Lymph node stromal cells acquire peptide–MHCII complexes from dendritic cells and induce antigen-specific CD4+ T cell tolerance

Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8⁺ T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4⁺ T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide–MHCII complexes from DCs and induce CD4⁺ T cell dysfunction by presenting transferred complexes to naive CD4⁺ T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4⁺ T cells through the presentation of self-antigens via transferred peptide–MHCII complexes of DC origin.Self-reactive T cells that escape thymic negative selection are kept in check by peripheral tolerance mechanisms that include T cell anergy and deletion. Research into how self-reactive T cells are tolerized in LNs has focused largely on DCs. Depending on their functional status, antigen presentation by DCs can indeed lead to different forms of T cell tolerance (Steinman et al., 2003; Helft et al., 2010). Recently, however, LN-resident radio-resistant cells, the LN stromal cells (LNSCs), have been suggested to contribute to peripheral T cell tolerance. These cells can be discriminated based on their lack of CD45 expression and the differential expression of podoplanin (gp38) and PECAM (CD31). Fibroblastic reticular cells (FRCs, gp38⁺CD31⁻) produce chemokines such as CCL19 and CCL21, thereby providing a scaffold on which the CC-chemokine receptor 7 (CCR7)⁺ T cells and DCs can migrate and establish contact (Turley et al., 2010). In LNs, blood endothelial cells (BECs, gp38⁻CD31⁺) lining the high endothelial venules are crucial for lymphocyte entry (Mueller and Germain, 2009). Afferent lymphatic endothelial cells (LECs, gp38⁺CD31⁺) promote DC entry (Johnson et al., 2006; Acton et al., 2012), as well as antigen delivery (Sixt et al., 2005; Roozendaal et al., 2009), into LNs, whereas efferent LECs regulate T cell egress from LNs (Cyster and Schwab, 2012). The function of so-called double-negative (DN) stromal cells (gp38⁻CD31⁻) remains unknown. For many years, LNSCs were thought to only play an architectural role in LN construction and homeostasis. More recently, however, studies have identified LNSCs as active players in modulating adaptive immune responses (Swartz and Lund, 2012). In vitro, DC adhesion to LECs leads to decreased levels of co-stimulatory molecules by DCs (Podgrabinska et al., 2009). Furthermore, FRCs inhibit the proliferation of newly activated T cells through a NOS2-dependent mechanism, but also indirectly affect T cell proliferation by suppressing DC functions (Khan et al., 2011; Lukacs-Kornek et al., 2011; Siegert et al., 2011). In addition, FRCs can suppress acute T cell proliferation both in vitro and in vivo (Siegert et al., 2011). Other studies have convincingly demonstrated a role for LNSCs in maintaining peripheral CD8⁺ T cell tolerance via direct presentation of self-antigens to self-reactive CD8⁺ T cells. Unlike DCs, which acquire antigens and subsequently cross-present self-peptides to CD8⁺ T cells in the draining LNs, LNSCs ectopically express and present PTAs (peripheral tissue antigens) to CD8⁺ T cells, and consequently induce clonal deletion of self-reactive CD8⁺ T cells (Lee et al., 2007; Nichols et al., 2007; Gardner et al., 2008; Magnusson et al., 2008; Yip et al., 2009; Cohen et al., 2010; Fletcher et al., 2010). In addition, we have recently shown that tumor-associated LECs can scavenge tumor antigens and cross-present them to cognate CD8⁺ T cells, driving their dysfunctional activation (Lund et al., 2012). The lack of expression of co-stimulatory molecules such as CD80/86, and high PD-L1 expression levels at the surface of LECs (Fletcher et al., 2010; Tewalt et al., 2012), were proposed as the major mechanisms by which these cells induce deletional CD8⁺ T cell tolerance.While accumulating evidence suggests that direct antigen presentation by LNSCs promotes CD8⁺ T cell deletion, it is unknown whether LNSCs can similarly contribute to CD4⁺ T cell tolerance. As previously described, FRCs, BECs, and LECs express MHCII under virally induced inflammatory conditions or IFN-γ treatment (Malhotra et al., 2012; Ng et al., 2012). However, little is known about the regulation of MHCII expression by LNSCs.Here, we show that endogenous MHCII expression by LNSCs is controlled by the IFN-γ–inducible promoter IV (pIV) of class II transactivator (CIITA). Due to basal pIV activity, LNSCs express low levels of MHCII upon steady state and up-regulate these molecules when exposed to IFN-γ. Unexpectedly, in addition to low endogenous basal expression, the majority of MHCII molecules detected at LEC, BEC, and FRC surface were acquired from DCs. Furthermore, antigen-presenting DCs transfer antigenic peptide–MHCII (pMHCII) complexes to LNSCs, in a process dependent on both cell–cell contact and DC-derived exosomes. Importantly, acquired pMHCII complexes were presented by LECs, BECs, and FRCs to CD4⁺ T cells and promoted cognate CD4⁺ T cell dysfunction by impairing their survival and response to further restimulation. These data suggest that LNSCs serve more diverse roles than previously thought in regulating CD4⁺ T cell immunity.     

