DNA Polymerase Theta Plays a Critical Role in Pancreatic Cancer Development and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.

Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model.

The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.

The Role of Palladin in Podocytes.

Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.

The Angiotensin-(1-7)/Mas Axis Improves Pancreatic ß-Cell Function in Vitro and in Vivo.

The angiotensin-converting enzyme 2/angiotensin (Ang)-(1-7)/Mas axis of the renin-angiotensin system often opposes the detrimental effects of the angiotensin-converting enzyme/Ang II/Ang II type 1 receptor axis and has been associated with beneficial effects on glucose homeostasis, whereas underlying mechanisms are mostly unknown. Here we investigate the effects of Ang-(1-7) and its receptor Mas on ß-cell function. Isolated islets from Mas-deficient and wild-type mice were stimulated with Ang-(1-7) or its antagonists and effects on insulin secretion determined. Islets' cytoplasmic calcium and cAMP concentrations, mRNA amounts of Ins1, Ins2, Pdx1, and Mafa and effects of inhibitors of cAMP downstream signaling were determined. Ang-(1-7) was also applied to mice by osmotic pumps for 14 days and effects on glucose tolerance and insulin secretion were assessed. Ang-(1-7) increased insulin secretion from wild-type islets, whereas antagonists and genetic Mas deficiency led to reduced insulin secretion. The Mas-dependent effects of Ang-(1-7) on insulin secretion did not result from changes in insulin gene expression or changes in the excitation-secretion coupling but from increased intracellular cAMP involving exchange protein activated directly by cAMP. Administration of Ang-(1-7) in vivo had only marginal effects on glucose tolerance in wild-type mice but still resulted in improved insulin secretion from islets isolated of these mice. Interestingly, although less pronounced than in wild types, Ang-(1-7) still affected insulin secretion in Mas-deficient islets. The data indicate a significant function of Ang-(1-7) in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signaling, modulating the accessory pathway of insulin secretion via increase in cAMP.

In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.

The hedgehog receptor patched1 in T cells is dispensable for adaptive immunity in mice.

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and Treg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo.

Healthy bone marrow cells reduce progression of kidney failure better than CKD bone marrow cells in rats with established chronic kidney disease.

Chronic kidney disease (CKD) is a major health care problem. New interventions to slow or prevent disease progression are urgently needed. We studied functional and structural effects of infusion of healthy and CKD bone marrow cells (BMCs) in a rat model of established CKD. CKD was induced by 5/6 nephrectomy (SNX) in Lewis rats, and disease progression was accelerated with L-NNA and 6% NaCl diet. Six weeks after SNX, CKD rats received healthy eGFP(+) BMCs, CKD eGFP(+) BMCs, or vehicle by single renal artery injection. Healthy BMCs were functionally effective 6 weeks after administration: glomerular filtration rate (GFR; inulin clearance) (0.48±0.16 vs. 0.26±0.14 ml/min/100 g) and effective renal plasma flow (RPF; PAH clearance) (1.6±0.40 vs. 1.0±0.62 ml/min/100 g) were higher in healthy BMC- versus vehicle-treated rats (both p < 0.05). Systolic blood pressure (SBP) and proteinuria were lower 5 weeks after treatment with healthy BMCs versus vehicle (SBP, 151±13 vs. 186±25 mmHg; proteinuria, 33±20 vs. 59±39 mg/day, both p < 0.05). Glomerular capillary density was increased, and less sclerosis was detected after healthy BMCs (both p < 0.05). Tubulointerstitial inflammation was also decreased after healthy BMCs. eGFP(+) cells were present in the glomeruli and peritubular capillaries of the remnant kidney in all BMC-treated rats. CKD BMCs also reduced SBP, proteinuria, glomerulosclerosis, and tubular atrophy versus vehicle in CKD rats. However, CKD BMC therapy was not functionally effective versus vehicle [GFR: 0.28±0.09 vs. 0.26±0.16 ml/min/100 g (NS), RPF: 1.15±0.36 vs. 0.78±0.44 ml/min/100 g (NS)], and failed to decrease tubulointerstitial inflammation and fibrosis. Single intrarenal injection of healthy BMCs in rats with established CKD slowed progression of the disease, associated with increased glomerular capillary density and less sclerosis, whereas injection of CKD BMCs was less effective.

