliminated

K first rule

kappa chain is the first to be used in a BCR, if it fails, the lamda light chain is used

Two light chains increases diversity

ProB/PreB

• VDJ heavy chain recombination is completed

• B cells undergo clonal expansion and proliferation

• VJ recombination of the light chain

Immature B cell

• expresses IgM

• Negative selection for cells that bind to self

• Cells undergo deletion, receptor editing, anergy

• undergoes tolerance

CXC112

Pre-pro B cells binds to CXCL12

Homing factor, keeps Pre/pro B cells in the bone marrow

IL7

B cell stromal factor

Pro survival signal

activates VDJ recombination

enables pro-B cell proliferation (allows clonal expansion)

What happens during somatic recombination mechanism?

• B cell shuffles gene segments and permantly removes DNA

• C region gets smaller

Epitope:

region of antiGEN that antibody binds to

E -pitope binds to antig-E-n

Paratope

antigen binding site, where the antigen binds to on a antibody

VDJ recombination of Heavy chain

1. Recombination signal sequences (RSS): DNA sequences that flank each Antibody gene segment

2. Gene segments are joined by Rag 1/2 recombinases (recombination activation gene)

3. Hydroxyl (OH-) at the nicked end and phosphate group forms a hairpin

4. Signal end joining ligates end

5. Ku70/80 recruited as scaffold for the enzymes

6. Artemis opens the hairpin

   1. happens asystemically, increases variance

7. P nucleotides added complementary overhands

8. exonucleus trims overhand

9. terminal deoxynucleotide transferase (TDT) adds nucleotides

10. ligase attaches DNA segments

VJ Recombination (Light chain specific)

1. Recombination signal sequences (RSS): DNA sequences that flank each Antibody gene segment

   1. RSS contains a heptomer, spacer (either 12 or 23 bp) and a nonamer

2. Gene segments are joined by Rag 1/2 recombinases (recombination activation gene)

3. Hydroxyl (OH-) at the nicked end and phosphate group forms a hairpin

4. Signal end joining ligates end

5. Ku70/80 recruited as scaffold for the enzymes

6. Artemis opens the hairpin

   1. happens asystemically, increases variance

7. P nucleotides added complementary overhands

8. ligase attaches DNA segments together

Junctional Diversity

variation of the number of nucleotides and how they come together

• Artemis variable hairpin opening

• exonuclease trimming

• Tdt adds p-nucleotides randomly

Combinatorial Diversity

diversity generated via ability to combine various genes

multiple gene segment for antibodies

different light and heavy chain combinations

Parts of RSS

heptomer, spacer (either 12 or 23 bp) and a nonamer

Central tolerance

happens during immature B stage in the bone marrow

self antigen in presented to immature B cells by other cells in bone marrow (especially stromal cells)

If no binding occurs, immature B proceeded to secondary lymphoid organs (most go to spleen)

If binding occurs, the cell undergoes clonal deletion, anergy, or receptor editing

Clonal deletion

cell death via apoptosis that occurs if BCR binds to self antigen during central tolerance

Anergy

permanent quiescence/unresponsiveness, cell cannot activate

Receptor editing

last ditch effort to save BCR if B cell binds to self antigen in central tolerance, genes are rearranged in the light chain using RAG 1 & 2 or switch to lambda chain if RAG 1 & 2 don’t work

Nemenzee Experiment

looks to see if receptor editing occurs

used transgenic mice with BCR against K^k antigen:

Group 1 had stromal cells that expressed K^d antigen, BCR didn’t bind and B cells in the periphery had a BCR specific to K^k

Group 2 had stromal cells that expressed K^d and K^k antigen, BCR did bind to stromal cells. Still, a small number of B cells found in periphery with a modified light chain that didn’t recognize k^k

Transition Phase

B cells enter the white pulp of the spleen

75% of B cells that passed central tolerance become transitional B cells; other 25% go to different secondary lymphoid organs or stay in the bone marrow

Order of transition phase

Immature B to T1 to T2

Differences between T1 and T2

T2 had higher IgD, less IgM, and increased BCR surface expression than T1

Peripheral tolerance categories

split into T1 and T2 tolerance

T1 Selection

removes 55-75% of cells

Negative selection: B cells that bind to self antigen are removed

occurs in the periateriolar sheath of the spleen

T2 Selection

Positive selection: low level signaling indicates survival

Indicative via tonic signaling and the upregulation of BAFF receptor

Tonic Signaling

low levels of BCR signaling in absence of an antibody binding; BCR stimulates cell via intracellular signaling