Antileishmanial 2-substituted quinolines: in vitro behaviour towards biological components.

Quinolines substituted on their carbon 2 have in vivo antileishmanial activity but some of them could not be detected in plasma when assayed for pharmacokinetic studies, suggesting a sequestration of the drugs by components of the blood compartment. The present study, performed on three quinolines (1, 2 and 3), showed strong affinity for two of them (2 and 3) with red blood cells (RBCs), whereas quinoline 1 did not react with them. This process was saturable, temperature dependant and positively correlated with the in vitro antileishmanial activity of the quinolines. In addition, a rapid and spontaneous reaction with thiol groups was demonstrated for unsaturated quinolines 2 and 3. The reactivity with RBCs could be part of the compounds targeting to the parasite. These results illustrate that derivatives of the quinoline series with similar antileishmanial in vivo activity have different behaviour in the blood compartment.     

Primary malignant vaginal melanoma treated with adriamycin and ifosfamide: a case report and literature review

BACKGROUND: Vaginal melanoma is a very rare but highly malignant gynecological disease, usually diagnosed in postmenopausal woman. The prognosis tends to be poor and it is associated with high rate of recurrence and short survival rates. CASE: The following paper describes a case report regarding a 72-year-old woman with a locally advanced malignant melanoma. The previous erroneous histopathological diagnosis was leiomyosarcoma. She underwent chemotherapy with 3 courses of doxorubicin and ifosfamide. The diagnosis of malignant melanoma was obtained after a repeated biopsy and further pathological investigations. She later underwent radical surgery and 2 additional cycles of the same chemotherapy. At present, 7 months after the last cycle, the patient was locally disease-free, but developed brain metastases, requiring chemotherapy treatment. CONCLUSION: In view of poor survival, this chemotherapy regimen may be an interesting alternative to the traditional treatment of vaginal melanoma.     

Gender gap in cerebrovascular accidents: comparison of the extent, severity, and risk factors in men and women aged 45-65

BACKGROUND AND OBJECTIVE: Few data are available on sex differences among relatively young adult stroke patients. The aim of the present study is to analyze such differences in mortality, principal risk factors, and outcome measures among patients aged 45-65 with acute stroke. The identification of these differences is indispensable for developing optimal strategies for the prevention and care of this disease. METHODS AND RESULTS: Retrospective study of 114 women and 190 men, aged 45-65 years, hospitalized from 1990 to 1998 in the Hadassah Medical Centers with confirmed CVA. Medical background, clinical presentation, imaging results, risk factors, lifestyle information, and rehabilitative status data were retrieved from medical records. No gender differences were observed in clinical presentation or imaging studies. In-hospital mortality rate among women was higher than men, 13.2% vs. 5.8%. A significant gender gap in comorbidity with diabetes, hypertension, and hypercholesterolemia was found: 29.1% of women vs. 14.3% of men. Men more than women had a history of ischemic heart disease (35.8% vs. 21.9%) and smoking and alcohol consumption (43.9% vs. 16.4%; 6.9% vs. 0.9%). Use of rehabilitative services was similar between the sexes. CONCLUSIONS: This study shows marked gender differences among younger adult stroke patients. The concomitance of multiple risk factors in the women may have contributed to the observed higher mortality rate. Characterization of risk factors for CVA in both sexes may aid in developing prevention strategies to reduce stroke incidence in this age group.     

CD8+ regulatory T cells generated by neonatal recognition of peripheral self-antigen

In comparison with CD4+ regulatory T cells, the generation and function of immunomodulatory CD8+T cells is less well defined. Here we describe the existence of regulatory anti-Kb-specific CD8+ T cells that are rendered tolerant during neonatal life via antigen contact exclusively on keratinocytes. These regulatory T cells maintain tolerance during adulthood as they prevent Kb-specific graft rejection by na?ve CD8+ T cells. Third-party immune responses remain unaffected. Up-regulation of TGF-beta1 and granzyme B in the regulatory CD8+ T cell population suggests the involvement of these molecules in common suppressive pathways shared with CD4+ regulatory T cells. In summary, CD8+ regulatory T cells can be induced extrathymically through antigen contact on neonatally accessible parenchymal cells and maintain tolerance throughout adult life.     

