Role of the membrane anchor in the regulation of Lck activity.

Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.

Anti-CD3 treatment up-regulates programmed cell death protein-1 expression on activated effector T cells and severely impairs their inflammatory capacity.

T cells play a key role in the pathogenesis of type 1 diabetes, and targeting the CD3 component of the T-cell receptor complex provides one therapeutic approach. Anti-CD3 treatment can reverse overt disease in spontaneously diabetic non-obese diabetic mice, an effect proposed to, at least in part, be caused by a selective depletion of pathogenic cells. We have used a transfer model to further investigate the effects of anti-CD3 treatment on green fluorescent protein (GFP)+ islet-specific effector T cells in vivo. The GFP expression allowed us to isolate the known effectors at different time-points during treatment to assess cell presence in various organs as well as gene expression and cytokine production. We find, in this model, that anti-CD3 treatment does not preferentially deplete the transferred effector cells, but instead inhibits their metabolic function and their production of interferon-γ. Programmed cell death protein 1 (PD-1) expression was up-regulated on the effector cells from anti-CD3-treated mice, and diabetes induced through anti-PD-L1 antibody could only be reversed with anti-CD3 antibody if the anti-CD3 treatment lasted beyond the point when the anti-PD-L1 antibody was washed out of the system. This suggests that PD-1/PD-L1 interaction plays an important role in the anti-CD3 antibody mediated protection. Our data demonstrate an additional mechanism by which anti-CD3 therapy can reverse diabetogenesis.

Foxp3 expression is required for the induction of therapeutic tissue tolerance.

CD4(+)Foxp3(+) regulatory T cells (Treg) are essential for immune homeostasis and maintenance of self-tolerance. They are produced in the thymus and also generated de novo in the periphery in a TGF-ß-dependent manner. Foxp3(+) Treg are also required to achieve tolerance to transplanted tissues when induced by coreceptor or costimulation blockade. Using TCR-transgenic mice to avoid issues of autoimmune pathology, we show that Foxp3 expression is both necessary and sufficient for tissue tolerance by coreceptor blockade. Moreover, the known need in tolerance induction for TGF-ß signaling to T cells can wholly be explained by its role in induction of Foxp3, as such signaling proved dispensable for the suppressive process. We analyzed the relative contribution of TGF-ß and Foxp3 to the transcriptome of TGF-ß-induced Treg and showed that TGF-ß elicited a large set of downregulated signature genes. The number of genes uniquely modulated due to the influence of Foxp3 alone was surprisingly limited. Retroviral-mediated conditional nuclear expression of Foxp3 proved sufficient to confer transplant-suppressive potency on CD4(+) T cells and was lost once nuclear Foxp3 expression was extinguished. These data support a dual role for TGF-ß and Foxp3 in induced tolerance, in which TGF-ß stimulates Foxp3 expression, for which sustained expression is then associated with acquisition of tolerance.

Activation rather than Foxp3 expression determines that TGF-ß-induced regulatory T cells out-compete naïve T cells in dendritic cell clustering.

Regulatory T (Treg) cells are critically important for the maintenance of immunological tolerance. Both centrally arising natural nTreg cells and those emerging in the periphery in response to TGF-ß, iTreg cells, play a role in the control of unwanted immune responses. Treg cells adopt multiple mechanisms to inhibit effector T cells, yet it is unclear whether these mechanisms are shared by nTreg cells and iTreg cells alike. Here, we show that iTreg cells, like nTreg cells, are able to out-compete naïve T cells in clustering around dendritic cells (DCs). However, using both a tamoxifen-responsive inducible Foxp3 retroviral construct and TGF-ß-induced iTreg cells from hCD2-Foxp3 knock in reporter mice, we show that it is prior antigen-induced activation rather than Foxp3 expression per se that determines the ability of iTreg cells to competitively cluster around DCs. We found no difference in the capacity of iTreg cells to displace naïve T cells around DCs to that of Tr1, Th1, Th2, or Th9 cells. An important difference was, however, that clustering of iTreg cells around DCs, just as for naïve T cells, did not effectively activate DCs.

Generation of anti-inflammatory adenosine by leukocytes is regulated by TGF-ß.

