2Factors that contribute to the increased threshold of activation in anergic B cells HEL and Ars/A1 models:Continuous signaling via self Ag/BCRDecreasd surface IgMChronic ERK and NFAT signalingIncreased intracellular Ca++Constitutive activation of SHIP-1 and DOK-1Inhibition of activation of Syk and Akt survival pathwayCompare 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:
3B cell signaling in response to acute and chronic stimulation Mono phosphorylation of ITAMSDual phosphorylationof ITAMSSHIP1 and DOK activated by LYN, modulate signalingAcute 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 stateAnergy reversible by removal of AgMost relevant to MD4XMD5 and Ars/A1 Tg modelsCambier JC, Nature Reviews, 2007
4Anergic 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 cellsFerry et.al Transplantation 81:
5Other mechanisms for rescuing autoreactive anergic B cells signaling threshold, negative signalingCD22-/-, SHIP1-/-, FcRIIb-/- mice exposure to auto AgTransfer autoreactive Tg B cells to auto Ag-free environmentCross anti-DNA and anti-Sm Tg mice to autoimmune MRL-lpr or B6-lpr miceReduce the affinity of the BCRDefects in cell death pathways (Fas, Bim)
6Stages of B cell development and repertoire editing: Checkpoint 2Checkpoint 1Newly formed B cellsCheckpoint 2Transitional B cellsCentral tolerancePeripheral toleranceCheckpoint 3GCCambier JC Nature, 2007
7Stages of transitional B cells BMSpleenTransitional B cellsT T T follicular B cellssIgM /++IgDCDCDCDCDRag1,Immature B cellssIgM +IgD-CD93+CD23-CD21-CDRag1, Rag2 +/-CD93 is C1q receptor, binds apoptotic cellsCD93 = AA4.1CD24= HSANon-dividing, half-life 2-4 days
9Differentiation of transitional B cells in the spleen Dependence on BAFF for survivalBMSPIncreasing BAFF-R expressionT1T2Naïve B cellMature B cellImmature B cellsAutoantigensAutoreactive naïve B cellRescueInduction of anergyAg removal and decreased stimulation thresholdSequential 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.T3Anergic B cellDeathOnly ~5% of immature B cells produced in BM enter the mature B cell pool.Significant cell lossJ. Cambier, Immunity 2006; M. Cancro, Immunol. Rev. 2004
10Properties of BAFFBAFF (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.
11BAFF 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.
12Mechanisms of BAFF-induced survival of B cells BAFF supports the survival of transitional and mature B cellswithout 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.
13Influence 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:
14Anergic B Cells are Susceptible to Fas-mediated Death
15Differentiation of transitional B cells in the spleen Dependence on BAFF for survivalBMSPIncreasing BAFF-R expressionT1T2Naïve B cellMature B cellImmature B cellsAutoantigensAutoreactive naïve B cellRescueInduction of anergyAg removal and decreased stimulation thresholdmT3Anergic B cellDeathOnly ~5% of immature B cells produced in BM enter the mature B cell pool.Significant cell lossJ. Cambier, Immunity 2006; M. Cancro, Immunol. Rev. 2004
16Evidence 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 exposureT1 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.
17Potential 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: ).
18Possible mechanisms for rescue and activation of autoreactive anergic B cells Increased availability of BAFFRemoval of self AgHigh avidity Ag stimulation + T cell helpTLR signalsDefects in Fas, Bim signalingAltered signaling thresholdRESCUEAUTOIMMUNITYFerry et.al Transplantation 81:
19Elimination of autoreactive B cells generated in GC CHECKPOINT 3:Elimination of autoreactiveB cells generated in GCAutoreactive B cells generated in germinal centers by somatic hypermutation normally are eliminated. Possible role for Fas/FasL.Lack of T cell help
20Defects 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)