1. B Cell Tolerance Wendy Davidson Ph.D. May 3, 2011
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2. 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
Compare normal and anergic. BAFF keeps B cells alive. This survival signal is overridden in anergic B cells if BAFF is present in normal amounts.
Ferry et.al Transplantation 81:

3. 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
Acute B cell signaling induces dual phosphorylation of ITAMS on Iga and Igb chains. Chronic signaling see monophos. and activation of SHIP and DOK that 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

4. 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 Transplantation 81:

5. 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)

6. Stages of B cell development and repertoire editing:
Checkpoint 2
Checkpoint 1
Newly formed B cells
Checkpoint 2
Transitional B cells
Central tolerance
Peripheral tolerance
Checkpoint 3 GC
Cambier JC Nature, 2007

7. Stages of transitional B cellsBM
Spleen
Transitional B cells
T T T follicular B cells
sIgM /++
IgD CD CD CD CD
Rag1,
Immature B cells
sIgM +
IgD-
CD93+
CD23-
CD21- CD
Rag1, Rag2 +/-
CD93 is C1q receptor, binds apoptotic cells
CD93 = AA4.1
CD24= HSA
Non-dividing, half-life 2-4 days

8. Proposed Model of Human B-Cell Development

9. 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
Sequential maturation of transitional B cells. Evidence that T1 give rise to T2. T3 likely anergic. If sort T3 cells they have all the properties of anergic cells. TR maturation is dependent on BAFF and BAFFR expression. Latter increases as cell mature. 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

10. 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.

11. 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.

12. 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.

13. 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 Immunity 20:

14. Anergic B Cells are Susceptible to Fas-mediated Death

15. 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 m 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

16. 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.

17. Potential dangers of having large numbers of anergized B cells produced during B cell selection.
The T3/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 Immunity 25: ).
Previous estimates suggest that ~5x106 new B cells arrive in the spleen every 4 days (Rolink et.al EJI 28: ).
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 Immunity 25: ).
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 Immunity 25: ).

18. 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 Transplantation 81:

19. Elimination of autoreactive B cells generated in GC
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

20. 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)