B cell tolerance
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B Cell Tolerance. Wendy Davidson Ph.D. May 3, 2011 Contact information: Email:[email protected] Tel:301-402-8399. What is B cell tolerance?. The adaptive immune system has evolved to generate Ab against any foreign protein.

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B Cell Tolerance

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B cell tolerance

B Cell Tolerance

Wendy Davidson Ph.D.

May 3, 2011

Contact information:

Email:[email protected]

Tel:301-402-8399


What is b cell tolerance

What is B cell tolerance?

  • The adaptive immune system has evolved to generate Ab against any foreign protein.

  • This unlimited repertoire will include autoreactive specificities with the potential to cause disease. 50-75% of B cells produced in BM are autoreactive.

  • B cell tolerance is the process by which autoreactive B cells are silenced.

  • Self tolerance is achieved by a series of checkpoints that target autoreactive B cells at different stages of development.

  • Central (BM) and peripheral tolerance.

  • B cells responsive to foreign Ag can be rendered tolerant by varying the dose of Ag. High vs low zone tolerance. IgE-mediated allergy.


B cell tolerance

Ig transgenic mice are useful models for studying mechanisms of B cell selection

  • Most common model: Anti-hen egg lyzozyme (HEL) transgenic mice crossed with mice where HEL is expressed as an endogenous soluble self antigen (sHEL) or a membrane bound self antigen (mHEL).

  • Other models: Anti-ssDNA, -dsDNA, -rheumatoid factor, Ars/A1, -Smith, -RBC and -insulin.

  • Models used to demonstrate mechanisms of B cell repertoire selection in the BM and periphery (deletion, receptor editing and anergy).


B cell tolerance

Single Cell B Cell cloning

  • Uses

  • BCR repertoire analysis- general and subset specific

  • B cell development

  • B cell selection

  • B cell response to infection or vaccines (clonality, affinity, protection, vaccine design)

  • Identification of Abs with specific properties (e.g. Flu vaccines)

  • BCR repertoire analysis in disease (e.g. SLE before and after treatment)


B cell tolerance

Comparison of anergic B cell phenotypes in BCR transgenic mice

Cambier JC 2007


B cell tolerance

Stages of B cell development and repertoire editing

 Ag independent 

Checkpoint 1

Newly formed B cells

Checkpoint 2

Transitional B cells

Checkpoint 3

GC

Central tolerance

Peripheral tolerance

Cambier JC Nature, 2007


B cell tolerance

Sequence of B cell selection and maturation


B cell tolerance

CHECKPOINT 1 (Central tolerance): Possible fates of newly formed immature autoreactive B cells in BM

  • Self antigens expressed in BM:

  • Receptor editing

  • Deletion

  • Anergy

  • Self antigens not expressed in BM:

  • Escape (Ignorance)


B cell tolerance

Possible Fates of Immature B Cells in the BM

Death by Bim-dependent pathway

e.g. anti-IgG (RF)

e.g. mHEL

e.g. sHEL


B cell tolerance

CHECKPOINT 1: Receptor editing

  • Major mechanism for eliminating autoreactive specificities from the developing B cell repertoire

  • 4-8x107 immature B cells generated/day and a high proportion (~75%) of these are autoreactive.

  • By single cell PCR analyses, ~2/3 of immature B cells in BM have secondary rearrangements of IgL genes, exhibit Ig gene rearrangements and continue to express RAG 1 and 2.

  • Many of these cells do not make it out of the BM, since only ~1/2 of immature and mature B cells in the periphery show evidence of receptor editing.

  • Receptor editing occurs predominantly in the BM.

  • (F. Melchers 2006. Immunity:25:864)


B cell tolerance

CHECKPOINT 1: Ig Receptor Editing in BM

Developmental arrest associated with inhibition of expression of homing receptors and BAFF-R and persistent expression of RAG-1,-2.

Bim-dependent apoptosis


B cell tolerance

Anergy in immature BM cells

  • Anergy is a potentially reversible state of tolerance (unresponsiveness) that develops when immature autoreactive BM B cells bind abundant low avidity or soluble Ag that does not produce a sufficiently strong signal to induce receptor editing or deletion.

  • Anergy is associated with developmental arrest, increased threshold for activation by Ag, altered migration and shortened lifespan in the periphery.

  • Anergy is plastic and mechanistically diverse.

  • Spectrum of anergic phenotypes - influenced by Ag reactivity.


B cell tolerance

Properties of anergic peripheral B cells in Ig

transgenic mice

  • Migrate to the spleen and reside in the extra-follicular T cell zones or red pulp and are generally excluded from the follicles.

  • Retain Ag-binding capacity but are Ag unresponsive.