Liposomal encapsulation of glucocorticoids alters their mode of action in the treatment of experimental autoimmune encephalomyelitis.

Glucocorticoids (GCs) are widely used to treat acute relapses of multiple sclerosis (MS). In this study, we demonstrate that liposomal encapsulation augments the therapeutic potency of GCs as they ameliorate experimental autoimmune encephalomyelitis (EAE) to the same extent as free GC, but at strongly reduced dosage and application frequency. Importantly, this is accompanied by an altered mode of action. Unlike free GCs, which mainly target T lymphocytes during EAE therapy, liposomal GCs only marginally affect T cell apoptosis and function. In contrast, liposomal GCs efficiently repress proinflammatory macrophage functions and upregulate anti-inflammatory genes associated with the alternatively activated M2 phenotype. The GC receptor (GR) per se is indispensable for the therapeutic efficacy of liposomal GC. In contrast to free GCs, however, the individual deletion of the GR either in T cells or myeloid cells has little effect on the efficacy of liposomal GCs in the treatment of EAE. Only the combined deletion of the GR in both cellular compartments markedly compromises the therapeutic effect of liposomal GCs on disease progression. In conclusion, encapsulation of GC does not only enhance their efficacy in the treatment of EAE but also alters their target cell specificity and their mode of action compared with free GCs.

Therapeutic efficacy of intranasally delivered mesenchymal stem cells in a rat model of Parkinson disease.

Safe and effective cell delivery remains one of the main challenges in cell-based therapy of neurodegenerative disorders. Graft survival, sufficient enrichment of therapeutic cells in the brain, and avoidance of their distribution throughout the peripheral organs are greatly influenced by the method of delivery. Here we demonstrate for the first time noninvasive intranasal (IN) delivery of mesenchymal stem cells (MSCs) to the brains of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. IN application (INA) of MSCs resulted in the appearance of cells in the olfactory bulb, cortex, hippocampus, striatum, cerebellum, brainstem, and spinal cord. Out of 1 × 106 MSCs applied intranasally, 24% survived for at least 4.5 months in the brains of 6-OHDA rats as assessed by quantification of enhanced green fluorescent protein (EGFP) DNA. Quantification of proliferating cell nuclear antigen-positive EGFP-MSCs showed that 3% of applied MSCs were proliferative 4.5 months after application. INA of MSCs increased the tyrosine hydroxylase level in the lesioned ipsilateral striatum and substantia nigra, and completely eliminated the 6-OHDA-induced increase in terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine, 5'-triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining of these areas. INA of EGFP-labeled MSCs prevented any decrease in the dopamine level in the lesioned hemisphere, whereas the lesioned side of the control animals revealed significantly lower levels of dopamine 4.5 months after 6-OHDA treatment. Behavioral analyses revealed significant and substantial improvement of motor function of the Parkinsonian forepaw to up to 68% of the normal value 40-110 days after INA of 1 × 106 cells. MSC-INA decreased the concentrations of inflammatory cytokines-interleukin-1ß (IL-1ß), IL-2, -6, -12, tumor necrosis factor (TNF), interferon-γ (IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF)-in the lesioned side to their levels in the intact hemisphere. IN administration provides a highly promising noninvasive alternative to the traumatic surgical procedure of transplantation and allows targeted delivery of cells to the brain with the option of chronic application.

T cell development critically depends on prethymic stromal patched expression.

