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Summary 3D outside diameter (OD) and wall thickness mapping of a casing joint consists of synchronized OD laser scanning and ultrasonic testing (UT) wall thickness measurement at a resolution of every 1 in. along the length and every 1° or 2° around the circumference. This full-length mapping data can then be used to construct a 3D finite element analysis (FEA) model that captures the geometric attributes, including OD, wall thickness, OD ovality, and wall eccentricity, of a casing joint. Together with the actual nonlinear material plasticity model and the measured as-manufactured residual stress, the 3D FEA model can predict the casing collapse strength with good accuracy. Also, the Klever-Tamano ultimate limit-state (KT ULS) collapse equation per API TR 5C3 (2018) together with a moving-average (MA) scheme can calculate the joint’s collapse strength. Results predicted by 3D FEA and KT ULS collapse models are compared to the actual full-scale collapse test results. A 3D FEA model generally produces more accurate results than the KT collapse equation. In addition, both 3D FEA and KT collapse models predict a collapse pressure slightly lower than the actual collapse pressure, which is therefore on the conservative side. The prediction of casing collapse strength can be beneficial to the end user in sorting and ranking the casing joints by collapse resistance, and therefore provides valuable information that can be used in the end user’s risk assessment. Furthermore, the end user may potentially select the joints with the highest predicted collapse strength for the portion of the casing string where the highest collapse resistance is desired. In addition, the OD and wall mapping data together with the actual material tensile properties (yield strength, ultimate tensile strength, and strain hardening parameters) can be used in estimating the internal yield, ductile rupture pressures, and other performance properties. This is outside the scope of this paper and can be pursued in future work. These predicted casing performance properties may then be used in sorting and ranking the joints based on a specific performance property of interest or a combination of performance properties.

Casing strings, consisting of hundreds of ∼40 feet long pipe joints connected by threaded connections, usually experience axial tension and/or torsion when rotationally running through the build sections during casing installation of horizontal wells. It is important to understand the influence of axial tension and torsion on the connection's fatigue performance. In this paper, fatigue performance of threaded OCTG connections subjected to cyclic dogleg bending deformation is evaluated using fatigue crack growth methodology and full-scale connection fatigue testing. Material constants in the fatigue crack growth model are first calibrated using a single set of full-scale connection fatigue test results on one connection. Several connection products (different combinations of connection design and size) are then analyzed using the calibrated fatigue model and compared to the full-scale fatigue-to-failure test results. Excellent agreement, in terms of both fatigue life and fatigue failure location, between the analysis and actual test results are observed. Effects of connection torque and axial tension on fatigue performance are also investigated using the calibrated fatigue crack growth model for a broad range of connection products. For each connection analyzed, four different torques with values in-between the connection's minimum required makeup torque and yield torque are considered. Four different axial tension levels, equivalent to 0, 10, 20, and 30 ksi axial tensile stress of the corresponding pipe, are also analyzed. Overall, different connection torques or axial tensions do not impact the connection's fatigue performance significantly except for the small size (e.g. 4.500"pipe OD) T&C connections.

Fatigue performance of OCTG threaded connections subjected to the cyclic rotational dogleg bending events during casing installation and well construction has become more and more crucial in horizontal well tubular design as its lateral becomes longer and longer for improving the well productivity. There are different methods for fatigue evaluation of structural components, such as full-scale fatigue testing and fatigue evaluation using analytic means (S-N fatigue, strain-based fatigue life, fatigue crack growth and fracture mechanics, and so on). In this paper, we implement fatigue crack growth and fracture mechanics method together with full-scale resonant bending fatigue testing for evaluating fatigue performance of OCTG connections. The fatigue evaluation procedure consists of the following steps: First, nonlinear elastic-plastic FEA model is utilized to simulate the connection makeup to its desired makeup torque (makeup position); Second, linear elastic FEA is performed to compute the alternating stress (i.e. fatigue driving stress) and static mean stress due to makeup and/or other external constant loads; Third, fatigue crack growth of a pre-existing crack-like surface circumferential flaw at critical locations is performed for selected dogleg severity (DLS), e.g.10, 15, 20, 25, 30, and 35 deg/100ft, etc. Finally, the connection fatigue curve in terms of fatigue life (number of cycles to fatigue failure,Nf) versus DLS (in deg/100ft) is constructed. Material parameters used in the fatigue crack growth model were calibrated to a single set of full-scale connection fatigue-to-failure tests for different DLS values on a single connection product (i.e., same OD, wall, grade, and connection design). By using this calibrated fatigue analysis model, we are able to achieve excellent agreement on the connection fatigue life for a broad range of DLS magnitudes (from 10 to 35 deg/100ft) between the analysis and the actual test results for several different connection products, i.e., different OD, different wall thickness, and different connection designs (API buttress-type thread, proprietary wedge thread, etc.). Moreover, the model is also able to predict the critical location at which a through-wall crack would develop and cause leak that are consistent with what were observed in the full-scale connection fatigue tests. Connection fatigue evaluation by means of fatigue crack growth and fracture mechanics presented in this paper is a value-added tool to the full-scale connection fatigue testing since it is extremely time and cost effective. The fatigue analysis tool can be used to calculate the fatigue life of any threaded OCTG connections subjected to cyclic loading (e.g., rotational dogleg bending, frac cycles, and so on). For instance, it can be very beneficial for assessing fatigue performance of a new connection product design and development, especially when the connection is intended to be used in the horizontal wells with long lateral for extended reach.

