Regulatory T cells (Tregs) play a fundamental role in the suppression of different immune responses; however, compartments at which they exert suppressive functions in vivo are unknown. Although many groups have described the presence of Tregs within inflammatory sites, it has not been shown that inflamed tissues are, indeed, the sites of active suppression of ongoing immune reactions. Here, by using ␣ E ؉ effector/memory-like Tregs from fucosyltransferase VII-deficient animals, which lack E/P-selectin ligands and fail to migrate into inflamed sites, we analyzed the functional importance of appropriate Treg localization for in vivo suppressive capacity in an inflammation model. Lack of suppression by Tregs deficient in E/P-selectin ligands demonstrates that immigration into inflamed sites is a prerequisite for the resolution of inflammatory reactions in vivo because these selectin ligands merely regulate entry into inflamed tissues. In contrast, control of proliferation of naive CD4 ؉ T cells during the induction phase of the immune response is more efficiently exerted by the naive-like ␣ E ؊ CD25 ؉ Treg subset preferentially recirculating through lymph nodes when compared with its inflammation-seeking counterpart. [21][22][23] or inflamed intestine in induced colitis. 24 Moreover, a few studies demonstrated the capacity of Tregs to down-regulate established immune reactions, [24][25][26][27] suggesting that regulation might also act locally in the inflammatory environment.The resulting questions-in which compartment suppression by Tregs occurs and how critical is an appropriate localization of Tregs for their in vivo suppressive capacity-have not been addressed directly, as yet.We have previously shown that by use of the marker ␣ E (CD103) the Treg compartment can be subdivided into a lineage or differentiation stage of natural, naive-like ␣ E Ϫ CD25 ϩ Tregs and into that of ␣ E ϩ Tregs (either CD25 ϩ or CD25 Ϫ ) with the phenotype of effector/memory cells. 28,29 Whereas ␣ E Ϫ CD25 ϩ Tregs express high levels of CD62L and CCR7 and recirculate through lymph nodes, effector/memory-like ␣ E ϩ Tregs express high levels of inflammatory chemokine receptors and multiple adhesion molecules as well as selectin ligands and efficiently migrate into inflamed sites. Importantly, the integrin ␣ E ␤ 7 itself only seems to play a role for the retention within peripheral tissues, 30,31 but is not involved in the extravasation process of CD4 ϩ T lymphocytes. 32 In the present study it rather serves as a surrogate marker to identify inflammation-seeking Tregs.Migration of CD4 ϩ effector T cells into inflamed skin has been previously reported to be critically dependent on the expression of selectin ligands, 33,34 which bind to the partially redundantly acting E-and P-selectins, expressed almost exclusively on endothelium of For personal use only. on May 9, 2018. by guest www.bloodjournal.org From inflamed tissues. Biosynthesis of functional selectin ligands is mediated by several glycosyltransferases. 35 Among these enzymes, fuco...

The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type-specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements. (Blood. 2011;117(10):2827-2838)

The differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.

We have previously shown that naïve CD8 ؉ T cells recognizing their cognate antigen within the liver are retained and undergo activation in situ, independent from lymphoid tissues. Intrahepatic primary T cell activation results in apoptosis and may play a crucial role in the ability of the liver to induce tolerance. Although adhesion molecules required for intrahepatic retention of T cells that have undergone previous extra-hepatic activation have been characterized, adhesive interactions involved in selective antigen-dependent intrahepatic retention of naïve CD8 ؉ T cells have not been investigated. By adoptively transferring radiolabeled T cell receptor (TCR)-transgenic CD8 ؉ T cells into recipient animals ubiquitously expressing the relevant antigen, we show that 40% to 60 % of donor antigenspecific naïve CD8 ؉ T cells were retained in the liver within 1 hour after transfer, despite ubiquitous expression of the antigen. Intravital microscopy showed that most donor naïve T cells slowed down and were irreversibly retained intrahepatically within the first few minutes after adoptive transfer, strongly suggesting that they were directly activated by liver cells in situ. This process was largely dependent on LFA-1 and ICAM-1, but was independent of blocking with antibodies against VCAM-1, ␣4 integrin, P-selectin, VAP-1, and ␤1 integrin. ICAM-2 seemed to play only a minor role in this process.

Summary Cellular infiltration is a classic hallmark of inflammation. Whereas the role of T cells in many types of inflammation is well established, the specific impact of antigen recognition on their migration into the site and on the accumulation of other effector cells are still matters of debate. Using a model of an inflammatory effector phase driven by T‐cell receptor (TCR) transgenic T cells, we found (i) that antigen‐specific T cells play a crucial role as ‘pioneer cells’ that condition the tissue for enhanced recruitment of further T effector cells and other leucocytes, and (ii) that the infiltration of T cells is not dependent on antigen specificity. We demonstrate that a small number of antigen‐specific T cells suffice to initiate a cascade of cellular immigration into the antigen‐loaded site. Although antigen drives this process, accumulation of T cells in the first few days of inflammation was not dependent on T‐cell reactivity to the antigen. Both transgenic and wild‐type T effector cells showed enhanced immigration into the site of antigen challenge after the initial arrival and activation of antigen‐specific pioneer cells. This suggests that bystander accumulation of non‐specific effector/memory T cells is a general feature in inflammation. Furthermore, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were identified as mediators that contribute to conditioning of the inflammatory site for high‐rate accumulation of T effector cells in this T‐cell‐driven model.