Ashley R. Sandy

A Distinct Subset of Proinflammatory Neutrophils Isolated from Patients with Systemic Lupus Erythematosus Induces Vascular Damage and Synthesizes Type I IFNs

Neutrophil-specific genes are abundant in PBMC microarrays from lupus patients because of the presence of low-density granulocytes (LDGs) in mononuclear cell fractions. The functionality and pathogenicity of these LDGs have not been characterized. We developed a technique to purify LDGs from lupus PBMCs and assessed their phenotype, function, and potential role in disease pathogenesis. LDGs, their autologous lupus neutrophils, and healthy control neutrophils were compared with regard to their microbicidal and phagocytic capacities, generation of reactive oxygen species, activation status, inflammatory cytokine profile, and type I IFN expression and signatures. The capacity of LDGs to kill endothelial cells and their antiangiogenic potential were also assessed. LDGs display an activated phenotype, secrete increased levels of type I IFNs, TNF-α, and IFN-γ, but show impaired phagocytic potential. LDGs induce significant endothelial cell cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells to differentiate into mature endothelial cells. LDG depletion restores the functional capacity of endothelial progenitor cells. We conclude that lupus LDGs are proinflammatory and display pathogenic features, including the capacity to synthesize type I IFNs. They may play an important dual role in premature cardiovascular disease development in systemic lupus erythematosus by simultaneously mediating enhanced vascular damage and inhibiting vascular repair.

Blockade of individual Notch ligands and receptors controls graft-versus-host disease

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell-mediated human disorders.

Notch signaling is a critical regulator of allogeneic CD4+ T-cell responses mediating graft-versus-host disease

Graft-versus-host disease (GVHD) remains the major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is caused by donor T cells that mediate host tissue injury through multiple inflammatory mechanisms. Blockade of individual effector molecules has limited efficacy in controlling GVHD. Here, we report that Notch signaling is a potent regulator of T-cell activation, differentiation, and function during acute GVHD. Inhibition of canonical Notch signaling in donor T cells markedly reduced GVHD severity and mortality in mouse models of allogeneic HSCT. Although Notch-deprived T cells proliferated and expanded in response to alloantigens in vivo, their ability to produce interleukin-2 and inflammatory cytokines was defective, and both CD4(+) and CD8(+) T cells failed to up-regulate selected effector molecules. Notch inhibition decreased the accumulation of alloreactive T cells in the intestine, a key GVHD target organ. However, Notch-deprived alloreactive CD4(+) T cells retained significant cytotoxic potential and antileukemic activity, leading to improved overall survival of the recipients. These results identify Notch as a novel essential regulator of pathogenic CD4(+) T-cell responses during acute GVHD and suggest that Notch signaling in T cells should be investigated as a therapeutic target after allogeneic HSCT.

Notch signaling in the hematopoietic system.

Because of its multiple effects in tissue homeostasis and cancer, Notch signaling is gaining increasing attention as a potential therapeutic target. Notch proteins belong to a family of highly conserved cell surface receptors. Ligand binding leads to proteolytic cleavage of Notch receptors by the γ-secretase complex, followed by translocation of the active intracellular Notch domain into the nucleus and transcriptional activation. Multiple genetic and pharmacological methods are available to inhibit or activate the Notch pathway, some of which are entering human clinical trials. In this review, we discuss our current understanding of Notch signaling in the hematopoietic system. Canonical Notch signaling is essential for the generation of definitive embryonic hematopoietic stem cells, but dispensable for their maintenance during adult life. Notch controls several early steps of T cell development, as well as specific cell fate and differentiation decisions in other hematopoietic lineages. In addition, emerging evidence indicates that Notch is a potent, context-specific regulator of T cell immune responses, including in several disease models relevant to patients. This knowledge will constitute a framework to explore Notch modulation as a therapeutic strategy and to understand potential hematopoietic side effects of systemic Notch inhibition.

T Cell–Specific Notch Inhibition Blocks Graft-versus-Host Disease by Inducing a Hyporesponsive Program in Alloreactive CD4+ and CD8+ T Cells

Graft-versus-host disease (GVHD) induced by donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). We previously reported that the pan-Notch inhibitor dominant-negative form of Mastermind-like 1 (DNMAML) markedly decreased the severity and mortality of acute GVHD mediated by CD4+ T cells in mice. To elucidate the mechanisms of Notch action in GVHD and its role in CD8+ T cells, we studied the effects of Notch inhibition in alloreactive CD4+ and CD8+ T cells using mouse models of allo-BMT. DNMAML blocked GVHD induced by either CD4+ or CD8+ T cells. Both CD4+ and CD8+ Notch-deprived T cells had preserved expansion in lymphoid organs of recipients, but profoundly decreased IFN-γ production despite normal T-bet and enhanced Eomesodermin expression. Alloreactive DNMAML T cells exhibited decreased Ras/MAPK and NF-κB activity upon ex vivo restimulation through the TCR. In addition, alloreactive T cells primed in the absence of Notch signaling had increased expression of several negative regulators of T cell activation, including Dgka, Cblb, and Pdcd1. DNMAML expression had modest effects on in vivo proliferation but preserved overall alloreactive T cell expansion while enhancing accumulation of pre-existing natural regulatory T cells. Overall, DNMAML T cells acquired a hyporesponsive phenotype that blocked cytokine production but maintained their expansion in irradiated allo-BMT recipients, as well as their in vivo and ex vivo cytotoxic potential. Our results reveal parallel roles for Notch signaling in alloreactive CD4+ and CD8+ T cells that differ from past reports of Notch action and highlight the therapeutic potential of Notch inhibition in GVHD.