Background: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)–expressing (c-Met + ) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. Methods: In-depth phenotyping of peripheral blood T cells, including c-Met + T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. Results: We show that c-Met + T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met + T cells are distinct from those of c-Met–negative (c-Met − ) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met + T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met + T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met + T cells. Conclusions: Our study demonstrates that the detection of circulating c-Met + T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

Introduction: Isolation and identification of circulating tumor cells (CTCs) offers a promising non-invasive diagnostic tool for cancer research, but significant technical hurdles have made practical application challenging. Recent studies have shown that CTCs undergo cellular changes, such as an epithelial-mesenchymal transition (EMT), with disease progression and metastasis, implying the heterogeneity of the tumor cell population in circulation. Systems for CTC isolation are often limited by a requirement for epithelial markers or separate systems for identification of CTCs. As such, an automated platform that successfully isolates both epithelial and mesenchymal cells and enables identification of cells is necessary for successful study of CTCs. The Genesis System from Bio-Rad™ Laboratories, Inc. isolates rare CTCs based on size and deformability. It also enables users to identify isolated cells through use of an on-slide immunofluorescence (IF) staining workflow, which minimizes sample manipulation and potential sample loss. Methods: In order to achieve high-throughput label-free CTC isolation, the Genesis System uses Celselect Slides™. Each slide contains 56,400 individual microchambers to capture CTCs while maintaining viability. This Celselect Slide technology can be used for either enrichment or enumeration protocols. For this study, enumeration protocols were performed to capture and identify CTCs using on-slide IF staining. Enrichment protocols were utilized to capture, enrich, and retrieve CTCs for downstream applications. The performance was evaluated using a breast cancer cell line, MCF-7, spiked into whole blood from healthy donors. Results: The slide isolated 83.95 ± 10.25% of spiked cancer cells in blood samples at a concentration of 10-250 cells/ml. During the CTC enrichment procedure, 99.92% of blood cells were eliminated. Afterwards, 70% of the isolated CTCs were successfully released from the slide and over 92% of the released cells remained viable. Discussion and Conclusion: We present a fully automated platform to isolate and identify CTCs using on-slide immunofluorescence staining, which minimizes inevitable cell loss during the workflow. Captured cells can be retrieved for downstream applications to facilitate discovery of unknown CTC functions and their clinical relevance in cancer research. The wide range of possible downstream applications include Droplet Digital™ PCR, quantitative PCR, western blot assays, bulk/single-cell genomics, cell culture, fluorescence in situ hybridization, and RNA-Seq. Additionally, the design of the system is amenable to other biofluids which may be utilized for liquid biopsies. The Genesis System with Celselect Slide technology offers a powerful diagnostic tool to monitor disease progression and personalized treatment. Citation Format: Yoon-Tae Kang, Errile Pusod, Ally Crudgington, Abiodun Bodunrin, Nick Bosio, Susana Chan, David Coe, Michelle Racey. Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6605.