Levels of anti-inflammatory extracellular adenosine are controlled by the sequential action of the ectonucleotidases CD39 and CD73, whose expression in CD4(+) T cells has been associated with natural regulatory T cells (nTregs). We here show that CD73 expression on activated murine CD4(+) T cells is induced by TGF-ß independently of Foxp3 expression, operates at the transcriptional level and translates into gain of functional capacity to generate adenosine. In the presence of AMP, CD73 induced by TGF-ß generates adenosine able to suppress proliferation of activated CD4(+) T cells in vitro. These effects are contextual and opposed by proinflammatory cytokines. CD73 is also upregulated by TGF-ß in CD8(+) T cells, DCs and macrophages, so providing an amplification mechanism for adenosine generation in tissue microenvironments. Together, these findings expose a novel anti-inflammatory role for TGF-ß.

Infectious tolerance via the consumption of essential amino acids and mTOR signaling.

Infectious tolerance describes the process of CD4(+) regulatory T cells (Tregs) converting naïve T cells to become additional Tregs. We show that antigen-specific Tregs induce, within skin grafts and dendritic cells, the expression of enzymes that consume at least 5 different essential amino acids (EAAs). T cells fail to proliferate in response to antigen when any 1, or more, of these EAAs are limiting, which is associated with a reduced mammalian target of rapamycin (mTOR) signaling. Inhibition of the mTOR pathway by limiting EAAs, or by specific inhibitors, induces the Treg-specific transcription factor forkhead box P3, which depends on both T cell receptor activation and synergy with TGF-beta.

SAP couples Fyn to SLAM immune receptors.

SAP (SLAM-associated protein) is a small lymphocyte-specific signalling molecule that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). Consistent with its single src homology 2 (SH2) domain architecture and unusually high affinity for SLAM (also called CD150), SAP has been suggested to function by blocking binding of SHP-2 or other SH2-containing signalling proteins to SLAM receptors. Additionally, SAP has recently been shown to be required for recruitment and activation of the Src-family kinase FynT after SLAM ligation. This signalling 'adaptor' function has been difficult to conceptualize, because unlike typical SH2-adaptor proteins, SAP contains only a single SH2 domain and lacks other recognized protein interaction domains or motifs. Here, we show that the SAP SH2 domain binds to the SH3 domain of FynT and directly couples FynT to SLAM. The crystal structure of a ternary SLAM-SAP-Fyn-SH3 complex reveals that SAP binds the FynT SH3 domain through a surface-surface interaction that does not involve canonical SH3 or SH2 binding interactions. The observed mode of binding to the Fyn-SH3 domain is expected to preclude the auto-inhibited conformation of Fyn, thereby promoting activation of the kinase after recruitment. These findings broaden our understanding of the functional repertoire of SH3 and SH2 domains.

MS4A4B is a GITR-associated membrane adapter, expressed by regulatory T cells, which modulates T cell activation.

In the aftermath of thymic negative selection, natural and adaptive regulatory T cells (Tregs) must acknowledge peripheral, "danger-free" self-Ag to ensure their sustained activity. In this paper, we show that natural and adaptive Tregs or T cells transduced with cDNA for Foxp3, just like Th1 cells, express members of the MS4A family of transmembrane molecules. Naive T cells transduced with MS4A4B become able to respond to lower levels of Ag. Using two family members, MS4A4B and MS4A6B, as baits in a yeast split-ubiquitin Treg library screen, we demonstrate their interaction with each other and with GITR, Orai1, and other surface receptors. Interaction of 4B with GITR augments GITR signaling and T cell IL-2 production in response to triggering with GITR ligand or anti-GITR Abs. This interaction provides a mechanism whereby MS4A family members, through lateral coassociation with costimulatory molecules, may amplify Ag signals. We propose that T cells preoccupied with immune defense use this MS4A family to enhance sensitivity to extrinsic Ag stimulation, ensuring its elimination, while Tregs use these adaptors to allow low level Ag signals to sustain regulatory function.

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function.

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

The cell surface receptor SLAM controls T cell and macrophage functions.

