Generation of a C57BL/6J mouse strain expressing the CD45.1 epitope to improve hematopoietic stem cell engraftment and adoptive cell transfer experiments.

Adoptive cell transfer between genetically identical hosts relies on the use of a congenic marker to distinguish the donor cells from the host cells. CD45, a glycoprotein expressed by all hematopoietic cells, is one of the main congenic markers used because its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, recent studies have identified genetic variations between these congenic strains and have notably highlighted a differential expression of cathepsin E (CTSE). The B6.SJL mouse presents a number of functional differences in hematopoietic stem cell engraftment potential and immune cell numbers compared with the B6 mouse. In this study, we showed that B6 and B6.SJL mice also differ in their CD8+ T cell compartment and CD8+ T cell responses to viral infection. We identified Ctse as the most differentially expressed gene between CD8+ T cells of B6 and B6.SJL and demonstrated that the differences reported between these two mouse strains are not due to CTSE. Finally, using CRISPR-Cas9 genome editing, we generated a CD45.1-expressing B6 mouse by inserting one nucleotide mutation (A904G) leading to an amino acid change (K302E) in the Ptprc gene of the B6 mouse. We showed that this new B6-Ptprcem(K302E)Jmar/J mouse resolves the experimental biases reported between the B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive cell transfer experiments in B6.

HPV38 impairs UV-induced transcriptional activation of the IL-18 pro-inflammatory cytokine.

IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging.

Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS-Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.

Modeling and characterization of inter-individual variability in CD8 T cell responses in mice.

To develop vaccines it is mandatory yet challenging to account for inter-individual variability during immune responses. Even in laboratory mice, T cell responses of single individuals exhibit a high heterogeneity that may come from genetic backgrounds, intra-specific processes (e.g. antigen-processing and presentation) and immunization protocols.To account for inter-individual variability in CD8 T cell responses in mice, we propose a dynamical model coupled to a statistical, nonlinear mixed effects model. Average and individual dynamics during a CD8 T cell response are characterized in different immunization contexts (vaccinia virus and tumor). On one hand, we identify biological processes that generate inter-individual variability (activation rate of naive cells, the mortality rate of effector cells, and dynamics of the immunogen). On the other hand, introducing categorical covariates to analyze two different immunization regimens, we highlight the steps of the response impacted by immunogens (priming, differentiation of naive cells, expansion of effector cells and generation of memory cells). The robustness of the model is assessed by confrontation to new experimental data.Our approach allows to investigate immune responses in various immunization contexts, when measurements are scarce or missing, and contributes to a better understanding of inter-individual variability in CD8 T cell immune responses.

Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature aging that recapitulates many normal aging characteristics. This disorder is caused by mutation in the LMNA gene leading to the production of progerin which induces misshapen nuclei, cellular senescence, and aging. We previously showed that the phospholipase A2 receptor (PLA2R1) promotes senescence induced by replicative, oxidative, and oncogenic stress but its role during progerin-induced senescence and in progeria is currently unknown. Here, we show that knockdown of PLA2R1 prevented senescence induced by progerin expression in human fibroblasts and markedly delayed senescence of HGPS patient-derived fibroblasts. Whole-body knockout of Pla2r1 in a mouse model of progeria decreased some premature aging phenotypes, such as rib fracture and decreased bone content, together with decreased senescence marker. Progerin-expressing human fibroblasts exhibited a high frequency of misshapen nuclei and increased farnesyl diphosphate synthase (FDPS) expression compared to controls; knockdown of PLA2R1 reduced the frequency of misshapen nuclei and normalized FDPS expression. Pamidronate, a FDPS inhibitor, also reduced senescence and misshapen nuclei. Downstream of PLA2R1, we found that p53 mediated the progerin-induced increase in FDPS expression and in misshapen nuclei. These results suggest that PLA2R1 mediates key premature aging phenotypes through a p53/FDPS pathway and might be a new therapeutic target.

Two-Photon Photosensitizer-Polymer Conjugates for Combined Cancer Cell Death Induction and Two-Photon Fluorescence Imaging: Structure/Photodynamic Therapy Efficiency Relationship.