Collapse performance of a casing string subjected to external hydraulic pressure is one of the most important parameters in the deep water well casing design. It often becomes one of the governing factors in a casing design program. This paper investigates the quantitative effect of inherent circumferential residual stress (RS) on casing collapse resistane to external pressure through parametric Finite Element Analysis (FEA). Nonlinear elastic-plastic FEA study on casing collapse calculations have been performed. Different magnitudes and orientations of the initial circumferential residual stress were considered. Modified Riks method was used to predict the casing onsite collapse pressure as well as the post-collapse response. Parameters considered in the analysis include: (i) two material grades (L80 and Grade 135); (ii) for each material grade, ratio of outside diameter to wall thickness (i.e. D/t) ranging from 10 to 50; (iii) for each D/t ratio, initial circumferential residual stress at casing inner diameter (ID) ranging from -40% to +50% of the material yield strength (YS). Analysis results indicate that there is a highly nonlinear, parabolic-like dependence of collapse strength on the initial circumferential residual stress. Comparison of the FEA results to the predictions by Klever-Tamano Ultimate Limit-State (KT ULS) equation indicates that the KT ULS equation in the current API Technical Report 5C3 (API TR 5C3) does not capture this nonlinear relationship. This is probably due to the limitation of the API historical collapse test data upon which the KT ULS equation was calibrated to. The majority of the API historical collapse tests had the compressive residual stress at the casing ID. Moreover, the relationship between casing collapse strength and residual stress may be used as a guideline for potentially developing new high collapse casing by full-length residual stress control in mill production.

Three-dimensional (3D) outside diameter (OD) and wall thickness mapping of a casing joint include synchronized OD laser scanning and wall UT (ultrasonic testing) measurement at a resolution of every 1-inch along the length and every 1 or 2 degrees around the circumference. This full-length mapping data can then be used to construct a 3D finite element analysis (FEA) model that captures the geometric attributes, including OD, wall thickness, OD ovality, and wall eccentricity of a casing joint. Together with the actual nonlinear material plasticity model and the measured residual stress, the 3D FEA model can accurately predict the casing collapse strength. Also, the Klever-Tamano ultimate limit-state (KT ULS) collapse equation per API TR 5C3 [1] together with a calibrated moving-average scheme was used to calculate the joint's collapse strength. Results predicted by 3D FEA and KT ULS collapse models were then compared to the actual full-scale collapse test results. A 3D FEA model generally produces more accurate results than the KT collapse equation. In addition, both 3D FEA and KT collapse models predict slightly lower than actual collapse pressure, which therefore is on the conservative side. The accurate prediction of casing collapse strength can be beneficial to the end user in sorting and ranking the casing joints by collapse resistance, and therefore provides valuable information that can be used in the end user's risk assessment with the consideration of the worst-case discharge scenario. Furthermore, the end user may potentially select the joints with the highest predicted collapse strength for the bottom portion of the casing string where the highest collapse resistance is desired. In addition, the OD and wall mapping data together with the actual material tensile properties (yield strength, ultimate tensile strength, and strain hardening parameters) can be used in estimating the internal yield and ductile rupture pressures and other performance properties. This is outside the scope of this paper and can be pursued in future work. These predicted casing performance properties may then be used in sorting and ranking the joints based on a specific performance property of interest or combination of performance properties.

Activation of T cells relies on the availability of intracellular cholesterol for an effective response after stimulation. We investigated the contribution of cholesterol derived from extracellular uptake by the low-density lipoprotein (LDL) receptor in the immunometabolic response of T cells. By combining proteomics, gene expression profiling, and immunophenotyping, we described a unique role for cholesterol provided by the LDLR pathway in CD8+ T cell activation. mRNA and protein expression of LDLR was significantly increased in activated CD8+ compared to CD4+ WT T cells, and this resulted in a significant reduction of proliferation and cytokine production (IFNγ, Granzyme B, and Perforin) of CD8+ but not CD4+ T cells from Ldlr −/− mice after in vitro and in vivo stimulation. This effect was the consequence of altered cholesterol routing to the lysosome resulting in a lower mTORC1 activation. Similarly, CD8+ T cells from humans affected by familial hypercholesterolemia (FH) carrying a mutation on the LDLR gene showed reduced activation after an immune challenge.

