Stephen H. Clarke

Single cell analysis shows decreasing FoxP3 and TGFβ1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes

Natural CD4+CD25+ regulatory T (CD4+CD25+ T reg) cells play a key role in the immunoregulation of autoimmunity. However, little is known about the interactions between CD4+CD25+ T reg cells and autoreactive T cells. This is due, in part, to the difficulty of using cell surface markers to identify CD4+CD25+ T reg cells accurately. Using a novel real-time PCR assay, mRNA copy number of FoxP3, TGFβ1, and interleukin (IL)-10 was measured in single cells to characterize and quantify CD4+CD25+ T reg cells in the nonobese diabetic (NOD) mouse, a murine model for type 1 diabetes (T1D). The suppressor function of CD4+CD25+CD62Lhi T cells, mediated by TGFβ, declined in an age-dependent manner. This loss of function coincided with a temporal decrease in the percentage of FoxP3 and TGFβ1 coexpressing T cells within pancreatic lymph node and islet infiltrating CD4+CD25+CD62Lhi T cells, and was detected in female NOD mice but not in NOD male mice, or NOR or C57BL/6 female mice. These results demonstrate that the majority of FoxP3-positive CD4+CD25+ T reg cells in NOD mice express TGFβ1 but not IL-10, and that a defect in the maintenance and/or expansion of this pool of immunoregulatory effectors is associated with the progression of T1D.

Autoreactive B Cell Regulation: Peripheral Induction of Developmental Arrest by Lupus-Associated Autoantigens

Anti-Sm and anti-ssDNA transgenic (Tg) mice were generated using the VH-D-JH rearrangement of an anti-Sm hybridoma of MRL/Mp-lpr/lpr origin. B cells of each specificity account for 15%-35% of the splenic repertoire, but no circulating anti-Sm or anti-ssDNA antibodies are detected. Most autoreactive cells exhibit an immature B cell phenotype and have short half-lives equivalent to those of non-Tg immature B cells. However, at least some anti-Sm B cells are functional, because immunization with murine snRNPs induces anti-Sm secretion. We propose that anti-Sm and anti-ssDNA are eliminated during the transition to mature B cells and that this late stage of tolerance induction is consequential to their spontaneous activation in murine lupus.

Lupus-Specific Antiribonucleoprotein B Cell Tolerance in Nonautoimmune Mice Is Maintained by Differentiation to B-1 and Governed by B Cell Receptor Signaling Thresholds

Systemic lupus erythematosus is an autoimmune disease characterized by the presence of autoantibodies. One of the unique targets of the immune system in systemic lupus erythematosus is Sm, a ribonucleoprotein present in all cells. To understand the regulation of B cells specific to the Sm Ag in normal mice, we have generated an Ig H chain transgenic mouse (2-12H Tg). 2-12H Tg mice produce B cells specific for the Sm that remain tolerant due to ignorance. We demonstrate here that anti-Sm B cells of 2-12H Tg mice can differentiate into Sm-specific peritoneal B-1 cells that remain tolerant. Differentiation to B-1 and tolerance are governed by the strength of B cell receptor signaling, since manipulations of the B cell receptor coreceptors CD19 and CD22 affect anti-Sm B cell differentiation and autoantibody production. These results suggest a differentiation scheme in which peripheral ignorance to Sm is maintained in mice by the differentiation of anti-Sm B cells to B-1 cells that have increased activation thresholds.

Early Preplasma Cells Define a Tolerance Checkpoint for Autoreactive B Cells

Ab-secreting plasma cells (PCs) are the effectors of humoral immunity. In this study, we describe regulation of autoreactive B cells specific for the ribonucleoprotein Smith (Sm) at an early pre-PC stage. These cells are defined by the expression of the PC marker CD138 and normal levels of CD19 and B220. They are present at a high frequency in normal mouse spleen and bone marrow, are Ag dependent, and are located predominantly along the T cell-B cell border and near bridging channels. Anti-Sm pre-PCs also occur at a high frequency in nonautoimmune mice and show additional phenotypic characteristics of PC differentiation. However, while some of these pre-PCs are Ab-secreting cells, those specific for Sm are not, indicating regulation. Consistent with this, anti-Sm pre-PCs have a higher turnover rate and higher frequency of cell death than those that do not bind Sm. Regulation of anti-Sm pre-PCs occurs upstream of the transcriptional repressor, B lymphocyte-induced maturation protein-1, expression. Regulation at this stage is overcome in autoimmune MRL/lpr mice and is accompanied by an altered B lymphocyte stimulator receptor profile. These data reveal a new B cell tolerance checkpoint that is overcome in autoimmunity.

Impaired Clearance of Apoptotic Cells Induces the Activation of Autoreactive Anti-Sm Marginal Zone and B-1 B Cells

Since apoptotic cell Ags are thought to be a source of self-Ag in systemic lupus erythematosus, we have examined the role of apoptotic cells in the regulation and activation of B cells specific for Sm, a ribonucleoprotein targeted in human and murine lupus. Using Ig-transgenic mice that have a high frequency of anti-Sm B cells, we find that apoptotic cell injection induces a transient splenic B cell response, while simultaneously causing extensive splenic and peritoneal anti-Sm B cell death. In contrast, mice deficient in the clearance of apoptotic cells develop a chronic anti-Sm response beginning at 1–2 mo of age. These mice have expanded marginal zone and B-1 B cell populations and anti-Sm B cells of both types are activated to form Ab-secreting cells. This activation appears to be Ag-specific, suggesting that activation is due to increased availability of apoptotic cell Ags. Since marginal zone and B-1 cells are positively selected, these data suggest a loss of ignorance rather than a loss of tolerance.