One of the challenges of photodynamic therapy is to increase the penetration depth of light irradiation in the tumor tissues. Although two-photon excitation strategies have been developed, the two-photon absorption cross sections of clinically used photosensitizers are generally low (below 300 GM). Besides, photosensitizers with high cross section values are often non-water-soluble. In this research work, a whole family of photosensitizer-polymer conjugates was synthesized via the covalent binding of a photosensitizer with a relatively high cross section along a biocompatible copolymer chain. The resulting photosensitizer-polymer conjugates were water-soluble and could be imaged in cellulo by two-photon microscopy thanks to their high two-photon absorption cross sections (up to 2600 GM in water, in the NIR range). In order to explore the structure/photodynamic activity relationship of such macromolecular photosensitizers, the influence of the polymer size, photosensitizer density, and presence of charges along the polymer backbone was investigated (neutral, anionic, cationic, and zwitterionic conjugates were compared). The macromolecular photosensitizers were not cytotoxic in the absence of light irradiation. Their kinetics of cellular uptake in the B16-F10 melanoma cell line were followed by flow cytometry over 24 h. The efficiency of cell death upon photoactivation was found to be highly correlated to the cellular uptake in turn correlated to the global charge of the macromolecular photosensitizer which appeared as the determining structural parameter.

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny.

Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated.

Poly-functional and long-lasting anticancer immune response elicited by a safe attenuated Pseudomonas aeruginosa vector for antigens delivery.

Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8+ T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy.

Immune signatures of protective spleen memory CD8 T cells.

Memory CD8 T lymphocyte populations are remarkably heterogeneous and differ in their ability to protect the host. In order to identify the whole range of qualities uniquely associated with protective memory cells we compared the gene expression signatures of two qualities of memory CD8 T cells sharing the same antigenic-specificity: protective (Influenza-induced, Flu-TM) and non-protective (peptide-induced, TIM) spleen memory CD8 T cells. Although Flu-TM and TIM express classical phenotypic memory markers and are polyfunctional, only Flu-TM protects against a lethal viral challenge. Protective memory CD8 T cells express a unique set of genes involved in migration and survival that correlate with their unique capacity to rapidly migrate within the infected lung parenchyma in response to influenza infection. We also enlighten a new set of poised genes expressed by protective cells that is strongly enriched in cytokines and chemokines such as Ccl1, Ccl9 and Gm-csf. CCL1 and GM-CSF genes are also poised in human memory CD8 T cells. These immune signatures are also induced by two other pathogens (vaccinia virus and Listeria monocytogenes). The immune signatures associated with immune protection were identified on circulating cells, i.e. those that are easily accessible for immuno-monitoring and could help predict vaccines efficacy.

TGF-ß inhibits the activation and functions of NK cells by repressing the mTOR pathway.

Transforming growth factor-ß (TGF-ß) is a major immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity through its antiproliferative and anti-inflammatory properties in various immune cell types. We provide genetic, pharmacologic, and biochemical evidence that a critical target of TGF-ß signaling in mouse and human natural killer (NK) cells is the serine and threonine kinase mTOR (mammalian target of rapamycin). Treatment of mouse or human NK cells with TGF-ß in vitro blocked interleukin-15 (IL-15)-induced activation of mTOR. TGF-ß and the mTOR inhibitor rapamycin both reduced the metabolic activity and proliferation of NK cells and reduced the abundances of various NK cell receptors and the cytotoxic activity of NK cells. In vivo, constitutive TGF-ß signaling or depletion of mTOR arrested NK cell development, whereas deletion of the TGF-ß receptor subunit TGF-ßRII enhanced mTOR activity and the cytotoxic activity of the NK cells in response to IL-15. Suppression of TGF-ß signaling in NK cells did not affect either NK cell development or homeostasis; however, it enhanced the ability of NK cells to limit metastases in two different tumor models in mice. Together, these results suggest that the kinase mTOR is a crucial signaling integrator of pro- and anti-inflammatory cytokines in NK cells. Moreover, we propose that boosting the metabolic activity of antitumor lymphocytes could be an effective strategy to promote immune-mediated tumor suppression.

Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.

Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.

Human natural killer cells promote cross-presentation of tumor cell-derived antigens by dendritic cells.

Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy.

T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow.

Trail(+)DX5(-)Eomes(-) natural killer (NK) cells arise in the mouse fetal liver and persist in the adult liver. Their relationships with Trail(-)DX5(+) NK cells remain controversial. We generated a novel Eomes-GFP reporter murine model to address this question. We found that Eomes(-) NK cells are not precursors of classical Eomes(+) NK cells but rather constitute a distinct lineage of innate lymphoid cells. Eomes(-) NK cells are strictly dependent on both T-bet and IL-15, similarly to NKT cells. We observed that, in the liver, expression of T-bet in progenitors represses Eomes expression and the development of Eomes(+) NK cells. Reciprocally, the bone marrow (BM) microenvironment restricts T-bet expression in developing NK cells. Ectopic expression of T-bet forces the development of Eomes(-) NK cells, demonstrating that repression of T-bet is essential for the development of Eomes(+) NK cells. Gene profile analyses show that Eomes(-) NK cells share part of their transcriptional program with NKT cells, including genes involved in liver homing and NK cell receptors. Moreover, Eomes(-) NK cells produce a broad range of cytokines, including IL-2 and TNF in vitro and in vivo, during immune responses against vaccinia virus. Thus, mutually exclusive expression of T-bet and Eomes drives the development of different NK cell lineages with complementary functions.

