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Regulatory T (T(reg)) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that T(reg) cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. T(reg) cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented T(H)1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted T(H)2-like polarization (increased expression of GATA-3, CCR4 and IL4). T(H)1-like T(reg) cells limited CD8(+) T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how T(reg) cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

Robust CD8+ T cell memory is essential for long-term protective immunity but is often compromised in cancer, where T cell exhaustion leads to loss of memory precursors. Immunotherapy via checkpoint blockade may not effectively reverse this defect, potentially underlying disease relapse. Here we report that mice with a CD8+ T cell–restricted neuropilin-1 (NRP1) deletion exhibited substantially enhanced protection from tumor rechallenge and sensitivity to anti-PD1 immunotherapy, despite unchanged primary tumor growth. Mechanistically, NRP1 cell-intrinsically limited the self-renewal of the CD44+PD1+TCF1+TIM3− progenitor exhausted T cells, which was associated with their reduced ability to induce c-Jun/AP-1 expression on T cell receptor restimulation, a mechanism that may contribute to terminal T cell exhaustion at the cost of memory differentiation in wild-type tumor-bearing hosts. These data indicate that blockade of NRP1, a unique ‘immune memory checkpoint’, may promote the development of long-lived tumor-specific Tmem that are essential for durable antitumor immunity. T cell exhaustion limits antitumor immune responses. Vignali and colleagues identify neuropilin-1 as a novel immune checkpoint that cell-intrinsically operates to limit memory cell formation and can be targeted to enhance antitumor responses.

SUMMARY Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth. We also show that T-reg-cell-specific inducible deletion of Tim-3 enhances anti-tumor immunity. Enhancement of Treg cell function by Tim-3 is strongly correlated with increased expression of interleukin-10 (IL-10) and a shift to a more glycolytic metabolic phenotype. Our data demonstrate that Tim-3+ Treg cells may be a relevant therapeutic target cell type for the treatment of cancer., In brief Regulatory T cells (Treg cells) limit the immune response to tumors, and tumor-infiltrating Treg cells are especially suppressive. However, the mechanisms underlying enhanced Treg cell function are poorly understood. Banerjee et al. show that Tim-3 expression is linked to increased Treg cell suppressive activity, possibly through the cytokine IL-10, in mouse models and people with cancer., Graphical Abstract

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3-mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)-dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

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Regulatory T cells (Treg) are critical mediators of self-tolerance but can also limit effective anti-tumor immunity. We and others previously reported that 40-60% percent of Treg-infiltrating head and neck cancer (HNC) and other tumors highly express Tim-3, compared with about 5% in lymphoid organs. Tumor-infiltrating Tim-3+ Treg also have enhanced suppressive function and display a more effector-like phenotype. Using a novel mouse model with cell type-specific Tim-3 expression, we show here that expression of Tim-3 by Treg is sufficient to drive Treg to a more effector-like phenotype, resulting in enhanced suppressive activity and increased tumor growth. These findings may help to reconcile previous reports that some Tim-3 antibodies enhance T cell responses in vivo, while expression of Tim-3 has a cell-intrinsic ability to enhance TCR signaling and T cell activation. Thus, we propose that Tim-3 regulates anti-tumor immunity at least in part through enhancement of Treg function. To our knowledge, this is the first example in which expression of a single co-stimulatory molecule is sufficient to drive differentiation of Treg in this manner.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works Mechanisms of resistance to cancer immunotherapy remain poorly understood. Lymphocyte activation gene–3 (LAG3) signaling is regulated by a disintegrin and metalloprotease domain-containing protein–10 (ADAM10)–and ADAM17-mediated cell surface shedding. Here, we show that mice expressing a metalloprotease-resistant, noncleavable LAG3 mutant (LAG3NC) are resistant to PD1 blockade and fail to mount an effective antitumor immune response. Expression of LAG3NC intrinsically perturbs CD4+ T conventional cells (Tconvs), limiting their capacity to provide CD8+ T cell help. Furthermore, the translational relevance for these observations is highlighted with an inverse correlation between high LAG3 and low ADAM10 expression on CD4+ Tconvs in the peripheral blood of patients with head and neck squamous cell carcinoma, which corresponded with poor prognosis. This correlation was also observed in a cohort of patients with skin cancers and was associated with increased disease progression after standard-of-care immunotherapy. These data suggest that subtle changes in LAG3 inhibitory receptor signaling can act as a resistance mechanism with a substantive effect on patient responsiveness to immunotherapy., NIH (Grant EB022433)

