Djemel Aït-Azzouzene

ΔBAFF, a Splice Isoform of BAFF, Opposes Full-Length BAFF Activity In Vivo in Transgenic Mouse Models

ΔBAFF is a novel splicing isoform of the regulator B cell-activating factor (BAFF, BLyS), a TNF family protein with powerful immunoregulatory effects. Overexpression of BAFF leads to excessive B cell accumulation, activation, autoantibodies, and lupus-like disease, whereas an absence of BAFF causes peripheral B cell immunodeficiency. Based on the ability of ΔBAFF to multimerize with full-length BAFF and to limit BAFF proteolytic shedding from the cell surface, we previously proposed a role for ΔBAFF in restraining the effects of BAFF and in regulating B lymphocyte homeostasis. To test these ideas we generated mice transgenic for ΔBAFF under the control of human CD68 regulatory elements, which target expression to myeloid and dendritic cells. We also generated in parallel BAFF transgenic mice using the same expression elements. Analysis of the transgenic mice revealed that ΔBAFF and BAFF had opposing effects on B cell survival and marginal zone B cell numbers. ΔBAFF transgenic mice had reduced B cell numbers and T cell-dependent Ab responses, but normal preimmune serum Ig levels. In contrast, BAFF transgenic mice had extraordinarily elevated Ig levels and increases in subsets of B cells. Unexpectedly, both BAFF and ΔBAFF appeared to modulate the numbers of B-1 phenotype B cells.

ΔBAFF, an Alternate Splice Isoform That Regulates Receptor Binding and Biopresentation of the B Cell Survival Cytokine, BAFF

The tumor necrosis family member BAFF is limiting for the survival of follicular B lymphocytes, but excessive BAFF signaling can lead to autoimmunity, suggesting that its activity must be tightly regulated. We have identified a conserved alternate splice isoform of BAFF, called ΔBAFF, which lacks 57 nt encoding the A–A1 loop and is co-expressed with BAFF in many mouse and human myeloid cells. Mouse ΔBAFF appears on the plasma membrane, but unlike BAFF it is inefficiently released by proteolysis. ΔBAFF can associate with BAFF in heteromultimers and diminish BAFF bioactivity and release. Thus, alternative splicing of the BAFF gene suppresses BAFF B cell stimulatory function in several ways, and ΔBAFF may promote other functions as well.

Basal B Cell Receptor-Directed Phosphatidylinositol 3-Kinase Signaling Turns Off RAGs and Promotes B Cell-Positive Selection

PI3K plays key roles in cell growth, differentiation, and survival by generating the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). PIP3 activates numerous enzymes, in part by recruiting them from the cytosol to the plasma membrane. We find that in immature B lymphocytes carrying a nonautoreactive Ag receptor, PI3K signaling suppresses RAG expression and promotes developmental progression. Inhibitors of PI3K signaling abrogate this positive selection. Furthermore, immature primary B cells from mice lacking the p85α regulatory subunit of PI3K suppress poorly RAG expression, undergo an exaggerated receptor editing response, and, as in BCR-ligated cells, fail to progress into the G1 phase of cell cycle. Moreover, immature B cells carrying an innocuous receptor have sustained elevation of PIP3 levels and activation of the downstream effectors phospholipase C (PLC)γ2, Akt, and Bruton’s tyrosine kinase. Of these, PLCγ2 appears to play the most significant role in down-regulating RAG expression. It therefore appears that when the BCR of an immature B cell is ligated, PIP3 levels are reduced, PLCγ2 activation is diminished, and receptor editing is promoted by sustained RAG expression. Taken together, our results provide evidence that PI3K signaling is an important cue required for fostering development of B cells carrying a useful BCR.

An immunoglobulin Cκ-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igκ–reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igκ-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.

Deletion of IgG-Switched Autoreactive B Cells and Defects in Faslpr Lupus Mice

During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered γ2a-reactive superantigen (γ2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Faslpr lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.

Paucity of V-D-D-J Rearrangements and VH Replacement Events in Lupus Prone and Nonautoimmune TdT−/− and TdT+/+ Mice

CDR3 regions containing two D segments, or containing the footprints of VH replacement events, have been reported in both mice and humans. However, the 12–23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for VH replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential VH replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the “cryptic heptamer” but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and VH replacement are rare occurrences in the mouse.

Tolerance-induced receptor selection: scope, sensitivity, locus specificity, and relationship to lymphocyte-positive selection

Summary:  Receptor editing is a mode of immunological tolerance of B lymphocytes that involves antigen‐induced B‐cell receptor signaling and consequent secondary immunoglobulin light chain gene recombination. This ongoing rearrangement often changes B‐cell specificity for antigen, rendering the cell non‐autoreactive and sparing it from deletion. We currently believe that tolerance‐induced editing is limited to early stages in B‐cell development and that it is a major mechanism of tolerance, with a low‐affinity threshold and the potential to take place in virtually every developing B cell. The present review highlights the contributions from our laboratory over several years to elucidate these features.

Peripheral B Cell Tolerance and Function in Transgenic Mice Expressing an IgD Superantigen

Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane-bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored by the increased numbers of IgM+ plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD.

Split Tolerance in Peripheral B Cell Subsets in Mice Expressing a Low Level of Igκ-Reactive Ligand

Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed κ-macroself Ag, a synthetic superantigen reactive to Igκ. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature κ+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of κ+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,κ. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.

Effect of cell:cell competition and BAFF expression on peripheral B cell tolerance and B‐1 cell survival in transgenic mice expressing a low level of Igκ‐reactive macroself antigen

In mice carrying a synthetic Igκ‐reactive superantigen (“κ macroself antigen”), low level expression induced split peripheral B cell tolerance in the sIgκ+ compartment, with striking reductions in follicular and marginal zone (MZ) B cells and the retention of significant numbers of sIgκ+ B‐1a but not B‐1b cells in the peritoneum. Here, we characterize the transgenic line pKκ with this split tolerance phenotype and assess the effects of B cell competition and the survival cytokine BAFF (B cell activating factor belonging to the TNF family) on peripheral tolerance. In pKκ mice the surviving peritoneal and splenic κ+ B cells were largely lost in mice carrying one copy of the human Cκ exon in place of the mouse version, a maneuver that generates additional antigen non‐reactive competitor B cells in this model. Furthermore, overexpression of BAFF suppressed κ‐macroself antigen‐induced deletion and promoted production of both IgM,κ and IgA,κ antibodies in mice with normal Igκ alleles but not in mice carrying one copy of the human Cκ allele. These findings suggest that BAFF overexpression has minimal effects on the survival of autoreactive B cells in a polyclonal immune system and that B cell:B cell competition plays a potent role in suppressing the survival of B‐1 and splenic B cells with excessive autoreactivity.