ICOS-expressing Regulatory T Cells Influence the Composition of Antitumor CTL Populations.

The role of ICOS in antitumor T cell responses and overall tumor progression has been controversial. In this study, we compared tumor progression in mice lacking ICOS selectively in regulatory T (Treg) cells or in all T cells. Using an experimental melanoma lung metastasis model, we found that Treg cell-specific ICOS knockout reduces the overall tumor burden compared with Cre control mice, with increased CD4+-to-Treg cell and CD8+-to-Treg cell ratios in the tumor. In contrast, there was no difference in the tumor burden in mice lacking ICOS in all of the T cell compartments. This suggests a dual role of ICOS costimulation in promoting protumor and antitumor T cell responses. Consistent with reduced tumor burden, we found that Treg cell-specific deletion of ICOS leads to an increase of CD8+ CTLs that express high levels of granzyme B and perforin. Moreover, single-cell transcriptome analysis revealed an increase of Ly108+Eomeshi CD8+ T cells at the cost of the Ly108+T-bethi subset in Treg cell-specific knockout mice. These results suggest that ICOS-expressing Treg cells suppress the CTL maturation process at the level of Eomes upregulation, a critical step known to drive perforin expression and cytotoxicity. Collectively, our data imply that cancer immunotherapies using ICOS agonist Abs may work better in Treg cell-low tumors or when they are combined with regimens that deplete tumor-infiltrating Treg cells.

ICOS costimulation is indispensable for the differentiation of T follicular regulatory cells.

ICOS is a T-cell costimulatory receptor critical for Tfh cell generation and function. However, the role of ICOS in Tfr cell differentiation remains unclear. Using Foxp3-Cre-mediated ICOS knockout (ICOS FC) mice, we show that ICOS deficiency in Treg-lineage cells drastically reduces the number of Tfr cells during GC reactions but has a minimal impact on conventional Treg cells. Single-cell transcriptome analysis of Foxp3+ cells at an early stage of the GC reaction suggests that ICOS normally inhibits Klf2 expression to promote follicular features including Bcl6 up-regulation. Furthermore, ICOS costimulation promotes nuclear localization of NFAT2, a known driver of CXCR5 expression. Notably, ICOS FC mice had an unaltered overall GC B-cell output but showed signs of expanded autoreactive B cells along with elevated autoantibody titers. Thus, our study demonstrates that ICOS costimulation is critical for Tfr cell differentiation and highlights the importance of Tfr cells in maintaining humoral immune tolerance during GC reactions.

Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy.

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

RNF13 Dileucine Motif Variants L311S and L312P Interfere with Endosomal Localization and AP-3 Complex Association.

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

Hippo Signal Transduction Mechanisms in T Cell Immunity.

Hippo signaling pathways are evolutionarily conserved signal transduction mechanisms mainly involved in organ size control, tissue regeneration, and tumor suppression. However, in mammals, the primary role of Hippo signaling seems to be regulation of immunity. As such, humans with null mutations in STK4 (mammalian homologue of Drosophila Hippo; also known as MST1) suffer from recurrent infections and autoimmune symptoms. Although dysregulated T cell homeostasis and functions have been identified in MST1-deficient human patients and mouse models, detailed cellular and molecular bases of the immune dysfunction remain to be elucidated. Although the canonical Hippo signaling pathway involves transcriptional co-activator Yes-associated protein (YAP) or transcriptional coactivator with PDZ motif (TAZ), the major Hippo downstream signaling pathways in T cells are YAP/TAZ-independent and they widely differ between T cell subsets. Here we will review Hippo signaling mechanisms in T cell immunity and describe their implications for immune defects found in MST1-deficient patients and animals. Further, we propose that mutual inhibition of Mst and Akt kinases and their opposing roles on the stability and function of forkhead box O and ß-catenin may explain various immune defects discovered in mutant mice lacking Hippo signaling components. Understanding these diverse Hippo signaling pathways and their interplay with other evolutionarily-conserved signaling components in T cells may uncover molecular targets relevant to vaccination, autoimmune diseases, and cancer immunotherapies.