BackgroundThe signaling pathways involving phosphoinositide-3-kinases (PI3Ks) are highly conserved and tightly regulated to influence the activation, proliferation, and survival of all cell types. PI3K signaling plays a major role in T cell responses to antigen due to its position directly downstream of T cell receptor (TCR)/CD28 ligation.1 2 Our lab has recently shown that the cell surface protein TrIP (Transmembrane Inhibitor of PI3K, gene name: Pik3ip1) has a distinctly high expression on T cells and is capable of downregulating PI3K signaling in CD4+ T cells, acting as a negative regulator of T cell immune responses.3 4 These studies revealed that CD4+ T cells lacking TrIP expression exhibit a more Th1 inflammatory phenotype compared to WT T cells, both in vivo and in vitro.3 These data have led us to propose that TrIP restricts the inflammatory activity of T cells more generally, including CD8+ T cells, and that targeting/knockout of this negative regulator may promote anti-tumor immunity.MethodsUsing a conditional TrIP knockout mouse model developed in our lab, we have performed syngeneic tumor challenges in CD8+ T cell-specific TrIP knockout mice (TrIPfl/flE8icre). We have also characterized the tumor immune infiltrate of these mice to understand the impact of T cell-specific TrIP deficiency on the immune landscape.ResultsOur data thus far show that CD8+ T cell-specific TrIP knockout mice (TrIPfl/flE8icre) are resistant to growth of syngeneic tumors. In addition to increased tumor resistance, we have also found that tumors harvested from our TrIPfl/flE8icre knockout mice contain twice as many infiltrating T cells compared to their WT counterparts. We also found that CD8+ T cells appeared to be the main drivers of this increased T cell infiltration, as their frequency was double that of the CD4+ population in tumors transplanted into TrIP KO mice.ConclusionsWe describe data demonstrating that TrIP, a relatively novel PI3K inhibitor, plays a significant role in the antitumor immune activity of CD8+ T cells. Our that CD8+ T cell-specific TrIP knockout mice are resistant to tumor challenge and show more robust tumor CD8+ T cell infiltrate. With these data, we are excited to propose TrIP as a potential future immunotherapeutic target worthy of continued investigation.ReferencesOkkenhaug K, Turner M, Gold MR. PI3K signaling in B cell and T cell biology. Front Immunol 2014;5:557. doi:10.3389/fimmu.2014.00557Kane LP, Weiss A. The PI-3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3. Immunol Rev 2003;192:7–20. doi:10.1034/j.1600-065X.2003.00008.xUche UU, Piccirillo AR, Kataoka S, et al. PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt. J Exp Med 2018;215:3165–3179. doi:10.1084/jem.20172018DeFrances MC, Debelius DR, Cheng J, Kane LP. Inhibition of T-cell activation by PIK3IP1. Eur J Immunol 2012;42:2754–2759. doi:10.1002/eji.201141653

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic T cell activation, including in chronic infection and solid tumors. We and others previously reported that Tim-3 exerts apparently paradoxical co-stimulatory activity in T cells (and other cells), including enhancement of ribosomal S6 protein phosphorylation (pS6). Here we examined the upstream signaling pathways that control Tim3-mediated increases in pS6 in T cells. We have also defined the localization of Tim-3 relative to the T cell immune synapse and impacts on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired Tim-3 co-stimulation of pS6. Strikingly, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in the context of a chimeric antigen receptor still allowed for robust T cell activation. Our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.One Sentence SummaryHere we define elements of signaling and localization associated with Tim-3 co-stimulatory function in T cells.

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The NF-κB essential modulator protein (NEMO) is required for activation of canonical NF-κB by the T cell antigen receptor (TCR). However, the subcellular localization of NEMO during this process is not well understood. By dynamically imaging fluorescent NEMO chimeras in live human T cells, we demonstrate that NEMO is rapidly recruited into TCR microclusters via domains previously implicated in the recognition of linear and K63-linked polyubiquitin. The recruitment of NEMO into TCR microclusters requires the activities of the tyrosine kinases Lck and ZAP-70, but not the adaptor proteins LAT or SLP-76. Thus, our findings reveal that the pathways leading from TCR to NF-κB bifurcate downstream of ZAP-70 to independently control the recruitment and activation of NEMO.

Summary Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-35 reporter mouse, we observed substantial enrichment of IL-35 + Treg cells in tumors. Neutralization with an IL-35-specific antibody or Treg cell-restricted deletion of IL-35 production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-35 enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-35 in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment.