Causes a higher concentration of DAG (diacylglycerol) in the inner leaflet of the plasma membrane of T2 cells

BAFF

BAFF receptor on B cells binds to BAFF; a pro survival signal

BAFF binding to BAFFR increases from T1 to T2

BAFF binding to BAFFR causes a decrease in DAG in inner leaflet of T1 cells

Effect of DAG on survival

low DAG leads to death; high DAG leads to survival

DAG concnetrations in T1 and T2 lead to phenotypic changes

Plasma Cells

immunoglobulin producing machines

have many ribosomes on rough ER

includes long-lived plasma cells/memory plasma cells that live in the bone marrow

B2 Cells/Mature follical cells

95% of mature B cells

highly migratory across all secondary lymphoid organs

high V region diversity due to lots of somatic recombination

high memory

T cell activation required

B1 Cells

5% of mature B cells

found mainly in the peritoneal and perivascular cavities

low V region diversity

moderate memory

independent of T cells

produces natural IgM, cytokines, and clears apoptic cells with IgM

Marginal Zone Cells

<1% of mature B cells

found in teh marginal zone of the spleen

low V region diversity

memory is unknown (newer cell discovered)

can be both T cell independent or dependent

responds to blood borne antigens and produce IgM

T cell dependent activation in B2 Cells steps

antigen binds to BCR. (3 steps)

T cells form an immunological synapse, activates B cell

Steps of BCR on B cell binding to antigen

1. follicular dendritic cells can present antien and secrete BAFF or B cells detect free floating antigen

2. antigen binding causes membrane changes, and the antigen in phagocytosed

3. process pieces of the antigen are displayed on MHC class II

Steps of immunological synapse with T cell

1. TCR binds to MHC class II with the amtigen togehter

2. CD4+ binds to only MHC class II (no antigen)

3. CD40 on a B cell binds to CD40L on a T cell

4. T cell releases cytokines to activate B cell

T Cell independent activation in B1 and Marginal Zone cells

Includes TI-1 and TI-2 (thymus independent antigens); TI1= extra activation signaling; TI-2 = extensive activation signaling

TI-1 Antigen

celled extra activation signaling

antigen binds to B cell receptor and PRR (commonly TLR4 or LPS)

TI-2 Antigen

called extensive activation signaling

antigen cross liks and binds with a large number of B cell receptors

What does activation of a B cell cause

clonal expansion, somatic hypermutation, afinity maturation, and isotype switching

Clonal Expansion

massive proliferation of B cell, occurs in dark zone of terminal center

Somatic hypermutation

AID (activation induced cytidine deaminase) causes point mutations in V regions of the BCR to form QUasi Clonal B cells

Affinity Maturation

Quasi clones enter the blood stream, process to the light zone of the germinal celnter

the BCR that binds best has increased survival and becomes a plasma B cell

BCR with weak or no binding die via apoptosis

Isotype switching

in plasma cells, the F_c portion changes to accomodate antibody function

Which Ig is secreted first from Plasma B cell

IgM is secreted first

Long Lived Plasma Cells

secrete antibodies for long period of time (up to a life time)

reside in the bone marrow

Memory B Cells

respond to previously encountered antigen

upon reexposure, the rapidly reactivate (reach peak antibody production in 5 days and produce 100 times more antibodies than the first response)

survive a lifetime due to anti-apoptosis gene

found in germinal centers, lymphoid tissue, blood, lymph, and MALTs

How can antibodies lead to pathogen clearance?

agglutination, neutralization, opsonization, complement activation (CDC), antibody dependent cell mediated cytotoxicity (ADCC)

Agglutination

clumps/holds the antigens together, prevents it from traveling and spreading

Neutralization

blocks attachment of pathogen to the cell (binds to the pathogen, which blocks it from binding to cells = reduced virulence)

Opsonization

labels antigens to be phagocytosed

Complement dependent cytotoxicity (CDC)

leads to MAC complex and cell lysis

Antibody dependent cell mediated cytotoxicity (ADCC)

causes antigen to be destroyed by macrophages, NK cells, and eosinophils

IgM

• pentamer

• secreted first, present at high levels at initial immune response

• high avidity and low affinity

• is both membrane bound and secreted

• cannot cross placenta

• sometimes coexpressed with IgD

IgA

• dimer, Fc regions are unexposed

• found in mucosa and fluids

• many people lack IgA

• protease resistant

• long half life

IgG

• monamer

• most common Ig (70-80% of Ig)