Signaling lymphocyte activation molecule (SLAM), a glycoprotein expressed on activated lymphocytes and antigen-presenting cells, has been shown to be a coregulator of antigen-driven T cell responses and is one of the two receptors for measles virus. Here we show that T cell receptor-induced interleukin (IL)-4 secretion by SLAM(-/-) CD4(+) cells is down-regulated, whereas interferon gamma production by CD4(+) T cells is only slightly up-regulated. Although SLAM controls production of IL-12, tumor necrosis factor, and nitric oxide in response to lipopolysaccharide (LPS) by macrophages, SLAM does not regulate phagocytosis and responses to peptidoglycan or CpG. Thus, SLAM acts as a coreceptor that regulates signals transduced by the major LPS receptor Toll-like receptor 4 on the surface of mouse macrophages. A defective macrophage function resulted in an inability of SLAM(-/-) C57Bl/6 mice to remove the parasite Leishmania major. We conclude that the coreceptor SLAM plays a central role at the interface of acquired and innate immune responses.

Viperin, through its radical-SAM activity, depletes cellular nucleotide pools and interferes with mitochondrial metabolism to inhibit viral replication.

Viperin (RSAD2) is an antiviral radical S-adenosylmethionine (SAM) enzyme highly expressed in different cell types upon viral infection. Recently, it has been reported that the radical-SAM activity of viperin transforms cytidine triphosphate (CTP) to its analogue 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Based on biochemical studies and cell biological experiments, it was concluded that ddhCTP and its nucleoside form ddhC do not affect the cellular concentration of nucleotide triphosphates and that ddhCTP acts as replication chain terminator. However, our re-evaluation of the reported data and new results indicate that ddhCTP is not an effective viral chain terminator but depletes cellular nucleotide pools and interferes with mitochondrial activity to inhibit viral replication. Our analysis is consistent with a unifying view of the antiviral and radical-SAM activities of viperin.

A Novel Role for Triglyceride Metabolism in Foxp3 Expression.

Lipid metabolism plays a key role in many cellular processes. We show here that regulatory T cells have enhanced lipid storage within subcellular lipid droplets (LD). They also express elevated amounts of both isoforms of diacylglycerol acyl transferase (DGAT1 & 2), enzymes required for the terminal step of triacylglycerol synthesis. In regulatory T-cells (Tregs), the conversion of diacylglycerols to triacylglycerols serves two additional purposes other than lipid storage. First, we demonstrate that it protects T cells from the toxic effects of saturated long chain fatty acids. Second, we show that Triglyceride formation is essential for limiting activation of protein kinase C via free diacyl glycerol moieties. Inhibition of DGAT1 resulted in elevated active PKC and nuclear NFKB, as well as impaired Foxp3 induction in response to TGFß. Thus, Tregs utilize a positive feedback mechanism to promote sustained expression of Foxp3 associated with control of LD formation.

CD4+ T Cell Fate Decisions Are Stochastic, Precede Cell Division, Depend on GITR Co-Stimulation, and Are Associated With Uropodium Development.

During an immune response, naïve CD4+ T cells proliferate and generate a range of effector, memory, and regulatory T cell subsets, but how these processes are co-ordinated remains unclear. A traditional model suggests that memory cells use mitochondrial respiration and are survivors from a pool of previously proliferating and glycolytic, but short-lived effector cells. A more recent model proposes a binary commitment to either a memory or effector cell lineage during a first, asymmetric cell division, with each lineage able to undergo subsequent proliferation and differentiation. We used improved fixation and staining methods with imaging flow cytometry in an optimized in vitro system that indicates a third model. We found that cell fates result from stochastic decisions that depend on GITR co-stimulation and which take place before any cell division. Effector cell commitment is associated with mTORC2 signaling leading to uropodium development, while developing memory cells lose mitochondria, have a nuclear localization of NFκB and depend on TGFß for their survival. Induced, T helper subsets and foxp3+ regulatory T cells were found in both the effector and memory cell lineages. This in vitro model of T cell differentiation is well suited to testing how manipulation of cytokine, nutrient, and other components of the microenvironment might be exploited for therapeutic purposes.

The Role of Lipid Metabolism in T Lymphocyte Differentiation and Survival.