Tim-3 is highly expressed on a subset of T cells during T cell exhaustion in settings of chronic viral infection and tumors. Using lymphocytic choriomeningitis virus (LCMV) Clone 13, a model for chronic infection, we found that Tim-3 was neither necessary nor sufficient for the development of T cell exhaustion. Nonetheless, expression of Tim-3 was sufficient to drive resistance to PD-L1 blockade therapy during chronic infection. Strikingly, expression of Tim-3 promoted the development of short-lived effector T cells, at the expense of memory precursor development, after acute LCMV infection. These effects were accompanied by increased Akt/mTOR signaling in T cells expressing endogenous or ectopic Tim-3. Conversely, Akt/mTOR signaling was reduced in effector T cells from Tim-3-deficient mice. Thus, Tim-3 is essential for optimal effector T cell responses, and may also contribute to exhaustion by restricting the development of long-lived memory T cells. Taken together, our results suggest that Tim-3 is actually more similar to costimulatory receptors that are up-regulated after T cell activation than to a dominant inhibitory protein like PD-1. These findings have significant implications for the development of anti-Tim-3 antibodies as therapeutic agents.

Tim-3 is highly expressed on a subset of T cells during T cell exhaustion, in settings of chronic viral infection and tumors (1, 2). Using LCMV Clone-13, a model for chronic infection, we have found that Tim-3 is neither necessary nor sufficient for the development of T cell exhaustion. Nonetheless, expression of Tim-3 was sufficient to drive resistance to PD-L1 blockade therapy during chronic infection. Strikingly, expression of Tim-3 promoted development of short-term effector T cells, at the expense of memory precursor development, after acute LCMV infection. These effects were accompanied by increased Akt/mTOR signaling in T cells expressing endogenous or ectopic Tim-3. Conversely, Akt/mTOR signaling was reduced in effector T cells from Tim-3 deficient mice. Thus, Tim-3 is essential for optimal effector T cell responses, but may also contribute to exhaustion, by restricting development of long-lived memory T cells, including PD-1int “stem-like” exhausted T cells that expand during PD-1 pathway blockade. Taken together, our results suggest that Tim-3 is actually more similar to co-stimulatory receptors that are upregulated after T cell activation, rather than a dominant inhibitory protein like PD-1. These findings have significant implications for the development of anti-Tim-3 antibodies as therapeutic agents.SignificanceDuring a chronic viral infection, prolonged exposure to viral antigens leads to dysfunction or “exhaustion” of T cells specific to the virus, a condition also observed in T cells that infiltrate tumors. The exhausted state is associated with expression of specific cell-surface proteins, some of which may inhibit T cell activation. Expression of Tim-3 is associated with acquisition of T cell exhaustion, although it is also expressed transiently during acute infection. Here we provide evidence that a major function of Tim-3 is to enhance T cell activation, during either acute or chronic viral infection. However, Tim-3 is not required for development of exhaustion. Thus, we propose that Tim-3 would be better described as a stimulatory, rather than inhibitory, protein.

Background Biologics are still considered by patients as a new and partially enigmatic tool for management of rheumatic diseases. Objectives The study was designed to evaluate educational needs and sources of knowledge in patients with rheumatic diseases treated with biologics. Methods Anonymous questionnaires were distributed in 23 Polish rheumatological centers involved in the treatment, 1231 questionnaires were used for analysis. Responses were received from 606 patients with rheumatoid arthritis, 427 with ankylosing spondylitis, 117 psoriatic arthritis, and 62 adult patients with juvenile idiopathic arthritis (in whom administration of the drugs had been introduced before they were 18-year-old), as well as 19 ones receiving the drugs due to other musculoskeletal disorders. The investigated group constituted about one-fifth of all rheumatic patients on biologics in Poland. Results Almost all the patients had learnt for the first time on biologics from their rheumatologist (93%). Few only patients had got such data from internet or from other patients. Likewise, most of the patients got majority of educational data on treatment with biologics from rheumatologist who was supervising the therapy (82%). Remaining sources included internet (8%) and other patients (5%). Relative low number of patients was educated by nurses (2%). Most of the patients (87%) were looking for more details on biological treatment. The patients with rheumatic disease lasting less than 10 yrs. were more interested in the management than those suffering longer. Most of the patients (94%) considered their rheumatologist as the main person responsible for their education on biologics. There was no difference between patients with various rheumatic diseases as well as no difference was found between female and male patients. Biological treatment attracted more interest in younger than older patients. Conclusions Education is still a challenge in patients receiving biologics. Most of the patients represented traditional attitude to health education, expecting almost all educational data to be provided by their physician. We were surprised that role of the nurses was found to be rather low. An increase in role of nurses seems to be the future aim of the educational efforts in Polish rheumatology. Disclosure of Interest None declared