Casing collapse under external pressure is a complex phenomenon and is one of the governing factors in the tubular design of oil and gas wells. Collapse strength of a casing depends on its outer diameter to wall thickness ratio (i.e. D/t), material Young's modulus, yield stress, shape of stress-strain curve, temperature, ovality, wall eccentricity, circumferential residual stress, as well as combined loadings. This paper studies the quantitative effects of two geometric impefections, namely (i) wear on the inside diameter (ID) of casing and (ii) wall eccentricity angle; as well as two combined loading scenarios: (iii) dogleg bending and (iv) axial compression. Although these scenarios are not covered in the industry Standards, such as API TR 5C3 (June 2018 edition) [1], they are important factors that must be considered in the tubular design of oil and gas wells. In order to quantify the effects of the four scenarios listed above, nonlinear parametric collapse Finite Element Analysis (FEA) studies were performed. Modified Riks method was utilized to predict the casing on-site collapse pressure as well as the unstable post-collapse response. Both material and geometric nonlinearities were taken into account. Elastic-plastic material property with strain hardening was incorporated in the collapse FEA models. Based on the results of the parametric FEA simulations, some important observations and conclusions can be made: (i) for the scenario of casing ID wear, reduction in worn casing collapse resistance is nearly proportional to the reduction in minimum worn thickness regardless of the casing dimensiosns and material grade; (ii) for the case of wall eccentricity irregularity, as the circumferential location of maximum wall thickness moves closer to the minimum wall thickness location, the casing collapse resistance decreases; (iii) casing collapse resistance decreases as the dogleg severity (DLS) increases. Moreover, the effect of dogleg bending on collapse resistance has a strong dependence on casing dimensions and material grade; (iv) in contrast to the effect of axial tension, dependence of collapse resistance on axial compression is quite nonlinear and non-monotonic. In other words, as axial compression increases, collapse resistance increases until the axial compression reaches approximately 50% to 60% (primarily depending on D/t ratio) of yield strength, and then decreases as axial compression increases further.

It has long been accepted that the “greenhouse effect”, where the atmosphere readily transmits short wavelength incoming solar radiation but selectively absorbs long wavelength outgoing radiation emitted by the earth, is responsible for warming the earth from the 255K effective earth temperature, without atmospheric warming, to the current average temperature of 288K. It is also widely accepted that the two main atmospheric greenhouse gases are H2O and CO2. What is surprising is the wide variation in the estimated warming potential of CO2, the gas held responsible for the modern concept of climate change. Estimates published by the IPCC for climate sensitivity to a doubling of CO2 concentration vary from 1.5 to 4.5°C based upon a plethora of scientific papers attempting to analyse the complexities of atmospheric thermodynamics to determine their results. The aim of this paper is to simplify the method of achieving a figure for climate sensitivity not only for CO2, but also CH4 and N2O, which are also considered to be strong greenhouse gases, by determining just how atmospheric absorption has resulted in the current 33K warming and then extrapolating that result to calculate the expected warming due to future increases of greenhouse gas concentrations. The HITRAN database of gaseous absorption spectra enables the absorption of earth radiation at its current temperature of 288K to be accurately determined for each individual atmospheric constituent and also for the combined absorption of the atmosphere as a whole. From this data it is concluded that H2O is responsible for 29.4K of the 33K warming, with CO2 contributing 3.3K and CH4 and N2O combined just 0.3K. Climate sensitivity to future increases in CO2 concentration is calculated to be 0.50K, including the positive feedback effects of H2O, while climate sensitivities to CH4 and N2O are almost undetectable at 0.06K and 0.08K respectively. This result strongly suggests that increasing levels of CO2 will not lead to significant changes in earth temperature and that increases in CH4 and N2O will have very little discernable impact.

Cholesterol homeostasis has a pivotal function in regulating immune cells. Here we show that apolipoprotein E (apoE) deficiency leads to the accumulation of cholesterol in the cell membrane of dendritic cells (DC), resulting in enhanced MHC-II-dependent antigen presentation and CD4+ T-cell activation. Results from WT and apoE KO bone marrow chimera suggest that apoE from cells of hematopoietic origin has immunomodulatory functions, regardless of the onset of hypercholesterolemia. Humans expressing apoE4 isoform (ε4/3–ε4/4) have increased circulating levels of activated T cells compared to those expressing WT apoE3 (ε3/3) or apoE2 isoform (ε2/3–ε2/2). This increase is caused by enhanced antigen-presentation by apoE4-expressing DCs, and is reversed when these DCs are incubated with serum containing WT apoE3. In summary, our study identifies myeloid-produced apoE as a key physiological modulator of DC antigen presentation function, paving the way for further explorations of apoE as a tool to improve the management of immune diseases., Cholesterol homeostasis can modulate immunity via multiple pathways. Here the authors show that apolipoprotein E, an important regulator of cholesterol, produced by myeloid cells can regulate T cell activation by controlling the antigen presentation activity of dendritic cells in both humans and mice.

