Local and systemic immunomodulatory mechanisms triggered by Human Papillomavirus transformed cells: a potential role for G-CSF and neutrophils

Cervical cancer is the last stage of a series of molecular and cellular alterations initiated with Human Papillomavirus (HPV) infection. The process involves immune responses and evasion mechanisms, which culminates with tolerance toward tumor antigens. Our objective was to understand local and systemic changes in the interactions between HPV associated cervical lesions and the immune system as lesions progress to cancer. Locally, we observed higher cervical leukocyte infiltrate, reflected by the increase in the frequency of T lymphocytes, neutrophils and M2 macrophages, in cancer patients. We observed a strong negative correlation between the frequency of neutrophils and T cells in precursor and cancer samples, but not cervicitis. In 3D tumor cell cultures, neutrophils inhibited T cell activity, displayed longer viability and longer CD16 expression half-life than neat neutrophil cultures. Systemically, we observed higher plasma G-CSF concentration, higher frequency of immature low density neutrophils, and tolerogenic monocyte derived dendritic cells, MoDCs, also in cancer patients. Interestingly, there was a negative correlation between T cell activation by MoDCs and G-CSF concentration in the plasma. Our results indicate that neutrophils and G-CSF may be part of the immune escape mechanisms triggered by cervical cancer cells, locally and systemically, respectively.Tis study was supported by Sao Paulo Research foundation: grants 2008/57889-1, 2010/20010-4, 2014/19326-6, by the Brazilian National Counsel of Technological and Scientifc Development: grant 573799/2008-3. KLFA and RAMR had PhD fellowships by Sao Paulo Research Foundation, CRSF has a Coordination for the Improvement of Higher Education Personnel PhD fellowship. We thank the Pathology Department of the School of Medicine, coordinated by Prof. Venâncio Avancini Ferreira Alves, Universidade de São Paulo for the slides containing histological samples from the biopsies used in this study. We thank Sandra Alexandre Alves for her technical support.info:eu-repo/semantics/publishedVersio

The PI3K p110δ regulates expression of CD38 on regulatory T cells.

The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and is required for full Treg-mediated suppression. To identify new genes involved in PI3K-dependent suppression, we compared the transcriptome of WT and p110δ(D910A) Tregs. Among the genes that were differentially expressed was the gene for the transmembrane cyclic ADP ribose hydrolase CD38. Here we show that CD38 is expressed mainly by a subset of Foxp3(+)CD25(+)CD4(+) T cells originating in the thymus and on Tregs in the spleen. CD38(high) WT Tregs showed superior suppressive activity to CD38(low) Tregs, which failed to upregulate CD73, a surface protein which is important for suppression. However, Tregs from heterozygous CD38(+/-) mice were unimpaired despite lower levels of CD38 expression. Therefore, CD38 can be used as a marker for Tregs with high suppressive activity and the impaired Treg function in p110δ(D910A) mice can in part be explained by the failure of CD38(high) cells to develop

Global identification of genes and pathways regulated by Akt during activation of T helper cells

We previously demonstrated that Akt differentially modulated a subset of NF-kB target genes during T cell activation. In the current study, we further explored the broader effects of Akt inhibition on T cell gene induction. Global microarray analysis was used to characterize T helper cell transcriptional responses following antigen receptor stimulation in the absence or presence of Akti1/2 (an allosteric inhibitor which targets Akt1 and Akt2), to identify novel targets dependent upon Akt and obtain a more comprehensive view of Akt-sensitive genes in Th2 helper T cells. Pathway analysis of microarray data from a CD4+ Th2 T cell line revealed effects on gene networks involving ribosomal and T cell receptor signaling pathways associated with Akti1/2 treatment. Using real-time PCR analysis, we validated the differential regulation of several genes in these pathways, including Ier3, Il13, Egr1, Ccl1 and Ccl4, among others. Additionally, transcription factor target gene (TFactS) analysis revealed that NF-kB and Myc were the most significantly enriched transcription factors among Akt-dependent genes after T cell receptor and CD28 stimulation. Akt activation elicited increases in the enrichment of NF-kB- and Myc-targeted genes. The present study has identified a diverse set of genes, and possible mechanisms for their regulation, that are dependent on Akt during T cell activation

Novel Foxo1-dependent transcriptional programs control Treg cell function

Regulatory T (T(reg)) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses. Foxp3 operates as a late-acting differentiation factor controlling T(reg) cell homeostasis and function, whereas the early T(reg)-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors. However, whether Foxo proteins act beyond the T(reg)-cell-commitment stage to control T(reg) cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulator of T(reg )cell function. T(reg) cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with T(reg)-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of T(reg) cells. Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt-Foxo1 signalling module controls a novel genetic program indispensable for T(reg) cell function