Inhibitory receptors are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (Tconv) cell proliferation, viability and function. However, the role of Inhibitory receptors on regulatory T (Treg) cells remains obscure, as they could be required for suppressive activity and/or limit Treg cell function. We evaluated the role of Lymphocyte Activation Gene-3 (LAG3, CD223) on Treg cells by generating mice in which LAG3 is absent on the cell surface of Treg cells in a murine model of Type 1 Diabetes. Surprisingly, mice that lacked LAG3 expression on Treg cells exhibited reduced autoimmune diabetes, consistent with enhanced Treg cell proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3-deficient Treg cells was largely comparable, substantial differences between intra-islet Treg cells were evident and involved a subset of genes and pathways that promote Treg cell maintenance and function. Consistent with these observations, Lag3-deficient Treg cells out-competed WT Treg cells in the islets but not in the periphery in co-transfer experiments due to enhanced IL2-Stat5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits Treg cell proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment and may contribute to Treg cell insufficiency in autoimmune disease.

The IκB kinase (IKK) complex mediates the activation of canonical NFκB isoforms following T cell receptor (TCR) ligation. This complex consists of the kinases IKKα and IKKβ and an essential adaptor subunit, the NFκB essential modulator protein (NEMO). Most models suggest that the IKK complex is activated within oligomeric Carma1/Bcl10/Malt1 (CBM) signalosomes. However, we observed that NEMO enters TCR microclusters before CBM complexes are assembled, within ~70 seconds of TCR engagement. NEMO also entered mobile vesicles and in larger membrane-bounded structures (hereafter ‘macroclusters’). The recruitment of NEMO into TCR microclusters is prevented by Src kinase inhibitors and by the catalytic inactivation of ZAP-70, but occurs in the absence of either SLP-76 or Carma1. Further, NEMO fails to co-localize with TCR-induced CBM polymers. Thus, the recruitment of NEMO to the TCR occurs via a CBM-independent mechanism. The deletion the zinc-finger (ZnF) domain of NEMO disables NFκB signaling and eliminates NEMO from microclusters, while preserving NEMO macroclusters. Since the ZnF domain interacts with polyubiquitin chains, we generated point mutations impacting the ubiquitin-binding site in the ZnF domain and two independent sites within the NEMO ubiquitin-binding ‘NUB’ domain. These mutations impair the ability of NEMO to capture K63-linked and/or linear polymers, hinder NFκB signaling, and eliminate NEMO from microclusters without disrupting NEMO macroclusters. These findings suggest that NEMO is rapidly recruited to polyubiquitin chains associated with the TCR, rather than the CBM complex, and that the CBM complex augments IKK-dependent NFκB signaling via a distinct, recruitment-independent mechanism.

Regulatory T cells (Tregs) can suppress a wide variety of cell types, in diverse organ sites and inflammatory conditions. Whereas Tregs possess multiple suppressive mechanisms, the number required for maximal function is unclear. Furthermore, whether any interrelationship or cross-regulatory mechanisms exist to orchestrate and control their utilization is unknown. In this study, we assessed the functional capacity of Tregs lacking the ability to secrete both IL-10 and IL-35, which individually are required for maximal Treg activity. Surprisingly, IL-10/IL-35 double-deficient Tregs were fully functional in vitro and in vivo. Loss of IL-10 and IL-35 was compensated for by a concurrent increase in cathepsin E (Ctse) expression, enhanced TRAIL (Tnfsf10) expression, and soluble TRAIL release, rendering IL-10/IL-35 double-deficient Tregs functionally dependent on TRAIL in vitro and in vivo. Lastly, whereas C57BL/6 Tregs are normally IL-10/IL-35 dependent, BALB/c Tregs, which express high levels of cathepsin E and enhanced TRAIL expression, are partially TRAIL dependent by default. These data reveal that cross-regulatory pathways exist that control the utilization of suppressive mechanisms, thereby providing Treg functional plasticity.

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim−/−, Bim−/−Puma−/−, Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3−/−) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4+ and CD8+ T cell homeostasis. Lag3−/− NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4+ T cells and, to a lesser extent, CD8+ T cells. Lag3−/− mice exhibited accelerated, invasive insulitis, corresponding to increased CD4+ and CD8+ T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4+ T cells and islet specific glucose-6-phosphatase-specific CD8+ T cells were significantly increased in the islets of Lag3−/− mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4+ and CD8+ T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.