Notch Signaling Regulates T Cell Accumulation and Function in the Central Nervous System during Experimental Autoimmune Encephalomyelitis

Systemic inhibition of Notch signaling was previously shown to attenuate experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis in mice. Different studies attributed these effects to decreased T-bet and IFN-γ expression, enhanced regulatory T cell function, reduced T cell chemotaxis to the CNS, or impaired Th9 cell differentiation. Interpretation of these heterogeneous findings is difficult because past experimental strategies did not ensure complete Notch inhibition in T cells and because many cell populations could be affected by systemic Notch blockade. To resolve the role of Notch in T cells during EAE, we used the pan-Notch inhibitor dominant-negative form of Mastermind-like 1 (DNMAML), as well as several complementary loss-of-function approaches specifically in myelin-reactive T cells. Notch inhibition in T cells profoundly decreased EAE incidence and severity. Notch-deprived myelin-reactive T cells had preserved activation and effector differentiation in secondary lymphoid tissues. However, Notch-deprived T cells failed to accumulate in the CNS after immunization. Parking wild-type and DNMAML T cells together in bone marrow chimeras increased accumulation of Notch-deprived T cells in the CNS after immunization but did not prevent EAE, indicating the absence of dominant suppression by DNMAML T cells. Analysis of CNS-infiltrating DNMAML T cells revealed markedly defective IL-17A and IFN-γ production, despite preserved T-bet expression. Collectively, our findings capture the profound overall effects of Notch signaling in myelin-reactive T cells and demonstrate that Notch controls the accumulation and pathogenic functions of CD4+ T cells within their target organ but not in lymphoid tissues during EAE.

Notch Signaling and Development of the Hematopoietic System

Notch signaling exerts multiple important functions in the hematopoietic system. Notch1-mediated signals are essential to induce the onset of definitive hematopoiesis within specialized domains of hemogenic endothelium in the fetal dorsal aorta. In contrast, Notch is dispensable for the subsequent maintenance of hematopoietic stem cells in the adult bone marrow. Notch is a key regulator of early T-cell development in the thymus. An expanding number of hematopoietic and lymphoid cell types have been reported to receive context-dependent inputs from the Notch pathway that regulate their differentiation and function. Progress in the field will continue to bring fundamental information about hematopoiesis and practical insights into the potential to modulate Notch signaling for therapeutic purposes.

TCR signaling and environment affect vasoactive intestinal peptide receptor-1 (VPAC-1) expression in primary mouse CD4 T cells

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).

Radical reversal of vasoactive intestinal peptide (VIP) receptors during early lymphopoiesis

Successful thymocyte maturation is essential for normal, peripheral T cell function. Vasoactive intestinal peptide (VIP) is a neuropeptide which is highly expressed in the thymus that has been shown to modulate thymocyte development. VIP predominantly binds two G protein coupled receptors, termed vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2, but their expression profiles in CD4(-)/CD8(-) (double negative, DN) thymocyte subsets, termed DN1-4, have yet to be identified. We hypothesized that a high VPAC1:VPAC2 ratio in the earliest thymocyte progenitors (ETP cells) would be reversed during early lymphopoiesis as observed in activated, peripheral Th(2) cells, as the thymus is rich in Th(2) cytokines. In support of this hypothesis, high VPAC1 mRNA levels decreased 1000-fold, accompanied with a simultaneous increase in VPAC2 mRNA expression during early thymocyte progenitor (ETP/DN1)→DN3 differentiation. Moreover, arrested DN3 cells derived from an Ikaros null mouse (JE-131 cells) failed to completely reverse the VIP receptor ratio compared to wild type DN3 thymocytes. Surprisingly, VPAC2(-/-) mice did not show significant changes in relative thymocyte subset numbers. These data support the notion that both VPAC1 and VPAC2 receptors are dynamically regulated by Ikaros, a master transcriptional regulator for thymocyte differentiation, during early thymic development. Moreover, high VPAC1 mRNA is a novel marker for the ETP population making it enticing to speculate that the chemotactic VIP/VPAC1 signaling axis may play a role in thymocyte movement. Also, despite the results that VPAC2 deficiency did not affect thymic subset numbers, future studies are necessary to determine whether downstream T cell phenotypic changes manifest themselves, such as a propensity for a Th(1) versus Th(2) polarization.

Gene expression profiling and network analysis of peripheral blood monocytes in a chronic model of allergic asthma.

The Aspergillus fumigatus mouse model of asthma mimics the characteristics of human fungal asthma, including local and systemic inflammation. Monocyte/macrophage lineage cells direct innate immune responses and guide adaptive responses. To identify gene expression changes in peripheral blood monocytes in the context of fungal allergy, mice were exposed to systemic and intranasal inoculations of fungal antigen (sensitized), and naïve and sensitized animals were challenged intratracheally with live A. fumigatus conidia. Microarray analysis of blood monocytes from allergic versus non‐allergic mice showed ≥ twofold modulation of 45 genes. Ingenuity pathway analysis revealed a network of these genes involved in antigen presentation, inflammation, and immune cell trafficking. These data show that allergen sensitization and challenge affects gene expression in peripheral monocytes.