We recently described that T cell specification in mice deficient in the Hedgehog (Hh) receptor Patched (Ptch) is blocked at the level of the common lymphoid progenitor in the bone marrow (BM). Adoptive transfer of wild-type BM in Ptch-deficient mice provides evidence that T cell development strictly depends on Ptch expression in the nonhematopoietic compartment. Transplantation experiments using BM deficient in the glucocorticoid receptor exclude any involvement of the stress hormone corticosterone in our model. Using cell-type-specific knockout mice, we show that T cell development is independent of T cell-intrinsic Ptch expression. Furthermore, Ptch expression by the thymus stroma is dispensable, as revealed by fetal thymus organ culture and thymus transplantation. In contrast, analysis of the earliest thymic progenitors in Ptch-deficient mice indicated that Ptch is required for the development or supply of thymic homing progenitors that give rise to earliest thymic progenitors. Collectively, our findings identified Ptch as an exclusive T cell-extrinsic factor necessary for proper development of T cells at their prethymic stage. This observation may be important for current considerations using Hh inhibitors upstream of Ptch in diseases accompanied by aberrant Hh signaling.

Impact of female sex hormones on the maturation and function of human dendritic cells.

PROBLEM: During pregnancy, the immune and the endocrine system cooperate to ensure that the fetal allograft develops without eliciting a maternal immune response. This is presumably in part achieved by dendritic cells (DCs) that play a dominant role in maintaining peripheral tolerance. In this study, we investigated whether female sex hormones, such as human chorionic gonadotropin (hCG), progesterone (Prog), and estradiol (E2), which are highly elevated during pregnancy, induce the differentiation of DCs into a tolerance-inducing phenotype. METHODS/RESULTS: Immature DCs were generated from blood-derived monocytes and differentiated in the presence of hCG, Prog, E2, or Dexamethasone (Dex) as a control. Unlike Dex, female sex hormones did not prevent the upregulation of surface markers characteristic for mature DCs, such as CD40, CD83, and CD86, except for hCG, which inhibited HLA-DR expression. Similarly, hCG, Prog, and E2 had any impact on neither the rearrangement of the F-actin cytoskeleton nor the enhanced chemokine secretion following DC maturation, both of which were strongly altered by Dex. Nevertheless, the T-cell stimulatory capacity of DCs was significantly reduced after hCG and E2 exposure. CONCLUSION: Our findings suggest that the female sex hormones hCG and E2 inhibit the T-cell stimulatory capacity of DCs, which may help in preventing an allogenic T-cell response against the embryo.

Stable silencing of the glucocorticoid receptor in myelin-specific T effector cells by retroviral delivery of shRNA: insight into neuroinflammatory disease.

Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.

The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene.

The muscle specific ubiquitin E3 ligase MuRF1 has been implicated as a key regulator of muscle atrophy under a variety of conditions, such as during synthetic glucocorticoid treatment. FOXO class transcription factors have been proposed as important regulators of MuRF1 expression, but its regulation by glucocorticoids is not well understood. The MuRF1 promoter contains a near-perfect palindromic glucocorticoid response element (GRE) 200 base pairs upstream of the transcription start site. The GRE is highly conserved in the mouse, rat, and human genes along with a directly adjacent FOXO binding element (FBE). Transient transfection assays in HepG2 cells and C(2)C(12) myotubes demonstrate that the MuRF1 promoter is responsive to both the dexamethasone (DEX)-activated glucocorticoid receptor (GR) and FOXO1, whereas coexpression of GR and FOXO1 leads to a dramatic synergistic increase in reporter gene activity. Mutation of either the GRE or the FBE significantly impairs activation of the MuRF1 promoter. Consistent with these findings, DEX-induced upregulation of MuRF1 is significantly attenuated in mice expressing a homodimerization-deficient GR despite no effect on the degree of muscle loss in these mice vs. their wild-type counterparts. Finally, chromatin immunoprecipitation analysis reveals that both GR and FOXO1 bind to the endogenous MuRF1 promoter in C(2)C(12) myotubes, and IGF-I inhibition of DEX-induced MuRF1 expression correlates with the loss of FOXO1 binding. These findings present new insights into the role of the GR and FOXO family of transcription factors in the transcriptional regulation of the MuRF1 gene, a direct target of the GR in skeletal muscle.