Although polyclonal regulatory T cells (Tregs) that once expressed Foxp3 (ex-Tregs) derived from Foxp3+ Tregs have been described in homeostatic and autoimmune settings, little is known regarding the influence of the tumor environment on ex-Treg development. After adoptive transfer of HY-specific green Tregs (peripheral or thymic) to Rag2−/− B6 female mice bearing syngeneic HY-expressing MB49 tumors, a significant fraction rapidly lost expression of Foxp3. On the second transfer to a Rag2−/− B6 male environment, these ex-Tregs expanded strongly, whereas Tregs that maintained expression of Foxp3 expression did not. Both FACS and quantitative real-time-PCR analysis revealed that ex-Tregs upregulated genes characteristic of a Th1 effector-memory phenotype including IFN-γ and downregulated a panel of Treg-specific genes. Peripheral HY-specific green Tregs were adoptively transferred to Rag2−/− B6 male mice, to dissect the factors regulating ex-Treg differentiation. Development of ex-Tregs was more efficient in the mesenteric lymph node (mLN) than peripheral lymph node environment, correlating with a much greater level of IL-6 mRNA in mLN. In addition, the preferential development of ex-Tregs in mLN was significantly impaired by cotransfer of HY-specific naive CD4 T cells. Collectively, our study not only demonstrates the plasticity of Ag-specific Tregs in the context of the tumor environment, but also defines key molecular and cellular events that modulate ex-Treg differentiation.

Summary In this study we investigated the impact of several factors on the expansion of natural regulatory T (nTreg) cells by tumours, including antigen specificity, transforming growth factor-β (TGF-β) signalling and the antigen-presenting cell subsets responsible for expansion. We found that antigen non-specific expansion of nTreg cells is tumour cell line-dependent. Although both antigen-specific and non-specific pathways can contribute to expansion, the migration of activated nTreg cells to tumour tissues is strictly antigen-dependent. Intact TGF-β signalling on nTreg cells is also essential for tumour-induced expansion. Finally, for stimulation of resting antigen-specific CD4 T cells, CD11c+ cells purified from tumour-draining lymph nodes were more potent than CD11b+ cells, suggesting that dendritic cells are the key antigen-presenting cell subset involved in cross-presentation of tumour antigens. This study not only provides an in vivo system in which cross-talk between nTreg cells and tumours can be explored but also reveals novel aspects of tumour immune evasion.

In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.

Complement activation is known to have deleterious effects on organ transplantation. On the other hand, the complement system is also known to have an important role in regulating immune responses. The balance between these two opposing effects is critical in the context of transplantation. Here, we report that female mice deficient in C1q (C1qa(-/-)) or C3 (C3(-/-)) reject male syngeneic grafts (HY incompatible) at an accelerated rate compared with WT mice. Intranasal HY peptide administration, which induces tolerance to syngeneic male grafts in WT mice, fails to induce tolerance in C1qa(-/-) or C3(-/-) mice. The rejection of the male grafts correlated with the presence of HY D(b)Uty-specific CD8(+) T cells. Consistent with this, peptide-treated C1qa(-/-) and C3(-/-) female mice rejecting male grafts exhibited more antigen-specific CD8(+)IFN-gamma(+) and CD8(+)IL-10(+) cells compared with WT females. This suggests that accumulation of IFN-gamma- and IL-10-producing T cells may play a key role in mediating the ongoing inflammatory process and graft rejection. Interestingly, within the tolerized male skin grafts of peptide-treated WT mice, IFN-gamma, C1q and C3 mRNA levels were higher compared to control female grafts. These results suggest that C1q and C3 facilitate the induction of intranasal tolerance.

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFβ while down-regulating IFN-γ, GM-CSF, IL5, and TNF-α production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFβ to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.

