Browsing by Subject "T cells"

LAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-γ1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LATm/m) mice, have drastically impaired thymocyte development; however, CD4+ T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LATm/m mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre+LATf/m mice). LATf/m mice develop a similar lymphoproliferative syndrome as LATm/m mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity.

Analysis of LATm/mRasGRP1-/- mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LATf/mRasGRP1-/- mice and observed that they developed disease similarly to LATf/m mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LATm/mGads-/- mice had an even more dramatic block in the DN stage of thymocyte development compared to LATm/m controls, although by four months of age CD4+ T cells had expanded. Following deletion of the wildtype LAT allele, LATf/mGads-/- mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads.

In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1d/d mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4+ T cells expanded and auto-antibodies were produced. RasGRP1d/d thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.

Total body irradiation (TBI) causes profound suppression of hematopoiesis and T cell depletion, increasing chances of morbidity associated with opportunistic infections in the lymphopenic condition. Currently, therapeutic options for improving recovery of the T cell compartment following radiation exposure are not available. Although mouse and nonhuman primate studies have demonstrated prolonged effects of TBI on T cell reconstitution, there is a lack of understanding in the kinetics and metabolic signatures of radioresistant T cells actively undergoing homeostatic proliferation. Furthermore, whether kinetics of systemic T cell recovery recapitulates T cell recovery in circulation remains unknown. In the current study, we performed comprehensive immunophenotyping and single-cell sequencing analyses of radioresistant T cells, as well as imaging of T cell recovery in vivo, to determine preferentially upregulated pathways during T cell recovery. We identified T cell populations unique to TBI treatment that upregulate components essential to support oxidative phosphorylation, a mitochondria-dependent metabolic process. We further investigated mechanisms of recovery in donor T cells following TBI exposure in the bone marrow transplant setting. We demonstrated that recovery of alloreactive donor T cells was highly dependent on aerobic glycolysis, which can be manipulated to reduce graft-versus-host-disease and preserve the functional recovery of non-alloreactive donor T cells. We then examined the effect of NT-I7, a long-acting recombinant human IL-7, in mediating T cell reconstitution due to its role in integrating metabolic requirements with pathways critical for T cell survival and growth. We found that NT-I7 led to accelerated T cell recovery following TBI through both thymic-dependent and independent pathways. More importantly, NT-I7 promoted functional T cell recovery. Taken together, these findings reveal unique kinetics and mechanisms of T cell recovery in response to radiation. The study also identified NT-I7 as a potential therapeutic treatment during T cell lymphopenia by supporting critical mechanisms utilized in T cell recovery.

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Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.

It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs.

In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.

Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.

Lymphocytes take on effector programs coordinated by lineage-defining transcription factors (LDTF), resulting in the production of cytokines that fight specific types of pathogens. Therefore, both adaptive and innate lymphocyte lineages can take on specialized effector programs; the type 1 program mediated by T-bet for killing intracellular pathogens and tumors, the type 2 program controlled by GATA3 for protection against helminths, and the type 3 program mediated by RORγt for fighting extracellular bacteria and fungi. While each program can be defined by a single LDTF, many context-dependent situations arise that lead to more than one LDTF being expressed in a cell at a given time. The dual expression of LDTFs can result in the switching of effector programs within a differentiated cell. Nevertheless, LDTFs work in a cooperative manner with signal-dependent TFs and other TFs that sense environmental cues to ultimately control effector fates.

Environmental signals can be sensed by various classes of cell-surface receptors that modulate the downstream signaling effectors and subsequent transcriptional output of a cell for differentiation, proliferation, maintenance, and effector function. Surface receptors, such as the T cell receptor (TCR), cytokine receptors, and costimulatory receptors, translate the environmental cues into downstream signaling cascades that act in concert to promote the differentiation of lymphocyte subsets. Cytokines fine-tune the activation and repression of lymphocytes through phosphorylation of signal transducer and activator of transcription (STAT) TFs that translocate into the nucleus, bind DNA, and regulate gene expression at key loci. Acting alongside STAT TFs, AP-1 TFs are basic leucine zipper (bZIP) TFs that help translate environmental cues into effector programming through binding to key TF and effector cytokine loci.

The ability of a differentiated cell to switch to an alternative fate is referred to as plasticity. Innate lymphoid cells (ILCs) are remarkably plastic at steady state and fate-mapping studies in the mouse intestine revealed that RORγt+ ILCs (ILC3s) can upregulate T-bet and shut down RORγt expression for full conversion to a type 1 ILC (ILC1). ILC3s help maintain healthy mucosal barriers through the production of IL-22 that promotes the release of antimicrobial peptides from epithelial cells. ILC3 to ILC1 plasticity therefore results in a shift from IL-22 to IFNγ production. While increased IFNγ production can be protective against viruses and intracellular pathogens, it can result in many autoimmune and inflammatory diseases when dysregulated. Notably, ILC3 plasticity is implicated in Crohn's disease.

Although the environmental cues regulating ILC3 plasticity were somewhat known, the molecular mechanisms governing ILC3 plasticity were undefined. Here, we identified the AP-1 TF c-Maf as an essential regulator of ILC3 homeostasis and plasticity that limits physiological ILC1 conversion. Phenotypic analysis of effector status in Maf-deficient CCR6- ILC3s using flow cytometry revealed a skewing towards T-bet and IFNγ production. To determine the molecular mechanisms by which c-Maf supported the type 3 program, we evaluated the global changes in transcriptome (RNA-seq), chromatin accessibility (ATAC-seq), and transcription factor motif enrichment. We found that c-Maf promoted ILC3 accessibility and supported RORγt activity and expression of type 3 effector genes. Conversely, c-Maf restrained T-bet expression and function, thereby antagonizing the type 1 program. We performed ATAC-seq on transitioning subsets in the CCR6- ILC3 compartment all the way through conversion to ILC1s to understand the chromatin landscape changes taking place during ILC3 plasticity. These results solidified c-Maf as a gatekeeper of type 1 regulatory transformation and a controller of ILC3 fate.