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BR3 and BCR signaling
The BCR is the main activator of the classical NF-« B pathway while BR3 primarily activates the alternative NF-« B2 pathway. As both pathways are required for B-cell development and survival, crosstalk between these two pathways is suggested. Supporting this hypothesis, a study demonstrated that p100, a substrate for NF-« B2, requires NF-« B1 activation for its expression in B cells (Stadanlick et al., 2008). In the absence of NF-« B1 activation, BR3 signaling quickly depletes avalaible p100 in the cell and is unable to sustain permanent NF-« B2 activation and the expression of downstream survival factors. However, as BR3 also weakly activates the NF-« B1 pathway, it is possible that BAFF signaling can allow some p100 expression to feed the NF-« B2 pathway in the absence of BCR-mediated NF-« B1 activation. In addition, BR3 expression is up-regulated by BCR ligation on mature B cells and it is the predominant receptor expressed on resting memory B cells (Avery et al., 2003).
The effects of BAFF on T lymphocytes
BR3 is also expressed on activated T cells and Treg cells (Mackay and Leung, 2006). Surprisingly, aberrant expression of BAFF in T cells of patients with SLE (Yoshimoto et al., 2006) or RA (Lai Kwan Lam et al., 2008) may engender some T helper-1 responses (Sutherland et al., 2005) and an anti-inflammatory Treg cell expansion (Walters et al., 2009). These observations show that BAFF has a dichotomous role in immune responses.
The effects of BAFF on innate immune response
BAFF is also an essential component of the innate immune response. BAFF synthesis is induced in myeloid DC by type I IFNs (Boule et al., 2004). BAFF up-regulates TLR expression, and together with IL-6 promotes Ig class-switching and plasma cell differentiation (Treml et al., 2007; Katsenelson et al., 2007). Activation of intracellular TLRs in B cells by immune complexes containing nucleic acids up-regulates expression of BAFF receptors, particularly TACI, and increases BCR-mediated signaling. The triad of TLRs, type I IFNs and BAFF creates an amplification loop that propages production of IgG auto-Abs to nucleic acids without any T-cell help (Groom et al., 2007). This mechanism may link the ìtype I IFN signatureî, BAFF excess and autoimmunity in pSS and SLE. DCs express BAFF receptors (mainly TACI), and when transduced with a siRNA that silences BAFF, remain in an immature state and fail to produce the IL-6 required for the differentiation of Th17 cells (Lai Kwan Lam et al., 2008). Human myeloid DCs stimulated with BAFF up-regulate co-stimulatory molecules, lose their phagocytic ability and produce inflammatory cytokines and chemokines including IL-1, IL-6, CCL2 and CCL5 inducing Th1 responses in vitro (Chang et al., 2008). These studies suggest that BAFF acts on DCs to help them in recruiting immune cells to inflammatory sites and to enhance the proinflammatory activity of T cells.
BAFF functions in immune tolerance
Fifty to 75% of the newly generated B cells in the BM have a self-reactive BCR. Thus, to avoid the generation of pathogenic auto-Abs, self-reactive B cells have to be deleted or anergised during successive checkpoints during B-cell development and maturation. Based on its receptor expression profile, BAFF has no effect on B-cell tolerance in the BM, but does act in the periphery (Figure 12). BAFF is needed for survival of T2 cells, which express high levels of BR3. Actually, BAFF is necessary to prevent T2 apoptosis, as has been demonstrated in BAFF or BR3 lacking models, where the B-cell ontogenesis is stopped at the T1 stage (Schiemann et al., 2001).
In BAFF and !BAFF (negative regulator of BAFF) transgenic mice models, interbred with transgenic mice for the site-directed 3H9 Ig H chain (whose specificity is derived from a DNA and chromatin-specifc hybridoma obtained form a MRL/lpr mouse), Ota et al., (Ota et al., 2010) analyzed how BAFF levels alter B-cell tolerance by characterizing changes in the B-cell repertoire that occur with over or under expression of BAFF. BAFF/3H9 mice model presented elevated number of B cells, including L chains generation high-affinity reactivity and auto-Ab production, demonstrating that BAFF signaling leads to tolerance escape and positive selection of autoreactive cells.
APRIL functions
The role of APRIL is more elusive that BAFF functions. Conflicting results emerged from two independently generated APRIL-/- mouse models: one showed no obvious phenotype (Varfolomeev et al., 2004) whereas the other showed impaired switching to IgA, bigger GCs and increased numbers of effector T cells (Castigli et al., 2004).
