Systemic lupus erythematosus (SLE) is a clinically heterogenous autoimmune disease that is caused by a combination of self-reactive B cells, T cells, and environmental factors. Although self-reactive lymphocytes are present in healthy individuals, it is not understood how the convergence of these cells with environmental factors leads to SLE (etiology). Hallmark features of SLE are circulating antibodies that recognize nuclear molecules and high levels of protein in the urine that results from antigen-antibody complex deposition in the kidneys, leading to organ failure. We now report that if a transcription factor called Kruppel-like factor 2 (KLF2) is eliminated from all B cells, the resulting conditional knockout mice (KLF2 cKO) develop SLE. This includes high concentrations of anti-nuclear antibodies in the sera and elevated protein levels in the urine. Preliminary data suggests that KLF2-deficient B1 cells are responsible for this breakdown in self-tolerance. Unlike conventional B cells that circulate in the blood and secondary lymphoid organs (e.g., spleen), B1 cells are typically confined to spaces surrounding the guts (peritoneal cavity) and lungs (pleural cavity). We have found that loss of KLF2 causes the B1 cells to leave the body cavities and home to the spleen, where they can interact with circulating T cells. Based on the expression pattern of KLF2 in normal B1 cells (resting B1 cells express high levels of KLF2; activated B1 cells do not express KLF2), we hypothesize that KLF2 is used to regulate B1 cell migration patterns in healthy individuals and a breakdown in this process can promote SLE.

We propose the following model of SLE etiology: Under normal circumstances, B1 cells that encounter commensal microbes in the guts or lungs become activated, leading to reduced expression of KLF2. In turn, these KLF2-deficient B1 cells home to the spleen, where they may present commensal antigens to T cells. This event is predicted to induce tolerance toward commensal antigens and thus prevent T cells from unnecessarily attacking innocuous molecules in the lungs and the guts. Once these B1 cells have finished interacting with the T cells, the B1 cells revert to a resting state and upregulate expression of KLF2. At this point, the KLF2-expressing B1 cells migrate back to the body cavities to start this process over again. In a sense, this migration pattern is akin to a treadmill. In the case of KLF2 cKO mice, however, the treadmill is now broken and the KLF2-deficient B1 cells are stuck in the spleen. Thus, a unique tolerizing mechanism that links B cells, T cells, and environmental factors is halted, which eventually leads to SLE. To test our hypothesis and associated model, we propose the following studies:

Aim 1: Test the hypothesis that B1 cells engage splenic CD4+ T cells to promote peripheral tolerance. B1 cells are found in body cavities, but removal of KLF2 using gene-targeted mouse models causes these cells to home to T cell-rich areas of the spleen. In Aim 1 studies, we will demonstrate that KLF2-deficient B1 cells directly interact with CD4^+ T cells in the spleen. Moreover, we will test if these interactions lead to the functional inactivation (tolerization) of antigen-specific CD4^+ T cells. Successful completion of these studies will prove that a primary role of B1 cells is to maintain tolerance toward innocuous commensal antigens. Not only would these results be paradigm-shifting in the field of immunology, but it would help explain how environmental factors (commensals) interface with self-reactive lymphocytes to promote SLE.

Aim 2: Test if KLF2-deficient B1 cells are directly responsible for SLE. KLF2 cKO mice present will hallmark symptoms of SLE, and defective B1 cell activity correlates with disease; however, it has yet to be proven that B1 cells are causal. To do so, we will generate chimeric mice that possess either KLF2-deficient B1 cells (and normal conventional B cells) or KLF2-deficient conventional B cells (and normal B1 cells). In this manner, we can determine which B cell lineage is responsible for SLE. Successful completion of this study will identify a B cell population that is amenable to therapeutic intervention.

Completion of these aims will define an event that contributes to SLE and thus addresses the Fiscal Year 2021 Lupus Research Program Focus Area, “Understanding the biological mechanisms of lupus disease including, but not limited to, studies of informative/rare patients.” I would like to stress that we are not suggesting direct targeting of KLF2 in human patients to treat SLE; instead, we propose targeting/augmenting a yet-to-be identified B cell function that requires KLF2 to prevent disease. For example, should we discover that defective B1 cell migration causes lupus in our KLF2 cKO mouse model, we will screen lupus patients’ circulating white blood cells to see if a similar mechanism is present in humans. In particular, cohorts of ethnic Chinese patients with low expression levels of KLF2 are more susceptible to SLE than patients with normal levels of KLF2. For this reason, we have established a collaboration with our colleagues who conducted the KLF2 study in SLE patients to verify that results discovered in our unique animal model translate to human patients. Once the basic tenets of SLE etiology have been established (i.e., identify the B cell lineage and mechanism responsible for SLE), therapeutics can be designed to augment KLF2-dependent B cell functions (e.g., adoptive transfer of “normal” B1 cells into lupus patients). It is presently unknown how universal this KLF2-deficient B cell event is to SLE patients. At the very least, it should benefit ethnic Chinese patients with low levels of KLF2. Alternatively, this KLF2-dependent mechanism may contribute to disease onset in a large percentage of lupus patients.