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.Infection and tissue damage cause an immediate inflammatory response that is characterized by the activation of innate immune cells and other local stromal cells, followed by rapid recruitment of additional immune cells to the affected site. This response serves to control the invading pathogen and to initiate reparative processes that restore tissue function. Certain surface-exposed immune receptors recognize several lipidated or proteinaceous activators that are foreign to the host, such as lipopeptides (TLR2), LPSs (TLR4), and flagellin (TLR5). In contrast, all nucleic acid–sensing immune receptors described to date are expressed in endolysosomal compartments (TLR3, TLR7–9; Takeda et al., 2003) or in the cytosol (RIG-I family members, AIM2, IFI16, and others; Hornung and Latz, 2010; Ranjan et al., 2009), and thus are sequestered away from the extracellular milieu. This internal location of nucleic acid sensing receptors has been proposed to limit receptor triggering by self-nucleic acids that are present in the extracellular space under homeostatic conditions. Indeed, experimental mislocalization of TLR9, a nonredundant endosomal signaling receptor for DNA, to the plasma membrane led to the recognition of self-DNA from the extracellular environment (Barton et al., 2006). At the same time, endosomal localization of TLR9 is required for efficient recognition of viral DNA, suggesting that nucleic acids become concentrated in endosomal compartments or that additional mechanisms of receptor processing are required for nucleic acid recognition in the endosome (Ewald et al., 2008, 2011; Park et al., 2008).Along with compartmentalization, other safeguards typically prevent self-nucleic acid recognition by innate immune receptors. For instance, digestion of DNA by extracellular and intracellular nucleases ensures that self-DNA released under normal conditions escapes detection by nucleic acid sensors (Napirei et al., 2000; Evans and Aguilera, 2003; Kawane et al., 2010). However, if nucleic acid concentrations exceed the nuclease capacity, such as during infections or in situations of increased cell damage, signaling receptors and their downstream inflammatory effects can be triggered. Chronic activation of inflammatory responses by nucleic acids can result in undesirable autoimmune syndromes and dramatic pathologies (Crow and Rehwinkel, 2009; Horton et al., 2010), yet the recognition of DNA during tissue destruction is an integral part of the host immune and repair responses (Gregorio et al., 2010). Hence, effective management of self-tolerance and damage sensing appears to require the coordinated delivery of extracellular DNA to intracellular sites of recognition.We were interested in defining cell surface receptor proteins that interact with extracellular nucleic acids. Here, we identify that the receptor for advanced glycation end-products (RAGE) binds directly to DNA and RNA and promotes their uptake into cells. RAGE can thereby sensitize cells to extracellular nucleic acids. A co-crystal structure of RAGE with DNA supports the concept that RAGE binds to nucleic acids via interaction with the charged sugar-phosphate backbones in a sequence-independent manner. While other receptors also play roles in nucleic acid recognition by immune cells, RAGE deficiency largely prevents an inflammatory response toward TLR9-stimulatory DNA in the lungs, suggesting that RAGE plays an important role in the control of immune responses to DNA in vivo.     

Sustained effector function of IL-12/15/18–preactivated NK cells against established tumors