BAFF heterogeneity
BAFF offers a lot of forms: mBAFF or sBAFF, monomer or trimers, homotrimers or heterotrimers, heterotrimer formation with APRIL, TWEAK or !BAFF, or even virus-like aggregates of 60 monomers (Daridon et al., 2008). The heterogeneity of BAFF may difficult the assessment of this cytokine. This question arises from the observation of normal or low levels of BAFF in patients with AIDs. Also intriguing is that raised serum concentrations of BAFF have been associated with auto-Abs production by some (Mariette et al., 2003) but not all investigators (Collins et al., 2006). Thus, receptor occupancy B cells by BAFF (Carter et al., 2005), its urinary excretion in case of renal failure (Collins et al., 2006) and sequestration in immune complexes (Gao et al., 2007) all hinder assessment of BAFF. In this respect, it is interesting that intravenous Igs contains natural anti-BAFF Abs (Le Pottier et al., 2007). Furthermore, on the basis that flaws in an ELISA might explain such variations, in our laboratory, we have set an inhouse ELISA, as a reliable tool to detect BAFF in these pathologies (Le Pottier et al., 2009).
Alternative splice isoforms
Four different mRNA from BAFF gene have been characterized in humans: the fulllength BAFF, the longer variant called )BAFF, an exon-3 lacking isoform (!BAFF or 3BAFF) and an exon-4 lacking isoform (!4BAFF). The larger transcript )BAFF was identified in the human cells lines HL-60 and U937, but sequencing proved this transcript to be non-functional because of incomplete splicing of intronic sequencing leading to premature termination codons (PTCs). An exon-3 (57 bp) lacking isoform of BAFF, referred to as !BAFF, has been identified in many mouse and human myeloid cells (Gavin et al., 2003). Based on its ability to multimerize with full-length BAFF, this truncated variant might restrain the effects of the full-length-cytokine, and thereby regulate B-cell homeostasis (Gavin et al., 2005). Currently, !BAFF protein has never been characterized. Recently, in our group, we detected an additional transcript (Results, Article 4: A new BAFF variant acting as a transcription factor in the regulation of immune response. Nat Genetics 2012; submitted) which was devoid of the 113 base pair segment encoded by the exon 4, and we called this new variant !4BAFF. The chromatin immunoprecipitation (ChIP) unveiled that the protein was associated with DNA, and, to our surprise, acted as a transcription factor for the fulllength form of BAFF. The discovery of this new variant and their role as a transcription factor are described in the article 4.
Table of contents :
ABBREVIATIONS
INTRODUCTION
1. Primary Sjˆgrenís Syndrome
1.1. Introduction
1.2. History of pSS
1.3. Epidemiology of pSS
1.4. Clinical picture
1.4.1. Sicca syndrome and glandular manifestations
1.4.2. Extraglandular manifestations
1.5. Diagnosis
1.5.1. Minor salivary gland biopsy
1.5.2. Whole Saliva Flow Collection
1.5.3. Ocular tests
1.5.4. Other diagnostic test in evaluation
1.5.4.1. SGs ultrasonography
1.5.4.2. Skin biopsy
1.5.4.3. Salivary biomarkers
1.6. Biologic abnormalities
1.6.1. Non-specific biologic abnormalities
1.6.2. Immunological abnormalities
1.6.3. Anti-SSA(Ro) and anti-SSB (La) Abs
1.6.4. Other auto-Abs
1.7. Measurement of chronicity and activity in pSS patients
1.8. Lymphoma in pSS patients
1.8.1. Epidemiology and clinical characteristics
1.8.2. Risk factors for lymphoproliferation
2. Pathophysiology of primary Sjˆgrenís syndrome
2.1. Introduction
2.2. Etiology
2.2.1. Genetic influence
2.2.2. Epigenetics
2.2.3. Environmental factors (Viruses)
2.2.4. Hormonal factors
2.3. Immunopathology of primary Sjˆgrení syndrome
