MICR 304 Immunology &

Serology

MICR 304 Immunology &

Serology

Lecture 10 B Lymphocytes

Chapter 6.8, 6.10, 6.17–6.18, 7.1–7.6, 7.23–7.28, 9.1 – 9.13

Fig. 6.18

Lecture 10 B Lymphocytes

Chapter 6.8, 6.10, 6.17–6.18, 7.1–7.6, 7.23–7.28, 9.1 – 9.13

Fig. 6.18

Overview of Today’s Lecture

• Cell signaling through BCR• Development of B cells• Positive and negative selection• Survival and maturation in

peripheral lymphoid tissue

Key Players in Immunology

Innate Adaptive

Cells PhagocytesEpithelial Cells

NK Cells

Lymphocytes(B-Ly, T-Ly)

Effector Molecules

ComplementAntimicrobial (Poly)PeptidesAntimicrobial

Lipids?

Antibodies

B Cell Receptor Complex• Membrane bound

immunoglobulin has no intrinsic signaling capacity

• Ig and Ig are invariant non-antigen specific transmembrane molecules that initiate signaling

• Contain tyrosine residues that can be phosphorylated by src kinases upon activation– ITAM (Immunoreceptor

Tyrosine-based Activation Motive)

• Ig and Ig are also important for transport of Ig to cell surface *Also present in TCR, NK receptors, Fc receptors

*

Cross-Linking is Required for B Cell Activation

• Binding of a monovalent antigen to a single BCR will not produce a signal.

• Receptor clustering caused by cross linking generates an intracellular signal.

Src Kinases Associate with BCR

• B cell receptor cross linking is required

• Src kinases Blk, Fyn, or Lyn associate with and phosphorylate Ig and Ig

• Tyrosine kinase Syk with 2 SH2 domains is recruited, binds to phosphorylated ITAM and is activated.

B Cell Co-Receptor Complex

• Binding of antigen is not enough for activation of naïve lymphocytes

• Simultaneous ligand binding to co-stimulatory receptors enhance antigen dependent signaling

• Co-receptor complex consists of– CD21 = complement receptor

CR2 for C3d fragment– CD19: becomes phosphorylated

and phosphorylated CD19 activates src kinases

– CD81 = TAPA-1 (target of antiproliferative antibody); role?

1. C3d

P2. 3. 3.

B Cell Activation through the BCR and Co-Receptors

B Cell Development

Main Phases of a B Cell’s Life HistoryB cell precursor

Rearrangement of Ig genes

Immature B cellNegative selection

Mature B cellMigration to peripheral lymphoid organs

Activated B cell

Plasma Cell Memory B cell

Main Phases of a B Cell’s Life History

B Cell Maturation is Coupled to Immunoglobulin

rearrangement1. Heavy chain rearrangement

– DJ– VDJ

2. Transient expression of heavy chain with surrogate light chain

– Important check point– Successful signaling involves Btk (Bruton’s tyrosine kinase)– 45% unsuccessful!– Switch to the other chromosome

3. Light chain rearrangement– VJ

4. IgM expressed on cell surface5. IgD expressed on cell surface in addition to IgM

B Cell Maturation is Coupled to Immunoglobulin

rearrangementCheck point

Ability to express Igon cell surface

Steps in Ig Gene Rearrangement at which Developing B Cells can be

lost

Development Dependent Expression of Surface Proteins

in B Cells

Expression of Proteins Involved in Gene Rearrangement and Production of pre-BCR and

BCR

Immature B Cells are Tested for Self Reactivity

Check pointAbility to express Ig

on cell surface

Check pointSelf reactivity

The Fate of Self Reactive B Cells

• Once complete IgM molecule is successfully expressed on B cell surface (sIgM) B cell has reached the immature B cell stage

• Ag receptor is now tested for tolerance to self antigen (central tolerance)

• Fate of immature B cell depends on the signal delivered from sIgM – No strong reactivity

– Strong reactivity

Further maturationAnergyIgnorance

Clonal deletionReceptor editing

Anergy

• In response to soluble self molecules with weak cross linking of IgM

• Down regulation of IgM and mainly expression of IgD

• Permanent block in signal transduction and unresponsiveness

• Migration to periphery• Anergic B cells cannot respond to

antigen anymore even under optimal conditions.

Ignorance

• Monovalent or low affinity soluble non cross-linking self antigens

• No signal transduction and activation in response to self antigen

• Migration to periphery• Potentially self reactive under certain

circumstances– High concentration of antigen– In the context of inflammation

• Pool for autoreactive cells in autoimmune diseases

Clonal Deletion• B Cells that respond to multivalent self

antigens are removed from repertoire• Premature activation of B cell initiates

apoptotic events.• The removed B cell cannot anymore

undergo clonal proliferation, hence clonal deletion.

