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Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.

CD8(+) T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8(+) T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at “re-invigorating” Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1(+) progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

Systemic lupus erythematosus severity correlates with elevated serum levels of type I IFNs, cytokines produced in large quantities by plasmacytoid dendritic cells (pDC) in response to engagement of TLR7 and TLR9 with endocytosed nucleic acids. B cell adaptor for PI3K (BCAP) promoted many aspects of TLR7-driven lupus-like disease, including Isg15 and Ifit1 expression in blood and an immature pDC phenotype associated with higher IFN production. BCAP−/− mice produced significantly less serum IFN-α than wild-type mice after injection of TLR9 agonist, and BCAP promoted TLR7 and TLR9-induced IFN-α production specifically in pDC. TLR-induced IFN-α production in pDC requires DOCK2-mediated activation of Rac1 leading to activation of IKKα, a mechanism we show was dependent on BCAP. BCAP−/− pDC had decreased actin polymerization and Rac1 activation and reduced IKKα phosphorylation upon TLR9 stimulation. We show a novel role for BCAP in promoting TLR-induced IFN-α production in pDC and in systemic lupus erythematosus pathogenesis.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically, and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data revealed the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

During an infection the antigen non-specific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T-cell receptor (TCR) signals that bystander activated, cytotoxic CD8 T-cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL mediated killing led to a significant defect in pathogen clearance. Together these data suggest a previously unappreciated, innate role for memory CD8 T-cells in the early immune response before the onset of a de-novo generated, antigen-specific CD8 T-cell response.

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr−/− mice (as we previously reported in Apoe−/− mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

Systemic lupus erythematosus (SLE) severity is correlated with elevated serum type I interferons (IFN), mainly IFNα, which is produced in large amounts by pDC in response to nucleic acid sensing by TLR7 and TLR9. TLR7 and TLR9-induced IRF7 translocation to the nucleus and subsequent IFNα production by pDC is dependent on phosphatidylinositol-3 kinase (PI3K), but how PI3K regulates this process remains undefined. We showed that B cell adaptor for PI3K (BCAP) links TLRs to PI3K activation in macrophages, thus we asked if BCAP plays a role in pDC IFNα production and SLE pathogenesis. We show BCAP promoted many aspects of TLR7-driven lupus-like disease including interferon-stimulated gene expression in blood. BCAP promoted TLR7 and TLR9-induced IFNα production in pDC, and BCAP−/− mice produced significantly less serum IFNα after injection of TLR9 agonist than WT mice, consistent with a pDC IFNα defect. TLR-induced IFNα production in pDC involves dual signaling pathways that run in parallel and converge upon the phosphorylation and activation of IKKα. There is a TLR-independent pathway initiated by nucleic acid recognition at the plasma membrane that involves Dock2-mediated activation of Rac1, required for phosphorylation of IKKα. The TLR-dependent pathway occurs upon nucleic acid recognition by endosomal TLR7 or 9 and leads to MyD88 activation. BCAP regulated IFNα production independently of the TLR-MyD88 pathway and both BCAP and PI3K were required for CpG DNA-induced early actin remodeling, a readout of Rac1 activation, and IKKα phosphorylation in pDC. Our data suggest BCAP and PI3K specifically regulate the TLR-parallel pathway in pDC. Overall, we show a novel role for BCAP in regulating IFNα production in pDC and SLE pathogenesis.

Objective— Enhanced plasminogen activation, mediated by overexpression of urokinase-type plasminogen activator (uPA), accelerates atherosclerosis in apolipoprotein E–null mice. However, the mechanisms through which uPA acts remain unclear. In addition, although elevated uPA expression can accelerate murine atherosclerosis, there is not yet any evidence that decreased uPA expression would retard atherosclerosis. Methods and Results— We used a bone marrow transplant (BMT) approach and apolipoprotein E–deficient ( Apoe −/− ) mice to investigate cellular mechanisms of uPA-accelerated atherosclerosis, aortic dilation, and sudden death. We also used BMT to determine whether postnatal loss of uPA expression in macrophages retards atherosclerosis. BMT from uPA-overexpressing mice yielded recipients with macrophage-specific uPA overexpression; whereas BMT from uPA knockout mice yielded recipients with macrophage-specific loss of uPA expression. Recipients of uPA-overexpressing BM acquired all the vascular phenotypes (accelerated atherosclerosis, aortic medial destruction and dilation, severe coronary stenoses) as well as the sudden death phenotype of uPA-overexpressing mice. Moreover, fat-fed 37-week-old recipients of uPA-null BM had significantly less atherosclerosis than recipients of uPA wild-type marrow (40% less aortic surface lesion area; P =0.03). Conclusions— The level of uPA expression by macrophages—over a broad range—is an important determinant of atherosclerotic lesion growth in Apoe −/− mice.