Constitutive resistance to cell death induced by inflammatory stimuli activating the extrinsic pathway of apoptosis is a key feature of vascular endothelial cells (ECs). Although this property is central to the maintenance of the endothelial barrier during inflammation, the molecular mechanisms of EC protection from cell-extrinsic, proapoptotic stimuli have not been investigated. We show that the Ig-family member CD31, which is expressed by endothelial but not epithelial cells, is necessary to prevent EC death induced by TNF-α and cytotoxic T lymphocytes in vitro. Combined quantitative RT-PCR array and biochemical analysis show that, upon the engagement of the TNF receptor with TNF-α on ECs, CD31 becomes activated and, in turn, counteracts the proapoptotic transcriptional program induced by TNF-α via activation of the Erk/Akt pathway. Specifically, Akt activation by CD31 signals prevents the localization of the forkhead transcription factor FoxO3 to the nucleus, thus inhibiting transcription of the proapoptotic genes CD95/Fas and caspase 7 and de-repressing the expression of the antiapoptotic gene cFlar. Both CD31 intracellular immunoreceptor tyrosine-based inhibition motifs are required for its prosurvival function. In vivo, CD31 gene transfer is sufficient to recapitulate the cytoprotective mechanisms in CD31(-) pancreatic β cells, which become resistant to immune-mediated rejection when grafted in fully allogeneic recipients.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.

It is now well established that the effector T cell (Teff) response is regulated by a series of metabolic switches. Quiescent T cells predominantly require ATP-generating processes, whereas proliferating Teff require high metabolic flux through growth-promoting pathways, such as glycolysis. Pathways that control metabolism and immune cell function are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell effector functions. Furthermore, functionally distinct T cell subsets have been shown to require distinct energetic and biosynthetic pathways to support their specific functional needs. In particular, naturally occurring regulatory T cells (Treg) are characterized by a unique metabolic signature distinct to that of conventional Teff cells. We here briefly review the signaling pathways that control Treg metabolism and how this metabolic phenotype integrates their differentiation and function. Ultimately, these metabolic features may provide new opportunities for the therapeutic modulation of unwanted immune responses.

Background. Regulatory T (Treg) cells, generated in vitro by Foxp3 gene transfer into naive CD4 + 25 - T cells, have been shown to inhibit the development of inflammation and autoimmune disease, but it is not known whether they are able to prevent allograft rejection. This study investigated whether Treg cells generated from naive CD4 + T cells by Foxp3 gene transfer could induce transplantation tolerance. Methods. HY-specific, T-cell receptor (TCR)-transgenic CD4 + 25 - T cells were retrovirally transduced with the Foxp3 gene. The phenotype, function, and cytokine profiles of the transduced cells were examined in vitro by fluorescence-activated cell sorter, T-cell proliferation assays, enzyme-linked immunosorbent assay, and intracellular cytokine staining. Adoptive transfer and skin grafting experiments were conducted to assess whether Foxp3-transduced HY-specific T cells could prevent the rejection of syngeneic male grafts. Results. CD4 + 25 - T cells retrovirally transduced with Foxp3 express a panel of cell surface and intracellular molecules closely associated with Treg activity. This Treg phenotype was stable during in vitro culture with some further maturation. In vitro, Foxp3-transduced cells were functionally anergic and suppressive T cells. In vivo adoptive transfer of Foxp3-transduced HY-specific TCR-transgenic CD4 + T cells protected male skin grafts from rejection by syngeneic females. Retroviral transduction of the Foxp3 gene into non-TCR-transgenic CD4 + 25 - T cells, however, had no influence on male skin graft rejection. Conclusion. This study provides the first evidence that Foxp3-transduced T cells can control the rejection of an allogeneic transplant and suggests that T-cell Foxp3 gene transfer may have therapeutic value in clinical transplantation.

Summary Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene—leading to increased GCK activity—had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration., Graphical Abstract, Highlights • Migration of regulatory T (Treg) cells requires glycolysis • This is mediated by the enzyme glucokinase induced by a PI3K-mTORC2 pathway • Treg cells lacking this pathway are unable to localize to inflammatory sites • A loss-of-function GCK regulator gene causes enhanced motility of human Treg cells, Regulatory T cell localization to inflammatory sites is key to their homeostatic function. Kishore and colleagues demonstrate that Treg cell migration requires the activation of glycolysis by the enzyme glucokinase induced via a Treg cell-selective PI3K-mTORC2 pathway.

SummaryEffector-T-cell-mediated immunity depends on the efficient localization of antigen-primed lymphocytes to antigen-rich non-lymphoid tissue, which is facilitated by the expression of a unique set of “homing” receptors acquired by memory T cells. We report that engagement of the hepatocyte growth factor (HGF) receptor c-Met by heart-produced HGF during priming in the lymph nodes instructs T cell cardiotropism, which was associated with a specialized homing “signature” (c-Met+CCR4+CXCR3+). c-Met signals facilitated T cell recruitment to the heart via the chemokine receptor CCR5 by inducing autocrine CCR5 ligand release. c-Met triggering was sufficient to support cardiotropic T cell recirculation, while CCR4 and CXCR3 sustained recruitment during heart inflammation. Transient pharmacological blockade of c-Met during T cell priming led to enhanced survival of heart, but not skin, allografts associated with impaired localization of alloreactive T cells to heart grafts. These findings suggest c-Met as a target for development of organ-selective immunosuppressive therapies.