Natural killer cell (NK cell)–based immunotherapy of cancer is hampered by the transient effector function of NK cells. Recently, mouse IL-12/15/18–preactivated NK cells were shown to persist with sustained effector function in vivo. Our study investigated the antitumor activity of such NK cells. A single injection of syngeneic IL-12/15/18–preactivated NK cells, but neither naive nor IL-15– or IL-2–pretreated NK cells, combined with irradiation substantially reduced growth of established mouse tumors. Radiation therapy (RT) was essential for the antitumor activity of transferred NK cells. IL-12/15/18–preactivated NK cells expressed high levels of IL-2Rα (CD25), and their rapid in vivo proliferation depended on IL-2 produced by CD4⁺ T cells. IL-12/15/18–preactivated NK cells accumulated in the tumor tissue and persisted at high cell numbers with potent effector function that required the presence of CD4⁺ T cells. RT greatly increased numbers and function of transferred NK cells. Human IL-12/15/18–preactivated NK cells also displayed sustained effector function in vitro. Our study provides a better understanding for the rational design of immunotherapies of cancer that incorporate NK cells. Moreover, our results reveal an essential role of CD4⁺ T cell help for sustained antitumor activity by NK cells linking adaptive and innate immunity.NK cells are potent antitumor effector cells (Cerwenka and Lanier, 2001; Ljunggren and Malmberg, 2007; Terme et al., 2008; Vivier et al., 2008). Accordingly, individuals with low NK cell activity display an increased risk to develop cancer (Imai et al., 2000), and high numbers of intratumoral NK cells are often correlated with improved prognosis for cancer patients (Coca et al., 1997; Villegas et al., 2002). Human tumors frequently express low levels of MHC class I molecules that interact with inhibitory NK cell receptors. For instance, alterations in the β2m gene can lead to an almost complete and irreversible lack of MHC class I in melanoma cells (D’Urso et al., 1991). In addition, many tumor cells express high levels of ligands for activating NK cell receptors (Raulet and Guerra, 2009), leading to efficient recognition by NK cells (Vivier et al., 2008; Pegram et al., 2011). So far, NK cell–based therapy was mainly successful in patients suffering from leukemia (Moretta et al., 2011). Acute myeloid leukemia patients that received haploidentical bone marrow grafts from Killer immunoglobulin receptor (KIR)–mismatched donors displayed a significantly increased 5-yr disease-free survival (Ruggeri et al., 2002). In addition, clinical benefits were observed upon infusion of KIR-mismatched NK cells after stem cell transplantation (Passweg et al., 2004; Miller et al., 2005; Geller and Miller, 2011; Geller et al., 2011). However, adoptive transfer of autologous IL-2–activated NK cells in patients suffering from solid tumors such as melanoma or renal cell carcinoma did not result in clinical benefits (Parkhurst et al., 2011). Thus, novel strategies are urgently needed to improve the antitumor activity of transferred NK cells against solid tumors.During certain viral infections (Sun et al., 2009a) and contact hypersensitivity reactions (O’Leary et al., 2006), persistent NK cell subpopulations mounting recall responses were detected, indicating previously unappreciated memory properties of NK cells (Paust and von Andrian, 2011; Sun et al., 2011; Vivier et al., 2011). In addition, NK cells preactivated with IL-12, IL-15, and IL-18 in vitro for 15 h were detectable at high numbers 3 wk after transfer into RAG-1^−/− mice and produced high levels of IFN-γ upon restimulation (Cooper et al., 2009). Much lower cell numbers and IFN-γ production were observed when IL-15–preactivated NK cells were transferred. Thus, the activation of NK cells with certain cytokines resulted in an NK cell population with enhanced effector function upon restimulation, indicating that NK cells are able to retain memory of prior activation.Because IL-12/15/18–preactivated NK cells were shown to persist with sustained effector function after restimulation (Cooper et al., 2009), we investigated whether application of IL-12/15/18–preactivated NK cells improves current protocols of immunotherapy of cancer. Our study reveals that a single injection of IL-12/15/18–preactivated NK cells, but neither naive nor of IL-15– or IL-2–pretreated NK cells, combined with radiation therapy (RT), substantially reduced growth of established mouse tumors. Our results raise the possibilities for the development of novel NK cell–based therapeutic strategies for clinical application.     

3-D Ultrasound Imaging Reliability of Measuring Dysplasia Metrics in Infants

Developmental dysplasia of the hip is a hip abnormality that ranges from mild acetabular dysplasia to irreducible femoral head dislocations. While 2-D B-mode ultrasound (US)-based dysplasia metrics or disease metrics are currently used clinically to diagnose developmental dysplasia of the hip, such estimates suffer from high inter-exam variability. In this work, we propose and evaluate 3-D US-derived dysplasia metrics that are automatically computed and demonstrate that these automatically derived dysplasia metrics are considerably more reproducible. The key features of our automatic method are (i) a random forest-based learning technique to remove regions across the coronal axis that do not contain bone structures necessary for dysplasia-metric extraction, thereby reducing outliers; (ii) a bone segmentation method that uses rotation-invariant and intensity-invariant filters, thus remaining robust to signal dropout and varying bone morphology; (iii) a novel slice-based learning and 3-D reconstruction strategy to estimate a probability map of the hypoechoic femoral head in the US volume; and (iv) formulae for calculating the 3-D US-derived dysplasia metrics. We validate our proposed method on real clinical data acquired from 40 infant hip examinations. Results show a considerable (around 70%) reduction in variability in two key 3-D US-derived dysplasia metrics compared with their 2-D counterparts.