2.3.1. Immunological findings and histopathology in exocrine glands
2.3.2. Epithelial cells as key regulators of autoimmune epithelitis
2.3.3. Neuro-exocrine junction
2.3.4. Innate immunity
2.3.5. T cells
2.3.6. B cells
2.3.6.1. Introduction
2.3.6.2. B-cell classification according to their ontogenic state
2.3.6.3. B lymphocytes in SGs
2.3.6.4. B lymphocytes in peripheral blood
2.3.6.5. Regulatory B cells
2.3.6.6. Memory B cells
2.3.6.7. Intrinsic B-cell defects
2.3.6.8. Autoreactivity-driven B lymphocyte proliferation
2.3.6.9. B cell-derived cytokines
2.3.6.10. IL-6
2.3.7. Cytokines and chemokines other than B-cell cytokines
2.3.7.1. T-cell cytokines
2.3.7.2. Interconnections between B- and T-cell cytokine networks
2.3.7.3. Type I IFN-s
2.3.7.4. Type II IFN-s
2.3.7.5. Lymphotoxins
2.3.7.6. IL-1
2.3.7.7. Chemokines and chemokine receptors
2.4. Conclusion: current model of the pathophysiology of primary Sjˆgrenís syndrome
3. Treatment of pSS
4. FLT3-Ligand
4.1. Introduction
4.2. FLT3-L
4.3. FLT3 and intracellular signaling
4.4. Distribution of FLT3
4.5. Functions of FLT3-L
4.5.1. Functions in hematological precursors
4.5.2. Functions in dendritic cells
4.6. FLT3 and FLT3-L in pathology
4.6.1. Hematological malignancies
4.6.2. Autoimmune diseases
5. BAFF
5.1. Introduction
5.2. BAFF generalities
5.2.1. Mice models
5.2.1.1. BAFF transgenic mice
5.2.1.2. Adaptor molecule Act-1 deficient mice
5.2.2. BAFF producing cells
5.2.3. BAFF-from gene to protein-
5.3. BAFF receptors
5.4. BAFF functions
5.4.1. The effects of BAFF on B lymphocytes
5.4.1.1. BAFF-signaling mediated B-cell survival
5.4.1.2. BR3 and BCR signaling
5.4.2. The effects of BAFF on T lymphocytes
5.4.3. The effects of BAFF on innate immune response
5.4.4. BAFF functions in immune tolerance
5.4.5. APRIL functions
5.5. BAFF heterogeneity
5.5.1. Alternative splice isoforms
5.6. BAFF in pathology
5.6.1. Hematological malignancies
5.6.2. Autoimmune diseases
5.6.3. BAFF in primary Sjˆgrenís syndrome
5.6.3.1. BAFF overexpression in saliva, sera and implications in circulating cells in pSS
5.6.3.2. BAFF in the salivary glands from pSS
5.6.3.2.1. Aberrant expression of BAFF in the SGs impacts B cells exclusively
5.6.3.2.2. Effect of BAFF on MZ-like B cells infiltrating the SGs
5.6.3.2.3. Autoreactive B cells and ectopic GCs
5.6.3.2.4. Effect of BAFF on memory B cells infiltrating the SGs
5.6.3.2.5. BAFF modulates repopulation of B cells in the blood and SGs of pSS patients treated with RTX 82
5.6.3.3. BAFF and genetic susceptibility to pSS
5.7. BAFF targeted therapy
5.8. Induction of BAFF
5.8.1. Induction of BAFF by cytokines
5.8.2. The transcription factors for BAFF
5.8.2.1. Nuclear factor-« B
5.8.2.2. Nuclear factor of activated T cells (NFAT)
5.8.2.3. CD40
5.8.2.4. BR3
5.8.2.5. !4BAFF
5.9. Alternative splice study
5.9.1. Alternative RNA splicing
5.9.2. Splicing machinery
5.9.3. Splicing regulatory elements
5.9.4. Serine/Arginine-rich protein splicing factors (SR proteins)
5.9.4.1. Nomenclature for Serine/arginine-rich protein splicing factors (SR proteins)
5.9.4.2. SR proteins localization
5.9.4.3. SC35 protein
5.9.5. Heterogeneous nuclear ribonucleoproteins (hnRNPs)
5.9.6. Minigene technique to study alternative splice
5.9.6.1. Minigene design
5.9.6.2. Identification of exonic and intronic elements that enhance or repress splicing
5.9.7. Alternative splicing of the immune system
5.9.7.1. Alternative splicing, immune system and regulatory proteins
5.9.7.2. Alternative splicing and immune activation of human T and B cells
5.9.8. Tissue-specific splicing
5.9.9. Consequences of aberrant spliced transcripts
5.9.10. Alternative splicing induced by IFN-#
OBJECTIVES
