Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase-and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved. (Blood. 2008;112:5026-5036) IntroductionWhen apoptosis occurs at high rates in mammalian tissues, apoptotic cells are almost invariably encountered in situ in association with macrophages. 1 These professional scavengers are attracted to the dying cells and engage in their safe, nonphlogistic disposal by phagocytosis. Examples of this innate immune response to dying cells are readily apparent during normal organogenesis, in normal adult tissues, such as the germinal centers of lymphoid follicles, in inflammatory responses, and in pathologic conditions including tumors. The efficient clearance of apoptotic cells by phagocytes is a homeostatic mechanism that militates against histotoxic, proinflammatory, or immunogenic effects that may result from persistence of apoptotic cells. [1][2][3] In recent years, much progress has been made in improving our understanding of the molecular mechanisms underlying the interactions between apoptotic cells and macrophages and the immunologic implications of those interactions. [1][2][3][4][5][6] Before the tethering/ engulfment phases of macrophage-mediated apoptotic-cell clearance, phagocytes are required to navigate effectively to sites of apoptosis. Active release of chemoattractant ("find-me") signals from apoptotic cells at an early stage after engagement of the cell-death program would be predicted to underpin this process, but knowledge of the molecules involved is currently limited. Lysophosphatidylcholine (LPC) is released from apoptotic cells and functions in soluble form as a chemoattractant for mononuclear phagocytes. 7 Significantly, no chemokine family members have previously been implicated in this chemotactic process. Here we show that the chemokine and adhesion molecule CX3CL1, 8,9 also known as neurotactin or fractalkine (FKN), together with its cognate receptor CX3CR1, 10,11 plays an active role in the chemotaxis of macrophages to apoptotic cells. FKN is a type I transmembrane protein, the extracellular portion of which comprises the chemokine domain attached to a mucin stalk. Well known for its roles in inflammatory processe...

Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

BACKGROUND. Type 1 diabetes (T1D) results from loss of immune regulation, leading to the development of autoimmunity to pancreatic β cells, involving autoreactive T effector cells (Teffs). Tregs, which prevent autoimmunity, require IL-2 for maintenance of immunosuppressive functions. Using a response-adaptive design, we aimed to determine the optimal regimen of aldesleukin (recombinant human IL-2) to physiologically enhance Tregs while limiting expansion of Teffs. METHODS. DILfrequency is a nonrandomized, open-label, response-adaptive study of participants, aged 18-70 years, with T1D. The initial learning phase allocated 12 participants to 6 different predefined regimens. Then, 3 cohorts of 8 participants were sequentially allocated dose frequencies, based on repeated interim analyses of all accumulated trial data. The coprimary endpoints were percentage change in Tregs and Teffs and CD25 (α subunit of the IL-2 receptor) expression by Tregs, from baseline to steady state. RESULTS. Thirty-eight participants were enrolled, with thirty-six completing treatment. The optimal regimen to maintain a steady-state increase in Tregs of 30% and CD25 expression of 25% without Teff expansion is 0.26 × 10 6 IU/m 2 (95% CI-0.007 to 0.485) every 3 days. Tregs and CD25 were dose-frequency responsive, Teffs were not. The commonest adverse event was injection site reaction (464 of 694 events). CONCLUSIONS. Using a response-adaptive design, aldesleukin treatment can be optimized. Our methodology can generally be employed to immediately access proof of mechanism, thereby leading to more efficient and safe drug development.