The mechanism and stimulatory requirements of regulatory T cell (Treg)-mediated suppression are still unclear. To assess the requirement for Treg stimulation by cognate peptide:MHC, we used T cells from OTII and AND TCR transgenic mice that are specific for and restricted by distinct, noncrossreactive peptide:MHC combinations. This allowed us to independently activate Tregs and their conventional T cell (Tconv) targets. Surprisingly, we found that suppression can occur in the absence of peptide:MHC-mediated stimulation of Tregs. This suppression was Treg dependent and not due to cold target inhibition. Using Rag1−/− TCR transgenic T cells, we show that regulation of Tconv proliferation by heterogeneous Tregs is not due to alloreactivity or crossreactivity. Finally, using anti-TCR-Vβ8-coated microbeads and Vβ8− Tregs, we show that TCR stimulation-independent suppression can occur in the absence of APCs. These data suggest that Tregs may possess constitutive regulatory activity that can be mediated in the absence of cognate peptide:MHC-TCR stimulation.

The effects of cationic substitution of niobium for titanium in (Ba 0.80 Sr 0.20 )TiO 3 composition on the structural and dielectric properties as well as on the electrical conductivity and Seebeck effect were investigated. X-ray diffraction study at room temperature and the real ( ɛ ′) and imaginary ( ɛ ″) parts of permittivity measurements in a wide temperature (from −200 to 450 °C) and frequency (0.1–100 kHz) range were carried out. The analysis of the results of electric conductivity and Seebeck coefficient leads to the conclusion that Nb 5+ ions, reducing the conductivity of about two orders, are playing role of donors. The correlation between electric characteristics and niobium concentration was confirmed and the evolution from normal ferroelectrics with diffuse phase transition to relaxor ferroelectrics has been found.

Nb-doped (Ba0.8Sr0.2)TiO3 ceramics were prepared using conventional mixed-oxide processing technique. Permittivity and loss factor were investigated as a function of temperature for various frequencies of the measuring field. The obtained results confirmed the relaxor ferroelectric behaviour of the studied ceramics, i.e. a strong frequency dispersion of the permittivity maximum and a visible shift of its temperature with frequency. Analysis of real part of permittivity allowed us to determine the value of the freezing temperature characterising the relaxor ferroelectrics. The physical processes, responsible for the relaxor behaviour of the studied ceramics are discussed.

The effect of antimony dopant on phase transition, dielectric response and relaxor behaviour of (Pb0.75Ba0.25)(Zr0.70Ti0.30)O3 ceramics was studied. Ceramic samples, with various Sb concentration from the range 1 to 4 at.%, were prepared by a conventional mixed oxide method. The crystal structure of the investigated ceramics was determined by an X-ray diffraction at room temperature that allowed to determine the unit cell parameters. Dielectric relaxation typical for ferroelectric relaxors was observed in the vicinity of diffuse ferroelectric–paraelectric phase transition. All parameters describing the relaxor behaviour determined from the Vogel–Fulcher relationship depend on the concentration of Sb dopant. The strong influence of antimony on grain structure and on remanent polarisation was confirmed as well.

The Ba1−x Sr x TiO3 materials have received increased attention as one of the most important materials for electroceramic components, such as high dielectric ceramic capacitors, tunable phase shifters and PTCR. In this paper the effect of 0.5; 1; 1.5; and 2 mole% of Bi2O3 addition on microstructure, structure, dielectric properties (i.e., dielectric constant ϵ′, and dielectric loss factor tan δ), as well as pyroelectric and thermally stimulated depolarization currents (TSDC) of (Ba0.8Sr0.2)TiO3 ceramics have been investigated. The distinct correlation between Bi2O3 content, wide maxima in TSDC, anomalies in ϵ′ (T) and tan δ (T) characteristics occurring within the range of the paraelectric phase was confirmed. Changes of electric conductivity and Seebeck's coefficient, caused by Bi2O3-additives, were also studied.

