PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

Receptor editing and marginal zone B cell development are regulated by the helix-loop-helix protein, E2A.

Previous studies have indicated that the E2A gene products are required to initiate B lineage development. Here, we demonstrate that E2A(+/-) B cells that express an autoreactive B cell receptor fail to mature due in part to an inability to activate secondary immunoglobulin (Ig) light chain gene rearrangement. Both RAG1/2 gene expression and RS deletion are severely defective in E2A(+/-) mice. Additionally, we demonstrate that E2A(+/-) mice show an increase in the proportion of marginal zone B cells with a concomitant decrease in the proportion of follicular B cells. In contrast, Id3-deficient splenocytes show a decline in the proportion of marginal zone B cells. Based on these observations, we propose that E-protein activity regulates secondary Ig gene rearrangement at the immature B cell stage and contributes to cell fate determination of marginal zone B cells. Additionally, we propose a model in which E-proteins enforce the developmental checkpoint at the immature B cell stage.

The scope of receptor editing and its association with autoimmunity.

Random assembly of antibody variable (V), diversity (D) and joining (J) gene segments creates a vast repertoire of antigen receptors, including autoreactive ones. Three ways that are known to reduce autoreactivity in the B-cell compartment include clonal deletion, functional inactivation and receptor editing, a mechanism involving a change in antigen receptor specificity through continued V(D)J recombination. New data suggest that editing can efficiently eliminate autoreactivity, yet, in an autoimmune context, secondary antibody gene rearrangements might also contribute to autoimmunity.

Peripheral B lymphocyte tolerance.

This lecture discusses two interrelated topics, B cell tolerance in the peripheral immune system and BAFF. Using the 3-83 antibody transgenic mouse bred to mice carrying cognate antigen in the liver, we previously found that clonal elimination drastically reduced the precursor frequency of autoreactive cells. The consensus model to explain this tolerance is the 2-signal hypothesis, which proposes that in the absence of T cell help BCR stimulation is a negative signal for B cells. However, this model fails to explain how these same B cells can respond to T-independent type II (TI-2) antigens, raising the question of how they distinguish TI-2 antigens from multimeric self determinants. We propose that B cells use NK-like missing self recognition to provide the needed specificity, as foreign antigens are unlikely to carry self markers. The model has implications for the evolution of the immune system, B lymphocyte signaling, tissue specificity of autoimmunity, and microbial subversion of the immune system. Overexpression of the critical B cell survival cytokine BAFF/BLyS has been associated with autoimmunity. We have discovered a novel splice isoform that regulates BAFF activity and may play a role in limiting B cell activity. The novel form, called DBAFF, is able to heteromultimerize with normal BAFF and can suppress receptor binding and proteolytic release from the cell surface. Preliminary studies from transgenic mice overexpressing wild type or DBAFF are consistent with a possible regulatory role for DBAFF, raising the possibility that the relative expression levels of BAFF and DBAFF regulates tolerance.

FGD2, a CDC42-specific exchange factor expressed by antigen-presenting cells, localizes to early endosomes and active membrane ruffles.

Members of the Fgd (faciogenital dysplasia) gene family encode a group of critical guanine nucleotide exchange factors (GEFs), which, by specifically activating Cdc42, control cytoskeleton-dependent membrane rearrangements. In its first characterization, we find that FGD2 is expressed in antigen-presenting cells, including B lymphocytes, macrophages, and dendritic cells. In the B lymphocyte lineage, FGD2 levels change with developmental stage. In both mature splenic B cells and immature bone marrow B cells, FGD2 expression is suppressed upon activation through the B cell antigen receptor. FGD2 has a complex intracellular localization, with concentrations found in membrane ruffles and early endosomes. Although endosomal localization of FGD2 is dependent on a conserved FYVE domain, its C-terminal pleckstrin homology domain mediates recruitment to membrane ruffles. FGD2 overexpression promotes the activation of Cdc42 and leads to elevated JNK1 activity in a Cdc42- but not Rac1-dependent fashion. These findings are consistent with a role of FGD2 in leukocyte signaling and vesicle trafficking in cells specialized to present antigen in the immune system.