Optimization of femtosecond laser-constructed clear corneal wound sealability for cataract surgery.

PURPOSE: To compare the sealability of femtosecond laser (FSL)-assisted corneal incisions (CIs) with that of triplanar manual (M)-CIs and to determine FSL wound parameters minimizing leakage. SETTING: Private practice. DESIGN: Phase IV, single-surgeon, retrospective cohort study. METHODS: One eye per patient was included. Two groups defined by the main wound (FSL-CI or M-CI) were compared for leakage, inferred by placement of a suture at the end of surgery. Leakage in FSL-CIs was analyzed as a function of customizable wound parameters: anterior plane depth (APD), posterior plane depth (PPD), anterior side-cut angle (ASCA), and posterior side-cut angle (PSCA). The risk of leakage of FSL-CIs with optimal and nonoptimal parameters was further compared with that of M-CIs. RESULTS: A total of 1100 eyes (757 [68.8%] FSL-CI; 343 [31.2%] M-CI) were included. Wound leakage occurred in 133 FSL-CI (17.6%) and 30 M-CI eyes (8.7%) (P < .001). FSL wound parameters associated with the lowest risk of leakage were 60% APD, 70% PPD, 120 degrees ASCA, and 70 degrees PSCA. FSL-CIs constructed with at least 3 optimal parameters (60% APD, 70% PPD, and 120 degrees ASCA) had a similar risk of leakage to M-CIs (odds ratio [OR], 1.1; 95% CI, 0.5-2.3). FSL-CIs with suboptimal parameters had twice the risk of leakage of M-CIs (OR, 2.0; 95% CI, 1.1-3.8). CONCLUSIONS: Overall, FSL-CIs leaked more than M-CIs. However, FSL-CIs with optimized wound profiles had an equivalent risk of leakage to M-CIs. Wound parameter customization is an asset of FSL technology that allows optimization of FSL-CI sealability.

Surgical support during the terrorist attacks in Paris, November 13, 2015: Experience at Bégin Military Teaching Hospital.

BACKGROUND: Recent conflicts have allowed the French Army Health Service to improve management quality for wartime-injured people during military operations. On November 13, 2015, it was in Paris that France was directly attacked and Bégin Military Teaching Hospital, like several hospitals in Paris, had to face a large number of gunshot victims. Thanks to our operational experience, injured people hospitalized in military hospitals benefited from a management based on triage and damage control (DC) principles. METHODS: Forty-five patients were taken care of in our hospital with an average age of 32 years. During triage, eight patients were categorized T1 (with four extreme emergencies) and 10 were classified T2 and 27 as T3. Twenty-two patients underwent emergency surgery, 15 for soft tissue lesions of limbs, 8 for ballistic fractures (one of which was a cervical wound), and 5 for abdominal wounds. Two patients classified T1 died early. RESULTS: In total, more than 50 operations were performed including iterative debridements, bone fixation, three amputations, and two flaps. After 9 months, all of the patients had healed. One woman with limb stiffness required an arthrolysis. CONCLUSION: This event showed that terrorist attacks and mass casualties with war wounds can occur in France. Acquired experience regarding war wounds by the French Army Health Service is precious. Everyone must understand the importance of triage and the principles of damage control. Every hospital must be ready to face this type of massive influx of injured people (white plan). LEVEL OF EVIDENCE: Epidemiological study, level V.

An Assessment of Demand for a Combined PharmD-MBA Program at the University of Saskatchewan.

