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David Nemazee

Publications and source records attributed to David Nemazee.

At least 19 recordsLinked to original sources
PubMed2006

Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling.

Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

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 V(H) 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 V(H) 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 V(H) 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 V(H) replacement are rare occurrences in the mouse.

Animals↗
PubMed2006

Split tolerance in peripheral B cell subsets in mice expressing a low level of Igkappa-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 kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. 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 kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.

Animals↗
PubMed2006

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 Igkappa-reactive macroself antigen.

In mice carrying a synthetic Igkappa-reactive superantigen ("kappa macroself antigen"), low level expression induced split peripheral B cell tolerance in the sIgkappa+ compartment, with striking reductions in follicular and marginal zone (MZ) B cells and the retention of significant numbers of sIgkappa+ B-1a but not B-1b cells in the peritoneum. Here, we characterize the transgenic line pKkappa 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 pKkappa mice the surviving peritoneal and splenic kappa+ B cells were largely lost in mice carrying one copy of the human Ckappa 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 kappa-macroself antigen-induced deletion and promoted production of both IgM,kappa and IgA,kappa antibodies in mice with normal Igkappa alleles but not in mice carrying one copy of the human Ckappa 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.

Animals↗
PubMed2006

Receptor editing in lymphocyte development and central tolerance.

The specificities of lymphocytes for antigen are generated by a quasi-random process of gene rearrangement that often results in non-functional or autoreactive antigen receptors. Regulation of lymphocyte specificities involves not only the elimination of cells that display 'unsuitable' receptors for antigen but also the active genetic correction of these receptors by secondary recombination of the DNA. As I discuss here, an important mechanism for the genetic correction of antigen receptors is ongoing recombination, which leads to receptor editing. Receptor editing is probably an adaptation that is necessitated by the high probability of receptor autoreactivity. In both B cells and T cells, the genes that encode the two chains of the antigen receptor seem to be specialized to promote, on the one hand, the generation of diverse specificities and, on the other hand, the regulation of these specificities through efficient editing.

Animals↗
PubMed2006

B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels.

Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and DeltaBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and DeltaBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and DeltaBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and DeltaBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.

Arthritis, Rheumatoid↗
PubMed2005

deltaBAFF, a splice isoform of BAFF, opposes full-length BAFF activity in vivo in transgenic mouse models.

DeltaBAFF 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 DeltaBAFF to multimerize with full-length BAFF and to limit BAFF proteolytic shedding from the cell surface, we previously proposed a role for DeltaBAFF in restraining the effects of BAFF and in regulating B lymphocyte homeostasis. To test these ideas we generated mice transgenic for DeltaBAFF 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 DeltaBAFF and BAFF had opposing effects on B cell survival and marginal zone B cell numbers. DeltaBAFF 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 DeltaBAFF appeared to modulate the numbers of B-1 phenotype B cells.

Alternative Splicing↗
PubMed2005

The design and implementation of the immune epitope database and analysis resource.

Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance is reflected in the vast amount of epitope-related information gathered, ranging from interactions between epitopes and major histocompatibility complex molecules determined by X-ray crystallography to clinical studies analyzing correlates of protection for epitope based vaccines. Our goal is to provide a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, "The Immune Epitope Database and Analysis Resource." We describe how we plan to capture, structure, and store this information, what query interfaces we will make available to the public, and what additional predictive and analytical tools we will provide.

Animals↗
PubMed2005

An immunoglobulin C kappa-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-Igkappa-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, Igkappa-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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2004

B cell selection defects underlie the development of diabetogenic APCs in nonobese diabetic mice.

One mechanism whereby B cells contribute to type 1 diabetes in nonobese diabetic (NOD) mice is as a subset of APCs that preferentially presents MHC class II-bound pancreatic beta cell Ags to autoreactive CD4 T cells. This results from their ability to use cell surface Ig to specifically capture beta cell Ags. Hence, we postulated a diabetogenic role for defects in the tolerance mechanisms normally blocking the maturation and/or activation of B cells expressing autoreactive Ig receptors. We compared B cell tolerance mechanisms in NOD mice with nonautoimmune strains by using the IgHEL and Ig3-83 transgenic systems, in which the majority of B cells recognize one defined Ag. NOD- and nonautoimmune-prone mice did not differ in ability to delete or receptor edit B cells recognizing membrane-bound self Ags. However, in contrast to the nonautoimmune-prone background, B cells recognizing soluble self Ags in NOD mice did not undergo partial deletion and were also not efficiently anergized. The defective induction of B cell tolerance to soluble autoantigens is most likely responsible for the generation of diabetogenic APC in NOD mice.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

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.

Animals↗
PubMed2003

DeltaBAFF, 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 deltaBAFF, which lacks 57 nt encoding the A-A1 loop and is co-expressed with BAFF in many mouse and human myeloid cells. Mouse deltaBAFF appears on the plasma membrane, but unlike BAFF it is inefficiently released by proteolysis. DeltaBAFF 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 deltaBAFF may promote other functions as well.

Alternative Splicing↗
PubMed2003

Visualization of the genesis and fate of isotype-switched B cells during a primary immune response.

The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment.

Adoptive Transfer↗