• The antibody molecule expressed by this B cell is irreversibly lost.

Receptor Editing

• Some B cells that respond to multivalent self antigen are rescued.

• B cell obtains a second chance.• Surface IgM is decreased.• RAG enzymes continue

rearrangement of light chain. • New light chain is coupled to

existing heavy chain and IgM is expressed at the cell surface.

• Retesting of self reactivity ensues.

Summary for Possible Outcomes of Self Reactivity

at Check Point

Survival and Maturation of B Cells

Main Phases of a B Cell’s Life HistoryB cell precursor:

Rearrangement of Ig genes

Immature B cell:Negative selection

Mature B cell:Migration to peripheral lymphoid organs

Activated B cell

Plasma Cell Memory B cell

TH

Homing of Naïve B Cells to Lymph Nodes

• High endothelial venules (HEV) and stromal cells secrete chemokine CCL21

• Dendritic cells are attracted by CCL21 and secrete chemokines CCL18 and CCL19

• B lymphocytes are attracted and stimulate follicular dendritic cells to secrete CXCL13 which attracts more B cells

Activation of B Cells by TH Cells

• B cells are APC.• Antigen bound sIgM is

internalized.• After fusion with lysosomes

antigen is processed and presented via MHC II to T cells.

• B cells express also costimulatory molecule CD40.

• Activated T cells that bind to antigen on MHC II activate B cells via binding to CD40 and by releasing cytokines that stimulate B cells to develop into plasma cells and memory cells.

CD40 LigandCD40

Activated B Cells Differentiate into Memory Cells and Plasma

Cells

Cognate TH cellUnder control of

BLIMP-1(B-ly induced maturation protein)

Somatic Hypermutation of B Cells Activated by TH Cells

Quickly engulfed by macrophages

Comparison of B Cells and Plasma Cells

• Plasma cells are terminally differentiated– Low expression of sIg– No MHC II, no more interaction with TH– High rate of Ig secretion– No more growth, somatic hypermutation or isotype switch

Cytokines Dictated Isotype Switch

Cytokine Induced Isotype

IL4 IgE

IL5 IgA

IFN IgG

TGF IgG and IgA

Memory B Cells

• Long lived descendants of cells that were once stimulated by antigen– > 70 years– Half time ~ 10 – 15 years

• Slow divisions if any• Low or absent Ig secretion• High level of MHC II• Increased expression of costimulatory

signals• High affinity for antigen

T Cell Independent B Cell Activation

• Response to bacterial polysaccharides, polymeric proteins, lipopolysaccharides

• Thymus independent antigens (TI)• TI-1 antigens

– Intrinsic activity and polyclonal B cell activation at high concentrations

– B-cell mitogens– E.g. LPS– Can activate immature and mature B cells

• TI-2 antigens– Highly repetitive structure– E.g. bacterial capsular polysaccharide– Activates only mature B cells– Costimulatory signals provided by dendritic cells and

macrophages

Innate B Lymphocytes

Today’s Take Home Message

(Student Contribution)• B cell maturation involves heavy chain rearrangement in which upon successful

completion, this chain will be expressed with a surrogate light chain. If this expression is successful, the heavy chain rearrangement will halt and the cell can transition to rearranging the light chain. IgM will be expressed on the surface to check if the immature B cell is reactive to self antigens. If it is not reactive then IgD will be expressed alongside with IgM.

• When the B cell is tested for tolerance to self antigen then no strong reaction to cross linking self antigens leads to further maturation; a response to soluble self molecules with weak cross linking leads to anergy; a response to monovalent or low affinity soluble non cross-linking self antigens leads to ignorance; a strong response to self antigen leads to receptor editing and if this does not reduce self reactivity to clonal deletion.

• B cells are activated by T cells after cognate T cells have bound via their TCR to MHC II presented antigen that B cells have captured with their surface immunoglobulin, and the costimulatory receptors CD40 (on the B cell) and CD40 L (on the T cell) have interacted which leads to further B cell maturation into plasma cells and memory cells.

• Plasma cells originate from T cell activated B cells but have become independent from T cells and have directed their cell function to high antibody production and secretion which is accompanied by a reduction of surface Ig and MHC II expression.

• T cell independent antigens like polysaccharides and LPS can activate B cells independent from T cells and innate B cells are fully functional without the aid by TH cells.