(1) Background: Combined MBA programs are becoming increasingly popular, and it is anticipated that there will be 60 combined pharmacy-MBA programs across North America in 2015. We aimed to see if there would be support for a combined PharmD-MBA program at the University of Saskatchewan. (2) Methods: A questionnaire was distributed to 1st, 2nd, and 3rd year pharmacy students at the University of Saskatchewan. A separate questionnaire was developed and all practicing pharmacists in Saskatchewan were emailed a link to SurveyMonkey® (Palo Alto, CA, USA) to fill it out online. In-person and phone interviews were conducted with pharmacy stakeholders in Saskatchewan and across the country. (3) Results: Of the 265 students, 193 (72.8%) were present on the days the questionnaires were distributed, and they all completed the questionnaires. When asked if they would have pursued a combined degree if the U of S had offered it when they entered the pharmacy program, 16.6% (32/193) and 37.3% (72/193) either strongly agreed or agreed and 29.0% (56/193) were unsure. When pharmacists were asked if an MBA would be valuable or applicable in their current job, 42.2% (128/303) agreed and 13.9% (42/303) strongly agreed. When asked if they felt students graduating with a combined degree would be at an advantage for certain job opportunities upon graduation, 33.6% (100/298) strongly agreed and 55.4% (165/298) agreed. A total of 8 interviews were conducted with key stakeholders from across Canada. Of these 8 stakeholders, only 2 were aware that other combined programs were offered. All of the stakeholders were in favour of the idea of a combined degree. Some felt it was important for the program to have a clear value proposition and healthcare related content would be desirable. (4) Conclusions: Overall, pharmacist, pharmacy student, and stakeholder input indicate that a combined program could be supported at the University of Saskatchewan.

The arthroscopic Latarjet procedure for the treatment of anterior shoulder instability.

Anterior instability is a difficult clinical problem that is treated by a variety of open and arthroscopic methods with good results. Bankart repair remains a popular option. However, in those situations involving irreparable ligamentous damage or bony deficiency, this technique may be insufficient to stabilize the shoulder. One of the principal methods of open treatment for this problem is the Latarjet procedure, as described in his article in 1954. It has proven to be a durable and reliable method of treatment for anteroinferior instability of the glenohumeral joint. Several authors have reported on the long-term outcomes of this procedure with satisfactory results. There has been no previous description of the Latarjet procedure being performed arthroscopically. We present the first report of a new surgical technique, the arthroscopic Latarjet procedure. This procedure is fully arthroscopic and combines the advantages of the open procedure with those of arthroscopic stabilization. This is a significant step forward in the development of arthroscopic shoulder reconstruction and enables shoulder surgeons to treat all cases of instability arthroscopically.

Molecular definition of an in vitro niche for dendritic cell development.

OBJECTIVE: Although dendritic cell (DC) precursors have been isolated from many lymphoid sites, the regulation and location of early DC development is still poorly understood. Here we describe a splenic microenvironment that supports DC hematopoiesis in vitro and identify gene expression specific for that niche. METHODS: The DC supportive function of the STX3 splenic stroma and the lymph node-derived 2RL22 stroma for overlaid bone marrow cells was assessed by coculture over 2 weeks. The DC supportive function of SXT3 was identified in terms of specific gene expression in STX3 and not 2RL22 using Affymetrix microchips. RESULTS: STX3 supports DC differentiation from overlaid bone marrow precursors while 2RL22 does not. A dataset of 154 genes specifically expressed in STX3 and not 2RL22 was retrieved from Affymetrix results. Functional annotation has led to selection of 26 genes as candidate regulators of the microenvironment supporting DC hematopoiesis. Specific expression of 14 of these genes in STX3 and not 2RL22 was confirmed by reverse transcription-polymerase chain reaction. CONCLUSION: Some genes specifically expressed in STX3 have been previously associated with hematopoietic stem cell niches. A high proportion of genes encode growth factors distinct from those commonly used for in vitro development of DC from precursors. Potential regulators of a DC microenvironment include genes involved in angiogenesis, hematopoiesis, and development, not previously linked to DC hematopoiesis.