Essential thrombocythemia

Essential thrombocythemia (ET) is an acquired myeloproliferative disorder (MPD) characterized by a sustained elevation of platelet number with a tendency for thrombosis and hemorrhage. The prevalence in the general population is approximately 30/100,000. The median age at diagnosis is 65 to 70 years, but the disease may occur at any age. The female to male ratio is about 2:1. The clinical picture is dominated by a predisposition to vascular occlusive events (involving the cerebrovascular, coronary and peripheral circulation) and hemorrhages. Some patients with ET are asymptomatic, others may experience vasomotor (headaches, visual disturbances, lightheadedness, atypical chest pain, distal paresthesias, erythromelalgia), thrombotic, or hemorrhagic disturbances. Arterial and venous thromboses, as well as platelet-mediated transient occlusions of the microcirculation and bleeding, represent the main risks for ET patients. Thromboses of large arteries represent a major cause of mortality associated with ET or can induce severe neurological, cardiac or peripheral artery manifestations. Acute leukemia or myelodysplasia represent only rare and frequently later-onset events. The molecular pathogenesis of ET, which leads to the overproduction of mature blood cells, is similar to that found in other clonal MPDs such as chronic myeloid leukemia, polycythemia vera and myelofibrosis with myeloid metaplasia of the spleen. Polycythemia vera, myelofibrosis with myeloid metaplasia of the spleen and ET are generally associated under the common denomination of Philadelphia (Ph)-negative MPDs. Despite the recent identification of the JAK2 V617F mutation in a subset of patients with Ph-negative MPDs, the detailed pathogenetic mechanism is still a matter of discussion. Therapeutic interventions in ET are limited to decisions concerning the introduction of anti-aggregation therapy and/or starting platelet cytoreduction. The therapeutic value of hydroxycarbamide and aspirin in high risk patients has been supported by controlled studies. Avoiding thromboreduction or opting for anagrelide to postpone the long-term side effects of hydrocarbamide in young or low risk patients represent alternative options. Life expectancy is almost normal and similar to that of a healthy population matched by age and sex

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics

Molecular definition of group 1 innate lymphoid cells in the mouse uterus

Determining the function of uterine lymphocytes is challenging because of the rapidly changing nature of the organ in response to sex hormones and, during pregnancy, to the invading fetal trophoblast cells. Here we provide the first genome-wide transcriptome atlas of mouse uterine group 1 innate lymphoid cells (g1 ILCs) at mid-gestation. The composition of g1 ILCs fluctuates throughout reproductive life, with Eomes-veCD49a+ ILC1s dominating before puberty and specifically expanding in second pregnancies, when the expression of CXCR6, a marker of memory cells, is upregulated. Tissue-resident Eomes+CD49a+ NK cells (trNK), which resemble human uterine NK cells, are most abundant during early pregnancy, and showcase gene signatures of responsiveness to TGF-β, connections with trophoblast, epithelial, endothelial and smooth muscle cells, leucocytes, as well as extracellular matrix. Unexpectedly, trNK cells express genes involved in anaerobic glycolysis, lipid metabolism, iron transport, protein ubiquitination, and recognition of microbial molecular patterns. Conventional NK cells expand late in gestation and may engage in crosstalk with trNK cells involving IL-18 and IFN-γ. These results identify trNK cells as the cellular hub of uterine g1 ILCs at mid-gestation and mark CXCR6+ ILC1s as potential memory cells of pregnancy.This work was funded by a Wellcome Trust Investigator Award 200841/Z/16/Z, the Centre for Trophoblast Research (CTR), and the Cambridge NIHR BRC Cell Phenotyping Hub to FC, the Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC) - Special Project 5x1000 no. 9962, AIRC IG 2017 Id.19920 and AIRC 2014 Id. 15283 to LM, and Ministero della Salute RF-2013, GR-2013-02356568 to PV. IF was funded by a CTR PhD fellowship

The Molecular Signature Underlying the Thymic Migration and Maturation of TCRαβ+CD4+CD8- Thymocytes

BACKGROUND: After positive selection, the newly generated single positive (SP) thymocytes migrate to the thymic medulla, where they undergo negative selection to eliminate autoreactive T cells and functional maturation to acquire immune competence and egress capability. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the genetic program underlying this process, we analyzed changes in gene expression in four subsets of mouse TCRαβ(+)CD4(+)CD8(-) thymocytes (SP1 to SP4) representative of sequential stages in a previously defined differentiation program. A genetic signature of the migration of thymocytes was thus revealed. CCR7 and PlexinD1 are believed to be important for the medullary positioning of SP thymocytes. Intriguingly, their expression remains at low levels in the newly generated thymocytes, suggesting that the cortex-medulla migration may not occur until the SP2 stage. SP2 and SP3 cells gradually up-regulate transcripts involved in T cell functions and the Foxo1-KLF2-S1P(1) axis, but a number of immune function-associated genes are not highly expressed until cells reach the SP4 stage. Consistent with their critical role in thymic emigration, the expression of S1P(1) and CD62L are much enhanced in SP4 cells. CONCLUSIONS: These results support at the molecular level that single positive thymocytes undergo a differentiation program and further demonstrate that SP4 is the stage at which thymocytes acquire the immunocompetence and the capability of emigration from the thymus