Mouse embryo assay to evaluate polydimethylsiloxane (PDMS) embryo-toxicity.

In vitro embryo culture to support In Vitro Fertilization (IVF) procedures is a well-established but still critical technique. In the last decade first attempts to use microfluidic devices in IVF have shown positive results, enabling to control the culture conditions and to preserve the quality of the embryos during their development. In this study we completed an industry standard mouse embryo assay (MEA) to exclude potential toxic effects of PDMS.

How does variability of immune system genes affect placentation?

Formation of the placenta is a crucial step in mammalian pregnancy. Apart from its function in ensuring an optimal supply of nutrients and oxygen to the fetus, the placenta is also the interface at which allo-recognition of invading trophoblast cells by the maternal immune system can potentially occur. We summarise here the "state of the art" on how variability of immune system genes that code for major histocompatibility complex (MHC) molecules and natural killer receptors (NKR) may impact on human placentation. MHC and NKR are the most polymorphic human genes. Our recent reports point out that specific combinations of fetal MHC and maternal NKR genes in humans correlate with the risk of pre-eclampsia, recurrent miscarriage (RM) and fetal growth restriction (FGR). Research in this field is still at an early stage and future studies in mouse and humans will be needed before the results can be translated to clinical applications. We discuss our recent work, as well as the opportunities offered by mouse genetics, to understand the cellular and molecular mechanisms underlying immune interactions at the maternal-fetal interface.

p110delta is required for innate immunity to transplantable lymphomas.

NK cell (natural killer cells) are lymphocytes of innate immunity that kill tumour cells and respond to infections, without prior stimulation. A balance of activating and inhibitory signals regulates NK cell cytotoxicity, but the molecular mechanisms are not fully understood. General inhibitors of PI3K (phosphoinositide 3-kinase) suppress cytotoxicity in human and mouse NK cells. However, which isoforms and how they regulate NK cell activation is unknown, and no data have been published on mice carrying PI3K mutations. p110delta expression is restricted to leucocytes, where it plays central roles in lymphocyte development and signalling. We have used mice carrying a catalytically inactive mutant form of p110delta in order to determine its role in NK cell biology. We show here that p110delta is not required to kill tumour cells, but unexpectedly p110delta mutant mice failed to fully reject transplanted lymphomas. Our results show for the first time a critical role for p110delta in NK cell biology in vivo.

Anatomy of a murder--signal transduction pathways leading to activation of natural killer cells.

Natural killer (NK) cells control the early phases of viral infections, modulate antigen-specific immune responses, and participate in the rejection of tumours and bone marrow grafts. A fine balance between inhibitory and activating receptors tightly regulates NK cell activation. Biochemical studies in human cell lines and primary cells have revealed some of the activating NK cell signalling pathways, however animal models are instrumental to understand the physiological implications of these findings for immune responses in vivo. Gene targeting in mice and biochemical studies in cells are helping to dissect out the various signal transduction pathways that control NK cell activation. A clearer view of these pathways may eventually help designing more effective immune therapies based on the use of NK cells.

Identification of committed NK cell progenitors in adult murine bone marrow.

Natural killer (NK) cells play important roles in innate immunity by lysing tumor and virally infected cells and by producing cytokines including interferon-gamma. While NK cell progenitors have been described in the fetal thymus, NK cell generation from hematopoietic stem cells (HSC) in the bone marrow (BM) occurs throughout life, and in athymic mice and humans. Interleukin (IL)-15 promotes NK development in vitro and is essential for the generation of normal numbers of NK cells in vivo. By characterizing BM cells expressing IL-15 receptor components, we found marked heterogeneity within the IL-2 receptor beta chain(+) (CD122(+)) subset, which included cells uniquely committed to the NK lineage. These CD122(+) NK cell precursors (NKP) are negative for markers used to identify mature NK cells, including NK1.1, DX5 and members of Ly-49 family, and fail to demonstrate natural cytotoxicity against susceptible target cells. In vitro culture of NKP generates mature lytic NK1.1(+) cells at high frequencies, while they do not give rise to T, B, myeloid or erythroid cells under appropriate conditions. NKP lack transcripts associated with early B and T cell differentiation (pTalpha, lambda5 and CD3epsilon), but express a group of genes (IL-15Ralpha, Id2, GATA-3 and Ets-1) and the 2B4 marker, which may define NK cell commitment. We propose that NKP represent the earliest adult BM precursor uniquely restricted to the NK cell lineage.

Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells.

PU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not known. The study shows that PU.1 is expressed in NK cells and that, on cell transfer into alymphoid Rag2/gammac(-/-) mice, hematopoietic progenitors of PU.1(-/-) fetal liver cells could generate functional NK cells but not B or T cells. Nevertheless, the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls. Moreover, PU.1(-/-) NK cells displayed reduced expression of the receptors for stem cell factor and interleukin (IL)-7, suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development. PU.1(-/-) NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12. Thus, despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells, PU.1 regulates NK cell differentiation and homeostasis.

CTLA-4-/- mice display T cell-apoptosis resistance resembling that ascribed to autoimmune-prone non-obese diabetic (NOD) mice.

The genes conferring susceptibility to autoimmune (insulin-dependent) diabetes mellitus (IDDM) are, in most cases, not defined. Among the loci so far identified as associated with murine IDDM (Idd1-19), only the nature of Idd1 has been assessed. Here we show that thymocytes and peripheral lymphocytes of the non-obese diabetic (NOD) mouse are relatively resistant to apoptosis induced by gamma-irradiation. By linkage analysis of F2 progeny mice, we map this trait to a locus on chromosome 1 containing the Idd5 diabetes susceptibility region. By the use of congenic mice, we confirm the linkage data and map this locus to a 6 cM region on proximal chromosome 1. Ctla4, being localized in this chromosomal region and mediating crucial functions in T cell biology, is a logical candidate gene in the Idd5 susceptibility region. In line with this, we demonstrate that T cells from Ctla4(-/-)deficient mice show a similar resistance to gamma-irradiation-induced apoptosis as observed in the NOD mice. This reinforces the notion that CTLA-4 contributes to the pathogenesis of autoimmune diabetes.

A new look at Syk in alpha beta and gamma delta T cell development using chimeric mice with a low competitive hematopoietic environment.

The Syk protein tyrosine kinase (PTK) is essential for B, but not T or NK, cell development, although certain T cell subsets (i.e., gamma delta T cells of intestine and skin) appear to be dependent on Syk. In this report, we have re-evaluated the role of Syk in T cell development in hematopoietic chimeras generated by using Syk-deficient fetal liver hematopoietic stem cells (FL-HSC). We found that Syk-/- FL-HSC were vastly inferior to wild-type FL-HSC in reconstituting T cell development in recombinant-activating gene 2 (RAG2)-deficient mice, identifying an unexpected and nonredundant role for Syk in this process. This novel function of Syk in T cell development was mapped to the CD44-CD25+ stage. According to previous reports, development of intestinal gamma delta T cells was arrested in Syk-/- -->RAG2-/- chimeras. In striking contrast, when hosts were the newly established alymphoid RAG2 x common cytokine receptor gamma-chain (RAG2/gamma c) mice, Syk-/- chimeras developed intestinal gamma delta T cells as well as other T cell subsets (including alpha beta T cells, NK1.1+ alpha beta T cells, and splenic and thymic gamma delta T cells). However, all Syk-deficient T cell subsets were reduced in number, reaching about 25-50% of controls. These results attest to the utility of chimeric mice generated in a low competitive hematopoietic environment to evaluate more accurately the impact of lethal mutations on lymphoid development. Furthermore, they suggest that Syk intervenes in early T cell development independently of ZAP-70, and demonstrate that Syk is not essential for the intestinal gamma delta T cell lineage to develop.

Role of Qa-1(b)-binding receptors in the specificity of developing NK cells.