  • Have high basal intracellular Ca++ levels, positive signaling via the BCR is blocked.

  • Short half life (2-3 days) and an increased dependence on BAFF for survival compared to functional B cells.

  • Can be rescued if BAFF is present in excess and competing non-Tg B cells are absent or reduced in number.

  • Maintenance of anergy is dependent on continuous binding of Ag and inhibitory BCR signaling.

  • Anergy can occur in immature and mature B cells and is reversible.

  • Escape from anergy can result in autoimmunity.


B cell tolerance

Anergic B cells have diverse phenotypes in BCR Tg mice

Cambier JC, Nature Reviews, 2007


B cell tolerance

Factors that contribute to the increased threshold of activation in anergic B cells

HEL and Ars/A1 models:

Continuous signaling via self Ag/BCR

Decreasd surface IgM

Chronic ERK and NFAT signaling

Increased intracellular Ca++

Constitutive activation of SHIP-1 and DOK-1

Inhibition of activation of Syk and Akt survival pathway

Ferry et.al. 2006. Transplantation 81:308-315


B cell tolerance

B cell signaling in response to acute and chronic stimulation

Mono phosphorylation of ITAMS

Dual phosphorylation

of ITAMS

SHIP1 and DOK activated by LYN, modulate signaling

  • Chronic BCR signaling required to maintain anergic state

  • Anergy reversible by removal of Ag

  • Most relevant to MD4XMD5 and Ars/A1 Tg models

Cambier JC, Nature Reviews, 2007


B cell tolerance

Anergic B cells can be rescued from death if sufficient BAFF is available

Fas/FasL and CD40/CD40L interactions also may contribute to the death of anergic B cells

Ferry et.al. 2006. Transplantation 81:308-315


B cell tolerance

Other mechanisms for rescuing autoreactive anergic B cells

  •  signaling threshold, negative signaling

  • CD22-/-, SHIP1-/-, FcRIIb-/- mice

  •  exposure to auto Ag

  • Transfer autoreactive Tg B cells to auto Ag-free environment

  • Cross anti-DNA and anti-Sm Tg mice to autoimmune MRL-lpr or B6-lpr mice

  • Reduce the affinity of the BCR

  • Defects in cell death pathways (Fas, Bim)


B cell tolerance

Stages of B cell development and repertoire editing:

Checkpoint 2

Checkpoint 1

Newly formed B cells

Checkpoint 2

Transitional B cells

Checkpoint 3

GC

Central tolerance

Peripheral tolerance

Cambier JC Nature, 2007


B cell tolerance

Stages of transitional B cells

BM

Spleen

Transitional B cells

T1 T2 T3 follicular B cells

sIgM ++ ++ + +/++

IgD - ++ ++ ++

CD93 ++ + + -

CD23 - + + +

CD21 - + + +

CD24 ++++ +++ ++ +

Rag1,2 - - - -

Immature B cells

sIgM +

IgD-

CD93+

CD23-

CD21-

CD24 ++++

Rag1, Rag2 +/-

CD93 = AA4.1

CD24= HSA

Non-dividing, half-life 2-4 days


B cell tolerance

Proposed Model of Human B-Cell Development


B cell tolerance

Differentiation of transitional B cells in the spleen

Dependence on BAFF for survival

BM

SP

Increasing BAFF-R expression

T1

T2

Naïve B cell

Mature B cell

Immature B cells

Autoantigens

Autoreactive naïve B cell

Rescue

Induction of anergy

Ag removal and decreased stimulation threshold

T3

Anergic B cell

Death

Only ~5% of immature B cells produced in BM enter the mature B cell pool.

Significant cell loss

J. Cambier, Immunity 2006; M. Cancro, Immunol. Rev. 2004


B cell tolerance

Properties of BAFF

  • BAFF (BLyS, TALL-1, THANK, zTNF4) and APRIL, a related cytokine, are members of the TNF super family.

  • BAFF interacts with three receptors BAFF-R (BR3), BCMA and TACI expressed predominantly on B lineage cells.

  • APRIL binds to BCMA and TACI only.

  • BAFF is produced predominantly by myeloid cells and acts on transitional, naive and mature B cells.

  • BAFF is essential for normal B cell development and survival in the periphery.

  • BAFF-deficient mice are deficient in B2 cells and MZ B cells but have normal numbers of B1 cells.

  • BAFF Tg mice have excess mature B cells, especially MZ B cells, and develop systemic autoimmunity and B cell lymphomas with age.


B cell tolerance

BAFF governs successful transitional B cell differentiation by enhancing survival

  • Interactions between BAFF and BAFF-R are essential for maturation of transitional B cells.