The differentiation and effector functions of both the innate and adaptive immune system are inextricably linked to cellular metabolism. The features of metabolism which affect both arms of the immune system include metabolic substrate availability, expression of enzymes, transport proteins, and transcription factors which control catabolism of these substrates, and the ability to perform anabolic metabolism. The control of lipid metabolism is central to the appropriate differentiation and functions of T lymphocytes, and ultimately to the maintenance of immune tolerance. This review will focus on the role of fatty acid (FA) metabolism in T cell differentiation, effector function, and survival. FAs are important sources of cellular energy, stored as triglycerides. They are also used as precursors to produce complex lipids such as cholesterol and membrane phospholipids. FA residues also become incorporated into hormones and signaling moieties. FAs signal via nuclear receptors and their channeling, between storage as triacyl glycerides or oxidation as fuel, may play a role in survival or death of the cell. In recent years, progress in the field of immunometabolism has highlighted diverse roles for FA metabolism in CD4 and CD8 T cell differentiation and function. This review will firstly describe the sensing and modulation of the environmental FAs and lipid intracellular signaling and will then explore the key role of lipid metabolism in regulating the balance between potentially damaging pro-inflammatory and anti-inflammatory regulatory responses. Finally the complex role of extracellular FAs in determining cell survival will be discussed.

Ureterorenoscopy training on cadavers embalmed by Thiel's method: simulation or a further step towards reality? Initial report.

INTRODUCTION: The technique of ureterorenoscopy has a significant learning curve. Cadavers embalmed by the Thiel method have been successfully used for simulation training in a number of surgical specialties. Here we present our experience of the first use of Thiel cadavers in a formal ureteroscopy training course. MATERIAL AND METHODS: The inaugural 'Masterclass in Flexible Ureterorenoscopy' was run with participants performing ureterorenoscopy on three Thiel cadavers under expert supervision. A qualitative questionnaire was delivered to the participants and faculty. Assessed domains were tissue characteristics of the cadaveric urinary tract, anatomical features and procedural aspects. A five-point Likert score was used to assess responses. Data regarding participant experience in endourology were also collected. RESULTS: 8 questionnaires were collected. All participants completed cadaveric ureterorenoscopy. Three-quarters reported the overall quality of tissue in the cadaveric bladder, ureters and pelvicalyceal system as high or excellent. Half reported the cadaveric bladder as being softer than in a live patient, whilst five out of eight thought that the cadaveric ureter was softer and more prone to trauma. Seven out of eight were satisfied with the overall quality of the cadaveric model. The quality of vision and irrigation in the upper urinary tracts was reported as high. CONCLUSIONS: Thiel cadavers have been shown to have excellent tissue characteristics, as well as being durable and reusable. We have described the first use of Thiel cadavers in a designated ureterorenoscopy course, with high levels of delegate satisfaction. Further work is required to develop the role of Thiel cadavers as part of an integrated, modular urology training.

A Bacterial Artificial Chromosome Reporter System for Expression of the Human FOXP3 Gene in Mouse Regulatory T-Cells.

The transcription factor FOXP3 plays key roles in the development and function of regulatory T cells (Treg) capable of preventing and correcting immunopathology. There has been much interest in exploiting Treg as adoptive cell therapy in man, but issues of lack of nominal antigen-specificity and stability of FoxP3 expression in the face of pro-inflammatory cytokines have been a concern. In order to enable fundamental studies of human FOXP3 (hFOXP3) gene regulation and to provide preclinical tools to guide the selection of drugs that might modulate hFOXP3 expression for therapeutic purposes, we generated hFOXP3/AmCyan bacterial artificial chromosome (BAC) transgenic mice and transfectants, wherein hFOXP3 expression was read out as AmCyan expression. Using the transgenic mice, one can now investigate hFOXP3 gene expression under defined experimental conditions used for mouse Foxp3 (mFoxp3) studies. Here, we demonstrate that hFOXP3 gene expression in BAC transgenic mice is solely restricted to CD4+ T-cells, as for mFoxp3 gene expression, showing that hFOXP3 expression in Treg cells depends on fundamentally similar processes to mFoxp3 expression in these cells. Similarly, hFOXP3 expression could be observed in mouse T-cells through TCR stimulation in the presence of TGF-ß. These data suggest that, at least in part, cell type-specific human and mouse foxp3 gene expression is regulated by common regulatory regions which for the human, are located within the 110-kb human FOXP3 BAC DNA. To investigate hFOXP3 gene expression further and to screen potential therapeutics in modulating hFOXP3 gene expression in vitro, we also generated hFOXP3/AmCyan expression reporter cell lines. Using the reporter cells and transcription factor inhibitors, we showed that, just as for mFoxp3 expression, inhibitors of NF-κB, AP1, STAT5, Smad3, and NFAT also block hFOXP3 expression. hFOXP3 induction in the reporter cells was also TGF-ß dependent, and substantially enhanced by an mTOR inhibitor, Torin1. In both the reporter transgenic mice and cell lines, histone H4 molecules in the hFOXP3 promoter and enhancers located in human CNS1 and CNS2 regions were highly acetylated in natural Treg and TCR/TGF-ß-induced Treg, indicating hFOXP3 gene expression is regulated by mechanisms similar to those previously identified for the mFoxp3 gene.