Systemic lupus erythematosus (SLE) severity is correlated with elevated serum levels of type I interferons (IFN), specifically IFNα. pDC are important in the pathogenesis and etiology of SLE due to their ability to produce large amounts of IFNα. TLR-induced INFα production in pDC occurs upon RNA or DNA recognition by TLR7 or 9 respectively. Two events are necessary to induce IFNα by pDC: 1) the binding of ligand to TLR7 or TLR9 within the early endosome, and 2) the phosphorylation and nuclear translocation of the transcription factor IRF7. TLR7 and TLR9-induced IRF7 translocation to the nucleus and subsequent IFN production by pDC is dependent on phosphatidylinositol-3 kinase (PI3K), but how PI3K regulates this process remains undefined. The PI3K pathway is important for many cellular processes, but it is unknown how PI3K activation is induced by TLR signaling in pDC. Our lab showed that the cytosolic signaling adapter B cell adaptor for PI3K (BCAP) links TLRs to PI3K activation in macrophages, resulting in inhibition of TLR-induced inflammatory cytokines. In contrast, here we show BCAP promotes TLR7 and TLR9-induced type I IFN production in pDC. We found that BCAP was required for optimal TLR9 and TLR7-induced IFNα production in pDC grown from BM in vitro in presence of Flt3L or sorted directly ex vivo, while inflammatory cytokine levels were less affected. Strikingly, BCAP−/− mice produced much less serum IFNα early after injection of TLR9 agonist than WT mice, consistent with a pDC IFNα defect. BCAP−/−pDC have reduced AKT phosphorylation when stimulated with TLR agonists, showing BCAP activates PI3K downstream of TLR in pDC. Overall, we show a novel role for BCAP in regulating IFNα production in pDC.

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr(-/-) mice (as we previously reported in Apoe(-/-) mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

B cell adaptor for PI3-kinase (BCAP) is a cytosolic adaptor protein involved in phosphatidylinositol-3 kinase (PI3K) activation in several immune lineages. In macrophages, we have shown that BCAP links TLR to phosphatidylinositol-3 kinase (PI3K) activation, thereby negatively regulating the production of proinflammatory cytokine production. BCAP is also expressed in plasmacytoid DC (pDC), in which PI3K signaling has been implicated in TLR responses. Therefore we investigated the role of BCAP in TLR7 and TLR9 responses in pDC. We confirmed high expression of BCAP in pDC by intracellular staining. We then examined pDC responses to TLR7 and TLR9 agonists. pDC grown from bone marrow in Flt3L from BCAP-/- mice secreted lower amounts of IFNa than wild type (WT) pDC in response to the TLR9 agonist CpG-C or TLR7 agonists R848 and ssRNA40. BCAP-/- pDC also had decreased Akt phosphorylation upon TLR7 and TLR9 stimulation, consistent with the function of BCAP to activate the PI3K pathway downstream of TLR in macrophages. BCAP-/- pDC secreted similar or higher levels of IL-6 than WT pDC, similar to inhibition of PI3K in human pDC. We found that BCAP-/- pDC directly isolated from bone marrow also showed reduced production of IFNa in response to CpG-C compared to WT pDC. Therefore, BCAP is a protein that specifically promotes TLR7/9-induced PI3K activation and IFNα production in pDC.

Recent studies illustrate that during an infection the antigen non-specific memory CD8 T cell compartment is not simply an inert pool of cells, but can participate in the immune response. This bystander activation of memory CD8 T cells occurs in the absence of cognate antigen recognition, but the overall role of T cell receptor (TCR) mediated signals has not been defined. Furthermore, it is unknown if and how these cells contribute to the clearance of an infection. We measured the strength of TCR signals that bystander activated CD8 T cells (BA-CTLs) receive in vivo and found evidence of TCR signaling, albeit limited. Given this marginal contribution of the TCR, we sought to determine how BA-CTLs efficiently kill target cells. We demonstrate that BA-CTLs recognize and directly eliminate target cells in an innate-like, granzyme-dependent manner. We next selectively inhibited BA-CTL mediated killing in vivo during the course of an infection, which led to a significant defect in pathogen clearance. Our data suggest that BA-CTLs play a previously unappreciated, essential role in the early immune response to pathogens before the onset of a de-novo generated, primary CD8 T cell response.