CCL4Running title: NR4A family renders B cells dependent upon a second signal ABSTRACT Ag stimulation (signal 1) triggers B cell activation and proliferation, and primes B cells to recruit, engage, and respond to T cell help (signal 2). However, failure to receive signal 2 within a defined window of time results in an abortive round of proliferation, followed by anergy or apoptosis. Although the molecular basis of T cell help has been extensively dissected, the mechanisms that restrain Ag-stimulated B cells, and enforce dependence upon co-stimulation, are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell receptor (BCR) stimulation, yet little is known about their function in humoral immune responses. Here we use germline and conditional loss-of-function mouse models to show that Nr4a1 and Nr4a3 play partially redundant roles to restrain both the survival and proliferation of B cells that receive signal 1 in the absence of co-stimulatory signals, and do so in part by repressing expression of BATF and consequently c-MYC. Correspondingly, Ab responses to TI-2 immunogens are enhanced in the absence of Nr4a1, but are unaltered in response to immunogens that incorporate co-stimulatory signals.Unexpectedly, we also identify a role for the NR4A family in restraining B cell access to T cell help by repressing expression of the T cell chemokines CCL3/4, as well as CD86 and ICAM1, and show that this is relevant under conditions of competition for limiting T cell help. Our studies collectively reveal a novel negative feedback loop mediated by the
NR4A family that increases B cell dependence upon T cell help and restrains stronglyAg-activated B cell clones from monopolizing limiting amounts of T cell help. We speculate that this imposes B cell tolerance and dampens immunodominance to facilitate preservation of clonal diversity during an immune response.were from The Jackson Laboratory 32-38 . TCRalpha-/-were obtained from the Weiss lab 39 . Nr4a3-/-mice were generated via electroporation of gRNA and Cas9 mRNA. In brief, Cas9 protein (40µM) and Nr4a3 gRNAs (80µM) were mixed and electroporated into C57BL/6 zygotes. The sequence of Nr4a3 exon 3 (containing start ATG) targeted for deletion is shown in Fig 4E. 15 founder lines with the targeted deletion were identified through a screen for PCR amplicon size, and confirmed via sequencing of cloned PCR products. Two founder lines were chosen for further analysis and were backcrossed for at least 4 generations onto the C57BL/6J genetic background. All other strains were fully backcrossed to the C57BL/6J genetic background for at least 6 generations. All mice were housed in a specific pathogen-free facility at UCSF according to University and National Institutes of Health guidelines.Antibodies and Reagents.
The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles upstream of Foxp3 to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to isolate the functions of this family in other immune cells. Here we take advantage of a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1-/- Nr4a3-/- (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional and phenotypic features of anergy accumulate in chimeric mice. Despite this, DKO T cells exhibit enhanced IL-2 production, implying a cell-intrinsic role for the NR4A family in peripheral T cell tolerance. These studies reveal roles for the NR4A family in multiple layered T cell tolerance mechanisms and demonstrate that each is essential to preserve immune homeostasis.
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