Localization of CD4+CD25+Foxp3+ regulatory T (Treg) cells to lymphoid and non-lymphoid tissue is instrumental for the effective control of immune responses. Compared with conventional T cells, Treg cells constitute a minute fraction of the T-cell repertoire. Despite this numeric disadvantage, Tregs efficiently migrate to sites of immune responses reaching an optimal number for the regulation of T effector (Teff) cells. The array and levels of adhesion and chemokine receptor expression by Tregs do not explain their powerful migratory capacity. Here we show that recognition of self-antigens expressed by endothelial cells in target tissue is instrumental for efficient Treg recruitment in vivo. This event relies upon IFN-γ-mediated induction of MHC-class-II molecule expression by the endothelium and requires optimal PI3K p110δ activation by the T-cell receptor. We also show that, once in the tissue, Tregs inhibit Teff recruitment, further enabling a Teff:Treg ratio optimal for regulation., Regulatory T cells (Tregs) are important for the maintenance of self-tolerance and this requires their trafficking to the lymph nodes and target tissues. Here, the authors show that the recognition of self-antigens expressed by endothelial cells in target tissue is instrumental for efficient Treg recruitment in vivo.

Pre-transplant conditioning regimens play a major role in triggering graft-versus-host disease (GVHD). This study investigated the effect of irradiation on donor T cell trafficking to lymphoid and non-lymphoid tissues by comparing the migration of carboxy-fluorescein diacetate succinimidyl ester-labelled, naive donor T lymphocytes in vivo in irradiated and non-irradiated syngeneic mice recipients. Recruitment of adoptively transferred naive T cells to secondary lymphoid organs was increased in irradiated mice and naive T cells also aberrantly localized to non-lymphoid tissues. Irradiation also induced aberrant effector memory T cell migration into lymph nodes and their localization to homing-privileged non-lymphoid sites, such as the gut. The presence of a minor histocompatibility mismatch further enhanced the aberrant accumulation of T cells in both lymphoid and non-lymphoid tissue, whilst their migratory pattern was not modified as compared to fully matched irradiated recipients. These effects correlated with decreased permeability of, and the secretion of chemotactic factors by the endothelium. Our findings are consistent with the possibility that excessive, dysregulated extravasation of T cells induced by irradiation promotes the development of GVHD.

Summary Previously we have shown that indoleamine 2,3-dioxygenase (IDO) and the tryptophan metabolite, 3-hydroxykynurenine (3HK) can prolong corneal allograft survival. IDO modulates the immune response by depletion of the essential amino acid tryptophan by breakdown to kynurenines, which themselves act directly on T lymphocytes. The tryptophan metabolite analogue N-(3,4-dimethoxycinnamonyl) anthranilic acid (DAA, ‘Tranilast’) shares the anthranilic acid core with 3HK. Systemic administration of DAA to mice receiving a fully MHC-mismatched allograft of cornea or skin resulted in significant delay in rejection (median survival of controls 12 days, 13 days for cornea and skin grafts, respectively, and of treated mice 24 days (P

Describes the various systems which have been developed for monitoring gaseous emissions from industrial chimney stacks and flues. Compares extractive and non‐extractive systems and outlines the four main types of analyzer used in continuous extractive systems. Gives a detailed description of the cross‐stack method and how it is used to determine carbon monoxide concentration in a typical process gas flue. Concludes that cross stack analyzers can be used to measure a variety of gases and that the adaptability and low maintenance requirements of this technology make it suitable for monitoring a comprehensive range of gaseous pollutants.

The bias of αβ T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFβ, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.

Objective— The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA) + cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). Methods and Results— BALB/c aortas (H-2 d ) transplanted into α-TFPI–transgenic (Tg) mice (H-2 b ) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2 d alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34 + (but not CD34 − ) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI–Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34 + cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45 + myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA + human TFPI + CD34 + cells recruited in Tg recipients were from a CD45 − lineage. WT CD34 + cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. Conclusion— Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA + CD34 + cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.