The strong influence of bismuth additions to the lead–barium–zirconate–titanate ceramics of Ba/Zr/Ti 25/70/30 composition on grain structure, dielectric properties and dc conductivity is confirmed. The Bi-modified ceramics exhibit classical relaxor ferroelectrics (RF) behaviour similar to the other lead containing perovskites such as lead–lanthanum–zirconate–titanate-type ceramics. Dielectric relaxation typical for the ferroelectric relaxors is observed in the vicinity of diffuse ferroelectric–paraelectric phase transition. Additional anomalies in e ′ (T) curves in the low frequency range, present in the paraelectric phase for the undoped ceramics, are eliminated by the Bi admixture which also reduces the dc conductivity by three orders of magnitude. A qualitative explanation of the experimental results is presented in the paper.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (

The effects of the external d.c. and a.c. electric field on the dielectric response and relaxor behaviour of lead–barium–zirconate–titanate (PBZT) ceramics of the composition Ba/Zr/Ti 25/70/30 have been studied. The significant influence of the strength of the d.c. bias field and the amplitude of the a.c. field on the dielectric constant maximum and frequency dispersion was determined. The magnitude of the dielectric response strongly decreases under the bias field and increases with the increase of the a.c. field amplitude. All parameters describing the relaxor behaviour of the studied ceramics changed under the external d.c. and a.c. electric field. The experimental results can be explained in terms of existing models of relaxors.

The main purpose of present studies is to learn the effect of sintering temperature on BaBi2Nb2O9 (BBN) ceramics properties. Grain structure analysis performed by the scanning electron microscope (SEM) exhibits differences in grain structure of the ceramics sintered at various temperatures for a constant time. Namely, the grain sizes increase with the increase of the sintering temperature. This fact is connected with changes in dielectric properties. The maximum of the real part of permittivity ( ) and corresponding temperature (T m) shifts to higher values. The properties characteristic for the ferroelectric relaxors also yield to change. The ceramics sintered in higher temperature revealed lower frequency dispersion of and T m. It should also be noticed that the freezing temperature and the Burn's temperature have changed. These results indicate that the behaviour of the relaxor ferroelectrics is weakened by the increase in the grain size.

From 1994 to 2003, fifty-five tropical cyclones entered the Canadian Hurricane Centre (CHC) Response Zone, or about 42% of all named Atlantic tropical cyclones in this ten-year period, and 2003 was the fourth consecutive year for a tropical cyclone to make landfall in Canada. The CHC forecasts all tropical cyclones that enter the CHC Response Zone and assumes the lead in forecasting once the cyclone enters its area of forecast responsibility. This study acknowledges the challenges of forecasting such tropical cyclones at extratropical latitudes. If a tropical cyclone has been declared extratropical, global models may no longer use vortex bogussing to carry the cyclone, and even if it is modeled, large model errors often result. The purpose of this study is to develop a new version of the Florida State University (FSU) hurricane superensemble with greater skill in tracking tropical cyclones, especially at extratropical latitudes. This has been achieved from the development of the synthetic superensemble, which is similar to the operational version of the multi-model superensemble that is used at FSU. The synthetic superensemble differs in that is has a larger set of member models consisting of regular member models, synthetic versions of these models, and the operational superensemble and its synthetic version. This synthetic superensemble is being used here to forecast hurricane tracks from the 2001, 2002, and 2003 hurricane seasons. The track forecasts from this method have generally less error than those of the member models, the operational superensemble, and the ensemble mean. This study shows that the synthetic superensemble performs consistently well and would be an asset to operational hurricane track forecasting.

Strong influence of niobium admixture added to the lead–barium–zirconate–titanate ceramics of a chosen composition Ba/Zr/Ti 25/70/30 on grain structure, dielectric, and pyroelectric properties was confirmed. The Nb-modified ceramics exhibit classical relaxor ferroelectric behaviour similar to other complex lead perovskites such as lead–lanthanum–zirconate–titanate-type ceramics. Additional anomalies in ɛ ′( T ) curves in low frequency range were observed in the paraelectric phase for undoped ceramics. These anomalies and some disturbances in regularities typical for the relaxor ferroelectric behaviour in the vicinity of diffuse ferroelectric–paraelectric phase transition in undoped PBZT ceramics are eliminated by the Nb admixture. An attempt at a quantitative explanation is presented in the paper.

The CD3ε proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3ε PRS, we have generated mice that lack this motif (CD3ε.PRSM). Pull-down experiments demonstrated the inability of Nck to bind to the CD3ε PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3ε.PRSM T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3ε PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 YxxØ motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the YxxØ motifs, with a clear hierarchy in their use (CD3δ > CD3γ ≥ CD3ζ ≫ CD3ε). Remarkably, a single, membrane-distal YxxØ motif in CD3δ could mediate ∼75% of receptor internalization, whereas its removal only reduced internalization by ∼20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3γ. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3δ. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.