• has several subclasses (G1 and G2)

• can cross blood vessels and placenta

• leads to opsonization, ADCC, CDC

  • CD16 on NK cells binds to he Fc Portion on antibodies

IgE

• monamer

• associated with mainly mast cells but also eosinophils and basophils

• involved in allergy and helminth production and worm infections

• Causes ADCC

• leads to the release of chemokines, cytokines, reactive oxygen species, antimicrobial peptides, histamine

• leads to smooth muscle contraction during anaphalaxis

IgD

• monamer

• coexpressed with IgM when B cell matures and leaves the bone marrow

• Important during B cell activation in T2 Stage

Why are blood cancers mostly B cell

60-70% of blood cancers are of the B cell

this is because of VDJ recombination and diversity —> DNA breaks and mutations cause some to be cencerous

Multiple myeloma

cancer of a plasma cell

leads to uncontrolled rgowth of cells and secretion of antibodies

B cell lymphomas

cencer in the lymphnodes or spleen

includes hodgkins (more specific and less common; predictable spread behavior) and non-hodgkins (more common and less specific; unpredictable)

Isotype Switching

• naive mature B cells start off with IgM and IgD on their surface

• if B cell becomes acctivated by T cell and cytokines anre present, B cell will isotype switch and secretes IgD, IgE, IgA, or IgG

• cytokines and environment determines which isotype the plasma cell will secrete

Autoimmune Encephalitis Pathology method

B cell response against the N-Methyl D aspartate receptor (NMDAR)

Encephylitis

inflammation in the brain

NMDAR

major receptor in the brain that is important in synaptic plasticity, learning, and memory

NMDA

binds to NMDAR, mimics glutamate (neuroexcitory)

In vitro

in cells

in vivo

in animal

ex vivo

extracted from animal

Anti-NMDAR Encephalitis

antibody against GluN1 subunit of NMDAR that leads to psychiatric and neurological symptoms

antibodies are found in cerebral spinal fluid

Epidemiology of Anti NMDAR encephalitis

4 times more likely in females, and more likely in women with teratomas

clinical triggers: ovarian teratomas and herpes simpler encephalitis

Vaccine used in the Primary Literature Paper parts

GluN1(359-378) peptide induces autoantibody production

complete Freund’s Adjuvant (CFA): an emulsion containing M. Tuberculosis, which enhances immune response and slowly releases antigens over time

Pertussis Toxin: disrupts the blood brain barrier to allow immune cells and anti GluN1 antibodies to impact central nervous system

Flow Cytometry

used to determine if a specific cell type expresses a marker of interest on its surface or determine the quantity of cells in a sample that express a specific marker

Dot Blot

Used to determine if a protein is present in a sample and its relative quantity, but cannot determine the size of the protein

Histological Immunofluorescence

used to determine if a target protein/marker is present in a sample, determine the location of the target (also target movement in live samples), and relative quantity

Split into direct and indirect

• direct: primary antibody with tag binds to antigen

• indirect: secondary antibody binds to the Fc region of an antibody binded to an antigen

Monoclonal antibody depletion

used to selectively eliminate specific cell types to find out the role of the eliminated cells

Antibodies do this by causing cell death through the CDC or ADCC pathway

Neuron Ca2+ assay

used to quantify ion channel and neuron signaling activity

does this by measuring intracellular Ca2+ using Fura-2-AM and video microscopy

Elevated O maze

Behavioral test to determine/identify anxiety

staying in closed regions means greater anxiety, while exploring open regions indicates less anxiety

Forced Swim Test

greater immobility duration indicates depressive-like despair

Y Maze

Exploring new areas indicates better spatial memory, while exploring the original arm indicates lower spatial memory

Actimitry:

measures horizontal and vertical motor activity

Immunization using GluN1(359-378) peptide key findings

vaccine blocks NMDA-dependent Ca2+

depletion of B cells using monoclonal antibody prevents memory loss and behavior despair, which is presented as a poteintial therapy

pathology is caused by B cell trafficking through meninges and ventricles through cerebral spinal fluid and more plasma CD138+ cells without T cell recruitment

Bepi prep linear epitope prediction

predicts surface accessibility, hydrophilicity, and amino acid propensity to guess which part of a protein an antibody/BCR will bind to on an antigen

Flair MRI

darkens cerebral spinal fluid, used to see lesions in the brain

Evans Blue

measure sthe blood brain barrier permeability, the bluer the brain the more permeable