A role for nuclear factor kappa B/rel transcription factors in the regulation of the recombinase activator genes.

In developing B cells, expression of surface immunoglobulin is an important signal to terminate recombinase activator gene (RAG) expression and V(D)J recombination. However, autoreactive antigen receptors instead promote continued gene rearrangement and receptor editing. The regulation by B cell receptor (BCR) signaling of RAG expression and editing is poorly understood. We report that in editing-competent cells BCR ligand-induced RAG mRNA expression is regulated at the level of RAG transcription, rather than mRNA stability. In immature B cells carrying innocuous receptors, RAG expression appears to be under rapidly reversible negative regulation. Studies involving transduction of a superrepressive (sr) I kappa B alpha protein indicate that NF-kappaB/Rel proteins promote RAG transcription. Interestingly, NF kappa B1-deficient cells overexpress RAG and undergo an exaggerated receptor editing response. Our data implicate NF kappa B transcription factors in the BCR-mediated regulation of RAG locus transcription. Rapidly activated NF kappa B pathways may facilitate prompt antigen receptor-regulated changes in RAG expression important for editing and haplotype exclusion.

Haplotype exclusion and receptor editing: irreconcilable differences?

Features of antibody genes and their regulation hinder two properties thought to be critical for clonal selection: haplotype exclusion and receptor diversity. These properties include: (1) the retention of multiple independent L-chain isotypes, which compounds the problem of allelic exclusion with one of isotype exclusion; (2) the process of receptor editing, in which recombination continues in cells already expressing antigen receptors; and (3) non-random associations and quasi-ordered rearrangements of the elements that generate light chain genes, which promote editing at the expense of allelic exclusion and receptor diversification. In contrast, heavy chain gene structure seems to promote haplotype exclusion and receptor diversity. It appears that requirements of receptor selection, such as the need for receptor editing as an immune tolerance mechanism and positive selection as a quality control checkpoint for receptor functionality, impose independent selections that shape the organization and regulation of the antibody genes. Despite these features, B cell development still achieves a significant level of phenotypic haplotype exclusion, suggesting that there is indeed significant selection for antibody monospecificity that is accommodated along with receptor editing. Thus, the immune system achieves both receptor selection and clonal selection, despite their partly antagonistic mechanisms.

CTCF functions as a critical regulator of cell-cycle arrest and death after ligation of the B cell receptor on immature B cells.

The WEHI 231 B cell lymphoma is used as a model of self-tolerance by clonal deletion because B cell receptor (BCR) ligation results in apoptosis. Two critical events precede cell death: an early rise and fall in expression of MYC and cell-cycle arrest associated with enhanced expression of p21, p27, and p53. CTCF is a transcription factor identified as a repressor of MYC recently shown to cause cell growth inhibition. The present studies demonstrate that BCR ligation of WEHI 231 as well as of normal immature B cells greatly increased expression of CTCF in association with down-regulation of MYC followed by growth arrest and cell death. Conditional expression of CTCF in WEHI 231 mimicked BCR ligation with activated cells showing repressed expression of MYC, enhanced expression of p27, p21, p53, and p19(ARF), and inhibition of cell growth and induction of apoptosis. In keeping with a central role for CTCF in control of B cell death, conditional expression of a CTCF antisense construct in WEHI 231 resulted in inhibition of p27, p21, p53, and p19(ARF) in association with enhanced expression of MYC. Activation of the endogenous CTCF locus by BCR ligation was also mimicked by three other routes to apoptotic death in WEHI 231: inhibition of the phosphoinositide 3-kinase or mTORFRAP signaling cascades and treatment with transforming growth factor (TGF)-beta. Rapid activation of CTCF by BCR ligation or treatment with TGF-beta was suppressed by ligation of CD40. These results demonstrate that CTCF is a common determinant to different pathways of death signaling in immature B cells.

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

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.