The sintering effect on transition parameters and dielectric characteristics of Ba0.8Sr0.2TiO3 (BST 80/20) ceramics prepared by the conventional mixed-oxide processing technique was studied. Grain structure analysis performed by a scanning electron microscope (SEM) and X-ray diffraction studies showed differences in grain size and contents of crystal phase within the ceramics sintered at constant temperature for a variety of sintering times. The phase transitions and dielectric properties were investigated by measuring the dielectric constant e′, the loss factor tan δ and the remanent polarization Pr as a function of temperature. The frequency dependence of e′(T) and tan δ(T) characteristics was also studied. It was shown that the maximal values of the dielectric constant and the remanent polarization are in general proportional to the content of the crystal phase in the volume of the studied ceramics.

Inhibitory receptors control immune responses preventing exacerbated T cell activation and limiting antitumor immunity. LAG3 co-expression with PD1 phenotypically marks functionally exhausted tumor-specific T cells, however LAG3 expression and function is itself regulated by ADAM metalloproteinase-mediated cell surface cleavage. To investigate the impact of LAG3 shedding on T cells within tumors, a conditional knock-in mouse was generated that results in a non-cleavable form of LAG3 (LAG3.NC). LAG3.NC CD4Cre, LAG3.NC ThPOKCreERT2 and LAG3.NC E8ICre mice (restricting LAG3.NC to all T cells, CD4+ T cells and CD8+ T cells, respectively) exhibit enhanced LAG3 expression on the respective T cell subsets infiltrating MC38 tumors. Upon therapeutic administration of anti-PD1, MC38 tumor-bearing wild-type mice show significant tumor regression and 40% become tumor-free, resulting in long-term survival. LAG3.NC CD4Cre and LAG3.NC ThPOKCreERT2 resist anti-PD1 therapy and succumb to tumor growth. However, this phenotype is not observed in LAG3.NC E8ICre mice, resulting in a similar frequency of tumor-free mice to controls. CD8+ TIL isolated from LAG3.NC CD4Cre mice do not show enhanced IFN-g/TNF-a production or proliferation as observed in controls or LAG3.NC E8ICremice following anti-PD1. Failure to mediate an antitumor immune response by LAG3.NC CD4Cre, but not LAG3.NC E8ICre mice, suggests that LAG3 cleavage on CD4+ T cells, including Tregs, is necessary for CD8+ TIL to elicit an effective immune response with anti-PD1. Selective ADAM10 inhibition in vivo, preventing LAG3 shedding, also reduces cytokine release, proposing that metalloproteinase-mediated LAG3 cleavage limits the efficacy of the antitumor immune response.

Dielectric and pyroelectric characteristics and changes of electric conductivity were investigated for Nb 2 O 5 -doped Pb(Zr 0.92 Ti 0.08 )O 3 ceramics. The influence of this dopant on the ceramics microstructure was also studied. Correlation between the investigated electric characteristics and grain structure was confirmed. Some progress in understanding the influence of Nb-dopant was reached in this way.

The research presented in this paper concerns BaBi2Nb2O9 (BBN) which is the member of the Aurivillius family and seems to be interesting from the point of view of its potential applications in storage media. Our investigations focused on temperature dependence crystal structure and mechanical properties of this ceramics as well as on the dielectric properties of samples. Correlation between positions of the maximum of the real part of electric permittivity and the behavior integral width of diffraction lines XRD versus temperature had been discussed based of the presence of polar nano-regions with orthorhombic distortion in macroscopic tetragonal matrix.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18–22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