Peripheral T cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis.

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral T(H)17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.

Unexpected features of acute T lymphoblastic lymphomas in Notch1IC transgenic rats.

Dysregulated Notch signaling accounts for the majority of acute T lymphoblastic leukemia/lymphoma (T-ALL) cases in humans. Here, we characterize lymphomas from Notch1IC transgenic rats, which develop T-ALL shortly after weaning, and show that they display a number of previously undocumented features. Starting from monoclonal thymic tumors, the CD4(+)CD8alphaalpha(+) lymphoma cells infiltrate the bone marrow and then spread to secondary lymphoid and non-lymphoid organs. However, major hallmarks of T-ALL cells in other murine models and human patients, such as constitutive NF-kappaB activity and increased levels of anti-apoptotic proteins, are remarkably absent in Notch1IC lymphomas. In contrast, CD30, a classic marker of Hodgkin lymphomas, is overexpressed in these tumors. Intriguingly, enforced Notch1 signaling up-regulates expression of Notch3, which has also been implicated in the pathogenesis of T-ALL. By blocking endogenous Notch signaling, we could demonstrate that Notch1IC is sufficient to induce sustained preTCR expression in transgenic thymocytes but not for their progression to the double-positive stage. This suggests that other Notch activities may also contribute to the phenotype of the transgenic rats. In summary, we anticipate this new animal model will help to further elucidate the role of Notch1 in the pathogenesis of T-ALL.

Tolerance induction by bone marrow transplantation in a multiple sclerosis model.

Experimental autoimmune encephalomyelitis (EAE) in rats is a highly valuable model of multiple sclerosis (MS) because it mimics major hallmarks of the human disease. EAE induced with myelin-oligodendrocyte-glycoprotein (MOG) in DA rats is relapsing/remitting, and lesions in the central nervous system show inflammation, demyelination, and axonal and neuronal loss. Recently, bone marrow transplantation (BMT) was introduced as a novel strategy to treat MS, but its efficiency and the underlying mechanism are debatable. In MOG-induced EAE we found that BMT at the peak of EAE but not in the chronic phase leads to disease attenuation. In both settings, rats receiving bone marrow (BM) transplants were protected from subsequently induced relapses. These findings could be confirmed by histopathology in which rats receiving BM transplants did not have lesions compared with controls not receiving transplants. Importantly, the protective effect was achieved by allogeneic, syngeneic, and BM grafts from diseased rats. BMT resulted in increased numbers of CD4(+)CD25(bright) regulatory T cells, increased Foxp3 expression, a shift in T-cell epitope recognition, and a strong reduction of autoantibodies even after rechallenge with MOG. Thus, our results indicate potential mechanisms of how BMT may contribute to the improvement of MS and provide a rationale for its application in patients suffering from various autoimmune diseases.

Resistance of single-positive thymocytes to glucocorticoid-induced apoptosis is mediated by CD28 signaling.

Glucocorticoids administered in pharmacological doses potently induce apoptosis in immature double-positive thymocytes. In contrast, single-positive thymocytes are completely resistant. We now provide evidence that this difference can be attributed to CD28 signaling. When taken into culture, single-positive thymocytes also become sensitive to glucocorticoid-induced apoptosis, which can be prevented by enforced CD28 engagement using a novel type of antibody. This is achieved, at least in part, by transcriptional regulation of apoptosis-related genes such as Bcl-X(L) via a calcium- and phosphatidylinositol 3 kinase-dependent pathway. Accordingly, deficiency of CD28 in genetically engineered mice leads to an increased sensitivity of single-positive thymocytes toward glucocorticoid-induced cell death in vivo. Taken together, we have identified CD28 signaling in the thymus as a key player in determining the differential sensitivity of double-positive and single-positive cells to glucocorticoid action.