Foxp3 drives oxidative phosphorylation and protection from lipotoxicity.

Tregs can adopt a catabolic metabolic program with increased capacity for fatty acid oxidation-fueled oxidative phosphorylation (OXPHOS). It is unclear why this form of metabolism is favored in Tregs and, more specifically, whether this program represents an adaptation to the environment and developmental cues or is "hardwired" by Foxp3. Here we show, using metabolic analysis and an unbiased mass spectroscopy-based proteomics approach, that Foxp3 is both necessary and sufficient to program Treg-increased respiratory capacity and Tregs' increased ability to utilize fatty acids to fuel oxidative phosphorylation. Foxp3 drives upregulation of components of all the electron transport complexes, increasing their activity and ATP generation by oxidative phosphorylation. Increased fatty acid ß-oxidation also results in selective protection of Foxp3+ cells from fatty acid-induced cell death. This observation may provide novel targets for modulating Treg function or selection therapeutically.

Induction of Immunological Tolerance as a Therapeutic Procedure.

A major goal of immunosuppressive therapies is to harness immune tolerance mechanisms so as to minimize unwanted side effects associated with protracted immunosuppressive therapy. Antibody blockade of lymphocyte coreceptor and costimulatory pathways in mice has demonstrated the principle that both naive and primed immune systems can be reprogrammed toward immunological tolerance. Such tolerance can involve the amplification of activity of regulatory T cells, and is maintained through continuous recruitment of such cells through processes of infectious tolerance. We propose that regulatory T cells create around them microenvironments that are anti-inflammatory and endowed with enhanced protection against destructive damage. This acquired immune privilege involves the decommissioning of cells of the innate as well as adaptive immune systems. Evidence is presented that nutrient sensing by immune cells acting through the mammalian target of rapamycin (mTOR) pathway provides one route by which the immune system can be directed toward noninflammatory and regulatory behavior at the expense of destructive functions. Therapeutic control of immune cells so as to harness metabolic routes favoring dominant regulatory mechanisms has offered a new direction for immunosuppressive therapy, whereby short-term treatment may be sufficient for long-term benefit or even cure.

Induced Foxp3(+) T Cells Colonizing Tolerated Allografts Exhibit the Hypomethylation Pattern Typical of Mature Regulatory T Cells.

Regulatory T cells expressing the transcription factor Foxp3 require acquisition of a specific hypomethylation pattern to ensure optimal functional commitment, limited lineage plasticity, and long-term maintenance of tolerance. A better understanding of the molecular mechanisms involved in the generation of these epigenetic changes in vivo will contribute to the clinical exploitation of Foxp3(+) Treg. Here, we show that both in vitro and in vivo generated antigen-specific Foxp3(+) Treg can acquire Treg-specific epigenetic characteristics and prevent skin graft rejection in an animal model.

Nutrient Sensing via mTOR in T Cells Maintains a Tolerogenic Microenvironment.

We have proposed that tolerance can be maintained through the induction, by Treg cells, of a tolerogenic microenvironment within tolerated tissues that inhibits effector cell activity but which supports the generation of further Treg cells by "infectious tolerance." Two important components of this tolerogenic microenvironment depend on metabolism and nutrient sensing. The first is due to the up-regulation of multiple enzymes that consume essential amino acids, which are sensed in naïve T cells primarily via inhibition of the mechanistic target of rapamycin (mTOR) pathway, which in turn encourages their further differentiation into FOXP3(+) Treg cells. The second mechanism is the metabolism of extracellular ATP to adenosine by the ectoenzymes CD39 and CD73. These two enzymes are constitutively co-expressed on Treg cells, but can also be induced on a wide variety of cell types by TGFß and the adenosine generated can be shown to be a potent inhibitor of T cell proliferation. This review will focus on mechanisms of nutrient sensing in T cells, how these are integrated with TCR and cytokine signals via the mTOR pathway, and what impact this has on intracellular metabolism and subsequently the control of differentiation into different effector or regulatory T cell subsets.