Kvalitetsbegreppet är svårdefinierat och mångdimensionellt. Därför bör det definieras för att ens kunna användas för mätning inom olika verksamheter. Syftet med uppsatsen är att undersöka hur Stockholms läns landsting, SLL, ställer för kvalitetskrav och utför sin uppföljning av vårdcentralerna. Vårdcentralen är den vårdenhet som först kommer i kontakt med patienter. Därför är det viktigt att de har en översikt över hela hälso- och sjukvården ifall de måste skicka patienter vidare för sjukvård som de själva inte kan ge. Under åren 1975-1995 drabbades Sverige av ekonomiska kriser och sjukvården fick mycket kritik för att inte hålla kvalitetsmåttet.[1] Den offentliga sektorn införde kvalitetsbegreppet och legitimerade den genom att sammanställa sju kvalitetskrav på vårdcentralerna. Dessa gjordes synbara för patienter och anställda hos vårdcentralerna genom att kvalitetskraven skall fortlöpande mätas. Denna studie genomfördes med en kvalitativ metod då insamlingen inte går att mätas eller analyseras med siffror. Empirin samlades in genom semi-strukturerade intervjuer som gjordes med tre anställda inom SLL för att öka våra kunskaper hur SLL går tillväga vid uppföljning av vårdcentraler. Data samlades även in genom Regelbok för husläkarverksamhet med basal hemsjukvård, som är lagstiftad för hela verksamheten och är grundpelaren för vårdavtalet. Med hjälp av en abduktiv ansats analyserades all insamlad data. Studien visar att SLL definierar kvaliteten genom att se detta begrepp kvalitativt med betoning på patientens perspektiv. De har även byggt upp mätningen av kvalitetskraven genom nyckeltal som är en förenklad verklighet då de underlättar jämförande analyser. Nyckeltal är ett försök att konkret beskriva komplexa förhållanden som är av innebörd för att se vårdcentralernas förmåga att fungera.[2] Detta gör att SLL:s uppföljning och kontroll av kvalitetskraven blir abstrakt och kan medföra att de missar värdefull information som inte tas upp av nyckeltalen. För att kunna kontrollera och följa upp kvalitetskraven hos vårdcentralerna använder sig SLL av både indirekta och direkta kontrollsystem. De indirekta kontrollsystemen finns i form av kapiterings- och besöksersättning (se begreppsförklaring) och de direkta kontrollsystemen är mätning av kvaliteten genom nyckeltal och belöningssystemet. Dessa kontrollsystem gör att patienten förvandlas till en kund som vårdcentraler skall tillfredsställa. [1] Åke Bergmark och Johan Fritzell, Välfärdens utveckling efter 1990-talets kris, Socialvetenskaplig tidskrift nr 2-3, 2007. [2] Hälso- och sjukvårdsnämndens förvaltning, Regelbok för husläkarverksamhet med basal hemsjukvård, Stockholms läns landsting 2011

Regulatory T cells (Tregs) are a critical subset of T cells that mediate peripheral tolerance. There are two types of Tregs: natural Tregs, which develop in the thymus, and induced Tregs, which are derived from naive CD4(+) T cells in the periphery. Tregs utilize a variety of mechanisms to suppress the immune response. While Tregs are critical for the peripheral maintenance of potential autoreactive T cells, they can also be detrimental by preventing effective anti-tumor responses and sterilizing immunity against pathogens. In this review, we will discuss the development of natural and induced Tregs as well as the role of Tregs in a variety of disease settings and the mechanisms they utilize for suppression.

T-cell receptor (TCR) transgenic (Tg) mice have revolutionized our understanding of many aspects of T-cell biology. Whereas they provide an almost unlimited source of T cells with a single specificity, breeding them onto different backgrounds and/or new knockout/knock-in mouse models is often time-consuming (6 months to several years), which can make the process costly and can significantly delay research. This protocol describes a new method for expressing defined TCR-alpha and TCR-beta proteins from a single 2A peptide-linked multicistronic retroviral vector in mice, using retrovirus-mediated stem cell gene transfer. We refer to these as 'retrogenic' (Rg) mice ('retro' from retrovirus and 'genic' from Tg) to avoid confusion with traditional transgenic mice. We have successfully used this approach to express over 50 different TCRs on several different mouse backgrounds in as little as 6 weeks.

Meeting abstracts Inhibitory receptors control immune responses preventing exacerbated T cell activation and the onset of autoimmunity; however, they also limit antitumor immunity. Enhanced co-expression of PD1 and LAG3 phenotypically mark functionally exhausted tumor-specific T cells, with dual

Future deep-space communications will require the collection and transmission of data from high-bandwidth links. NASA's Jet Propulsion Laboratory (JPL) is investigating the utility of laser communications for future missions to Mars and for future communication stations on the moon. Cloud cover impacts the availability of space to ground optical communications. Mitigating these impacts requires a geographically diverse network of ground communication. Selecting the number and location of stations for a network requires an optimization algorithm that can distinguish and rank site availability based on multi-year cloud climatologies for many locations around the globe. The optimization algorithm must also consider the movement and location of a space-borne probe. In this JPL-funded study, the TASC Lasercom Network Optimization Tool (LNOT) is used to determine optimal networks of receiving stations by analyzing cloud mask data from the continental United States, Hawaii, South America, Europe, northern and southern Africa, the Middle East, central and eastern Asia, and Australia. To generate cloud masks, raw visible and infrared radiance data from GOES (Geostationary Operational Environmental Satellite) and Meteosat satellites are compared to predicted clear sky background values. Several threshold tests in the Cloud Mask Generator (CMG) involving radiance-derived cloud identification tools (e.g., fog product, albedo product) are used to estimate the probability of cloud cover for a given pixel of a satellite image. When stations are chosen from a list of sites of interest, six stations are needed to achieve a network availability of 90 % or better.