Low-Affinity Anti-Smith Antigen B Cells Are Regulated by Anergy as Opposed to Developmental Arrest or Differentiation to B-1

Understanding the regulation of B lymphocytes specific for self-Ags targeted in human and murine systemic lupus erythematosus, such as the ribonucleoprotein Smith Ag (Sm), is crucial to understanding the etiology of this autoimmune disease. To address the role of B cell receptor affinity in the regulation of anti-Sm B cells, we generated low-affinity anti-Sm transgenic mice by combining the anti-Sm 2-12H transgene with a Vκ8 transgene. In contrast to 2-12H transgenic mice, in which anti-Sm B cells are predominantly splenic transitional, and peritoneal B-1, low-affinity anti-Sm B cells are long-lived B-2 cells and are found in the spleen, lymph nodes, and peritoneum. However, they are unresponsive to LPS in vitro, indicating that they are anergic, although they do not down-regulate IgM and are not excluded from follicles even in the presence of nonautoreactive B cells. Thus, low-affinity anti-Sm B cells appear to have a partial form of anergy. Interestingly, these cells have elevated levels of MHC class II and CD95, but not CD40, CD80, or CD86, suggesting that they are poised to undergo deletion rather than activation upon T cell encounter. These data identify anergy as a mechanism involved in anti-Sm B cell regulation.

Positive selection focuses the VH12 B-cell repertoire towards a single B1 specificity with survival function.

Summary:  B cells of varying antigen specificities are consistently present in the unmanipulated repertoire. These B cells appear to belong to the marginal zone (MZ) and B1 B‐cell subsets and provide protection to the blood and lymph, respectively. Some are specific for self‐antigens, suggesting that they are selected based on specificity for self but have a protective role against foreign pathogens. One of these specificities is for phosphatidylcholine (PtC). Anti‐PtC B cells comprise 5–8% of the B1 repertoire and are protective against bacterial pathogens. In general, they are restricted to the expression of two VH/Vκ combinations, VH11/Vκ9 and VH12/Vκ4/5H. This review focuses on the differentiation of VH12 anti‐PtC B cells. They undergo a series of positive selection events beginning at the pre‐B‐cell stage that enriches for those with a VHCDR3 and L chain required for PtC binding and eliminating the majority of VH12 B cells that lack the ability to bind PtC. Thus, positive selection focuses the VH12 repertoire toward PtC, ensuring that anti‐PtC VH12 B cells are a significant component of the B1‐cell repertoire in all individuals.

Anti-Sm B Cell Differentiation in Ig Transgenic MRL/Mp-lpr/lpr Mice: Altered Differentiation and an Accelerated Response

To determine the regulation of B cells specific for the ribonucleoprotein Sm, a target of the immune system in human and mouse lupus, we have generated mice carrying an anti-Sm H chain transgene (2-12H). Anti-Sm B cells in nonautoimmune 2-12H-transgenic (Tg) mice are functional, but, in the absence of immunization, circulating anti-Sm Ab levels are not different from those of non-Tg mice. In this report, we compare the regulation of anti-Sm B cells in nonautoimmune and autoimmune MRL/Mp-lpr/lpr (MRL/lpr) and bcl-2-22-Tg mice. Activation markers are elevated on splenic and peritoneal anti-Sm B cells of both nonautoimmune and autoimmune genetic backgrounds indicating Ag encounter. Although tolerance to Sm is maintained in 2-12H/bcl-2-22-Tg mice, it is lost in 2-12H-Tg MRL/lpr mice, as the transgene accelerates and increases the prevalence of the anti-Sm response. The 2-12H-Tg MRL/lpr mice have transitional anti-Sm B cells in the spleen similar to nonautoimmune mice. However, in contrast to nonautoimmune mice, there are few if any peritoneal anti-Sm B-1 cells. These data suggest that a defect in B-1 differentiation may be a factor in the loss of tolerance to Sm and provide insight into the low prevalence of the anti-Sm response in lupus.

Identification of a Precursor to Phosphatidyl Choline-Specific B-1 Cells Suggesting That B-1 Cells Differentiate from Splenic Conventional B Cells In Vivo: Cyclosporin A Blocks Differentiation to B-1

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vκ4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vκ4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.

EBV Latent Membrane Protein 2A Induces Autoreactive B Cell Activation and TLR Hypersensitivity

EBV is associated with systemic lupus erythematosus (SLE), but how it might contribute to the etiology is not clear. Since EBV-encoded latent membrane protein 2A (LMP2A) interferes with normal B cell differentiation and function, we sought to determine its effect on B cell tolerance. Mice transgenic for both LMP2A and the Ig transgene 2-12H specific for the ribonucleoprotein Smith (Sm), a target of the immune system in SLE, develop a spontaneous anti-Sm response. LMP2A allows anti-Sm B cells to overcome the regulatory checkpoint at the early preplasma cell stage by a self-Ag-dependent mechanism. LMP2A induces a heightened sensitivity to TLR ligand stimulation, resulting in increased proliferation or Ab-secreting cell differentiation or both. Thus, we propose a model whereby LMP2A induces hypersensitivity to TLR stimulation, leading to activation of anti-Sm B cells through the BCR/TLR pathway. These data further implicate TLRs in the etiology of SLE and suggest a mechanistic link between EBV infection and SLE.