Regulation of mouse NK cell development and function by cytokines.

Natural Killer (NK) cells are innate lymphocytes with an important role in the early defense against intracellular pathogens and against tumors. Like other immune cells, almost every aspects of their biology are regulated by cytokines. Interleukin (IL)-15 is pivotal for their development, homeostasis, and activation. Moreover, numerous other activating or inhibitory cytokines such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-18, IL-21, Transforming growth factor-ß (TGFß) and type I interferons regulate their activation and their effector functions at different stages of the immune response. In this review we summarize the current understanding on the effect of these different cytokines on NK cell development, homeostasis, and functions during steady-state or upon infection by different pathogens. We try to delineate the cellular sources of these cytokines, the intracellular pathways they trigger and the transcription factors they regulate. We describe the known synergies or antagonisms between different cytokines and highlight outstanding questions in this field of investigation. Finally, we discuss how a better knowledge of cytokine action on NK cells could help improve strategies to manipulate NK cells in different clinical situations.

Nanocarriers with ultrahigh chromophore loading for fluorescence bio-imaging and photodynamic therapy.

We describe the design of original nanocarriers that allows for ultrahigh chromophore loading while maintaining the photo-activity of each individual molecule. They consist in shells of charged biocompatible polymers grafted on gold nanospheres. The self-organization of extended polymer chains results from repulsive charges and steric interactions that are optimized by tuning the surface curvature of nanoparticles. This type of nano-scaffolds can be used as light-activated theranostic agents for fluorescence imaging and photodynamic therapy. We demonstrate that, labeled with a fluorescent photosensitizer, it can localize therapeutic molecules before triggering the cell death of B16-F10 melanoma with an efficiency that is similar to the efficiency of the polymer conjugate alone, and with the advantage of extremely high local loading of photosensitizers (object concentration in the picomolar range).

Contrasting Cu, Fe, and Zn isotopic patterns in organs and body fluids of mice and sheep, with emphasis on cellular fractionation.

We report Cu, Fe, and Zn natural isotope compositions in organs, body fluids, diets and feces of mice and sheep. Large and systematic isotope variability is observed, notably in the δ(66)Zn in liver and δ(65)Cu in kidneys, but significant differences exist between mice, sheep and humans, especially in the δ(66)Zn value of blood. The results are interpreted with reference to current knowledge of metal trafficking and redox conditions in cells. In general, the light isotopes preferentially fractionate into 'softer' bonds involving sulfur such as cysteine and glutathione, whereas heavy isotopes fractionate into 'harder' bonds involving nitrogen (histidine) and even more oxygen, notably hydroxides, phosphates, and carbonates. Bonds involving the reduced forms Cu(+) and Fe(2+) are enriched in the light isotopes relative to bonds involving the oxidized Cu(2+) and Fe(3+) forms. Differences in blood Zn isotope abundances between mice, sheep and humans may reflect a different prevalence of Zn ZIP transporters. The isotopically heavy Cu in the kidneys may reflect isotope fractionation during redox processes and may be relevant to ascorbate degradation into oxalate.

TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.

Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

Tumor promotion by intratumoral plasmacytoid dendritic cells is reversed by TLR7 ligand treatment.

Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.

S1PR5 is pivotal for the homeostasis of patrolling monocytes.

Patrolling Ly6C(-) monocytes are blood-circulating cells that play a role in inflammation and in the defense against pathogens. Here, we show that similar to natural killer (NK) cells, patrolling monocytes express high levels of S1PR5, a G-coupled receptor for sphingosine-1 phosphate. We found that S1pr5(-/-) mice lack peripheral Ly6C(-) monocytes but have a normal number of these cells in the bone marrow (BM). Various lines of evidence exclude a direct contribution of S1PR5 in the survival of Ly6C(-) monocytes at the periphery. Rather, our data support a role for S1PR5 in the egress of Ly6C(-) monocytes from the BM. In particular, we observed a reduced frequency of patrolling monocytes in BM sinusoids of S1PR5 KO mice. Unexpectedly, S1P was not a chemoattractant for patrolling monocytes and had no significant effect on their viability in vitro. Moreover, the disruption of S1P gradients in vivo did not alter Ly6C(-) monocyte trafficking and viability. These data suggest that S1PR5 regulates the trafficking of monocytes via a mechanism independent of S1P gradients.