  • BAFF-R-deficient mice have severely impeded transitional B cell differentiation and mature B cells with significantly shortened lifespans. TACI and BCMA KO mice exhibit normal transitional B cell differentiation.

  • BAFF-R expression increases as T1 cells differentiate into T2 cells.

  • Competition for available BAFF dictates the lifespan of anergic B cells and resting transitional, naïve and mature B cells.


B cell tolerance

Mechanisms of BAFF-induced survival of B cells

  • BAFF supports the survival of transitional and mature B cells

  • without inducing proliferation.

  • BAFF contributes to B cell survival by inducing the expression of pro-survival members of the Bcl-2 family and interfering with the nuclear translocation of pro-apoptotic PKC to the nucleus.

  • BCR ligation upregulates expression of BAFF-R, but not TACI or BCMA, on late transitional and mature B cells.

  • Both BCR- and BAFF-R mediated signals appear to be essential for repertoire selection and survival of mature B cells.


B cell tolerance

Influence of excess BAFF on the selection of self-reactive B cells

Models 1 and 2: Excess BAFF does not rescue cells deleted early in development.

Models 3 and 4: Responsiveness to excess BAFF corresponds to a maturational change in T2 cells involving expression of BAFF-R.

Thien et. al. 2004. Immunity 20:785-798.


B cell tolerance

Anergic B Cells are Susceptible to Fas-mediated Death


B cell tolerance

Differentiation of transitional B cells in the spleen

Dependence on BAFF for survival

BM

SP

Increasing BAFF-R expression

T1

T2

Naïve B cell

Mature B cell

Immature B cells

Autoantigens

Autoreactive naïve B cell

Rescue

Induction of anergy

Ag removal and decreased stimulation threshold

T3

Anergic B cell

Death

Only ~5% of immature B cells produced in BM enter the mature B cell pool.

Significant cell loss

J. Cambier, Immunity 2006; M. Cancro, Immunol. Rev. 2004


B cell tolerance

Evidence that anergic B cells and T3 cells have similar properties

  • Both have a similar surface phenotype (CD93+ CD23+ IgMlo, CD24inter, IgDhi) and lifespan.

  • Non-autoreactive BCR transgenic mice have few T3 cells suggesting that this population may not represent a developmental stage between T2 and naïve B cells.

  • Maintenance of the T3 phenotype requires continuous antigen exposure

  • T1 and T2 B cells are only found in the blood and spleen. Anergic B cells with the phenotype of T3 cells are detectable in spleen, LN and blood.

  • Anergic B cells and T3 cells have similar abnormalities in BCR signaling and have constitutively activated ERK and DOK-1.

  • T3 cells from normal mice are enriched for autoreactive specificities and have a similar gene expression profile to HEL Tg anergic B cells.


B cell tolerance

Potential dangers of having large numbers of anergized B cells produced during B cell selection.

  • TheT3/anergic B cell population in normal mice ranges from 1-5x106 cells/spleen and ~50% of these are replaced every 4 days (Merrell et.al. 2006.Immunity 25:953-962).

  • Previous estimates suggest that ~5x106 new B cells arrive in the spleen every 4 days (Rolink et.al. 1998. EJI 28:3738-3748).

  • Therefore, a significant proportion (upto ~50%) of the B cells entering the spleen every four days from BM may be anergic or become anergic (Merrell et.al. 2006.Immunity 25:953-962).

  • Since anergy is potentially reversible, having large numbers of anergic autoreactive B cells in the periphery poses a significant risk of autoimmunity. However, under normal circumstances, this danger is likely kept in check by the short half life of anergic B cells. (Merrell et.al. 2006.Immunity 25:953-962).


B cell tolerance

Possible mechanisms for rescue and activation of autoreactive anergic B cells

Increased availability of BAFF

Removal of self Ag

High avidity Ag stimulation + T cell help

TLR signals

Defects in Fas, Bim signaling

Altered signaling threshold

RESCUE

AUTOIMMUNITY

Ferry et.al. 2006. Transplantation 81:308-315


B cell tolerance

CHECKPOINT 3:

Elimination of autoreactive

B cells generated in GC

Autoreactive B cells generated in germinal centers by somatic hypermutation normally are eliminated. Possible role for Fas/FasL.

Lack of T cell help


B cell tolerance

Defects in the induction and control of B cell tolerance lead to systemic autoimmunity

  • Topics for lecture 2:

  • Sources of autoreactive B cells (incompletely tolerized cells, ignorant B cells)

  • Sites and mechanisms of activation of autoreactive B cells (antigens, TLRs, extrafollicular responses)

  • Contributions of autoreactive B cells to disease (effectors and APC)


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