Immune deficient mice, reconstituted with human stem cells, have been used to analyze human immune responses in vivo. Although they have been used to study immune responses to xenografts, allografts, and pathogens, there have not been models of autoimmune disease in which the mechanisms of the pathologic process can be analyzed. We have found that reconstituted “humanized” mice treated with anti-CTLA-4 antibody (ipilimumab) develop autoimmune disease characterized by hepatitis, adrenalitis, sialitis, ANAs, and weight loss. Induction of autoimmunity involved activation of T cells and cytokine production and increased infiltration of antigen presenting cells. When anti-CTLA-4 mAb treated mice were co-treated with anti-CD3 mAb (teplizumab) hepatitis and ANAs were no longer seen and weight loss did not occur. The anti-CD3 blocked proliferation and activation of T cells, release of IFNγ and TNF, macrophage infiltration and release of IP-10 that was induced with anti-CTLA-4 mAb. We also found increased levels of Tregs (CD25+CD127-) in the spleen and mesenteric lymph nodes in the mice treated with both antibodies and greater constitutive phosphorylation of STAT5 in Tregs in spleen cells compared to mice treated with anti-CTLA-4 mAb alone. We describe the first model of human autoimmune disease in vivo. Humanized mice may be useful for understanding the mechanisms of biologics that are used in patients. Hepatitis, lymphadenopathy, and other inflammatory sequelae are side effects of ipilumimab treatment in humans and the present study may provide insights into this pathogenesis and the effects of immunologics on autoimmunity.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function—a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.

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IntroductionType 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing β cells in the pancreatic islets, leading to insulinopenia and hyperglycaemia. Genetic analyses indicate that alterations of the interleukin-2 (IL-2) pathway mediating immune activation and tolerance predispose to T1D, specifically the polymorphic expression of the IL-2 receptor-α chain (CD25) on T lymphocytes. Replacement of physiological doses of IL-2 could restore self-tolerance and prevent further autoimmunity by enhancing the function of CD4+ T regulatory cells (Tregs) to limit the activation of auto reactive T effector cells (Teffs). In this experimental medicine study, we use an adaptive trial design to determine the optimal dosing regimen for IL-2 to improve Treg function while limiting activation of Teffs in participants with T1D.Methods and analysisThe Adaptive study of IL-2 dose frequency on Tregs in type 1 diabetes(DILfrequency) is a mechanistic, non-randomised, repeat dose open-label, response-adaptive study of 36 participants with T1D. The objective is to establish the optimal dose and frequency of ultra-low dose IL-2: to increase Treg frequency within the physiological range, to increase CD25 expression on Tregs, without increasing CD4+ Teffs. DILfrequency has an initial learning phase where 12 participants are allocated to six different doses and frequencies followed by an interim statistical analysis. After analysis of the learning phase, the Dose and Frequency Committee will select the optimal targets for Treg frequency, Treg CD25 expression and Teff frequency. Three groups of eight participants will be treated consecutively in the confirming phase. Each dose and frequency selected will be based on statistical analysis of all data collected from the previous groups.EthicsEthical approval for DILfrequency was granted on 12 August 2014.ResultsThe results of this study will be reported, through peer-reviewed journals, conference presentations and an internal organisational report.Trial registration numbersNCT02265809, ISRCTN40319192, CRN17571.

Background: Serious infections of the head and neck cause lymphedema that can lead to airway compromise and oropharyngeal obstruction. Lymphangiogenesis occurs in the head and neck during infection and after immunization. The goal of this project was to develop tools to image lymphatic vessels in living animals and to be able to isolate individual lymphatic endothelial cells in order to quantify changes in single cells caused by inflammation. Methods: The ProxTom transgenic red-fluorescent reporter mouse was developed specifically for the purpose of imaging lymphatic vessels in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in development and for the maintenance of lymphatics in adulthood. Mice were immunized and their lymphatic vessels in lymph nodes were imaged in vivo. Individual lymphatic endothelial cells were isolated by means of their fluorescence. Results: The ProxTom transgene has the red-fluorescent reporter td-Tomato under the control of Prox1 regulatory elements. tdTomato was faithfully expressed in lymphatic vessels coincident with endogenous Prox1 expression. We show lymphangiogenesis in vivo after immunization and demonstrate a method for the isolation of lymphatic endothelial cells by their tdTomato red-fluorescence. Conclusions: The faithful expression of the red-fluorescent reporter in the lymphatic vessels of ProxTom means that these mice have proven utility for in vivo study of lymphatic vessels in the immune response. ProxTom has been made available for distribution from the Jackson