NK cells acquire the ability to recognize MHC class I molecules during development. Studies with Qa-1(b) tetramers (Qa-1 tetramers) showed that nearly all NK1.1(+) cells from newborn C57BL/6 mice express Qa-1-binding receptors. Cytotoxic activity of these cells is fully inhibited by Qa-1 ligands on target cells. In contrast, neither receptors for H-2K(b) nor H-2D(b) were observed on NK1.1(+) cells from newborn mice. After birth, frequencies of Qa-1 tetramer(+)/ NK1.1(+) cells gradually decrease as the number of Ly49(+) /NK1.1(+) cells increases. Cell transfer studies showed that Qa-1 tetramer(+) cells from newborn mice do not lose expression of Qa-1 receptors, but that they further acquire expression of Ly49 molecules. Acquisition of Qa-1-binding receptors appears largely independent of host MHC class I molecules, as observed in studies using beta2-microglobulin-deficient (beta2m(-/-)) mice as well as K(b)/ D(b-/-) and K(b)/D(b)/beta2m(-/-) mice. The present results suggest that Qa-1-binding receptors play an important role in the specificity of developing NK cells, and suggest that these cells rely mainly on inhibitory receptors specific for non-classical MHC class I molecules to maintain self tolerance during the first weeks of life.

Tyrosine kinase SYK: essential functions for immunoreceptor signalling.

The tyrosine kinase SYK plays critical roles in signalling through immune receptors. Gene-targeting studies have identified the cell types that require SYK for development and function, and the receptors that use SYK as well as their downstream signalling effectors. There is also evidence of a role for SYK in non-immune cells and in the maintenance of vascular integrity.

The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells.

Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit(-/-) (W/W) mice. Although c-kit(-/-)NK cells were generated in W/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)(+) cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit(+) NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.

Redundant role of the Syk protein tyrosine kinase in mouse NK cell differentiation.

Syk and ZAP-70 subserve nonredundant functions in B and T lymphopoiesis. In the absence of Syk, B cell development is blocked, while T cell development is arrested in the absence of ZAP-70. The receptors and the signaling molecules required for differentiation of NK cells are poorly characterized. Here we investigate the role of the Syk protein tyrosine kinase in NK cell differentiation. Hemopoietic chimeras were generated by reconstituting alymphoid (B-, T-, NK-) recombinase-activating gene-2 x common cytokine receptor gamma-chain double-mutant mice with Syk-/- fetal liver cells. The phenotypically mature Syk-/- NK cells that developed in this context were fully competent in natural cytotoxicity and in calibrating functional inhibitory receptors for MHC molecules. Syk-deficient NK cells demonstrated reduced levels of Ab-dependent cellular cytotoxicity. Nevertheless, Syk-/- NK cells could signal through NK1. 1 and 2B4 activating receptors and expressed ZAP-70 protein. We conclude that the Syk protein tyrosine kinase is not essential for murine NK cell development, and that compensatory signaling pathways (including those mediated through ZAP-70) may sustain most NK cell functions in the absence of Syk.

Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation.

NK lymphocytes participate in both innate and adaptive immunity by their prompt secretion of cytokines including IFN-gamma, which activates macrophages, and by their ability to lyse virally infected cells and tumor cells without prior sensitization. Although these characteristics of NK cells are well documented, little is known about the genetic program that orchestrates NK development or about the signaling pathways that trigger NK effector functions. By crossing NK-deficient common gamma-chain (gammac) and recombinase activating gene (RAG)-2 mutant mice, we have generated a novel alymphoid (B-, T-, and NK-) mouse strain (RAG2/gammac) suitable for NK complementation in vivo. The role of the c-abl proto-oncogene in murine NK cell differentiation has been addressed in hemopoietic chimeras generated using RAG2/gammac mice reconstituted with c-abl-/- fetal liver cells. The phenotypically mature NK cells that developed in the absence of c-abl were capable of lysing tumor targets, recognizing "missing self," and performing Ab-dependent cellular cytotoxicity. Taken together, these results exclude any essential role for c-abl in murine NK cell differentiation in vivo. The RAG2/gammac model thereby provides a novel approach to establish a genetic map of NK cell development.

Natural killer and T cells of innate and adaptive immunity: lymphoid compartments with different requirements for common gamma chain-dependent cytokines.

A group of cytokines, including interleukin-2, -4, -7, -9 and -15, are related through the usage of a shared receptor subunit, the common cytokine receptor gamma chain, gamma c. gamma c-dependent cytokines critically affect the development and maintenance of the lymphoid system. This review will highlight our current knowledge on the gamma c-dependent cytokine network and on the non-redundant roles that these cytokines play in the development and homeostasis of T and natural killer cells involved in innate and adaptive immunity.

Diabetes induction in C57BL/6 mice reconstituted with lymphocytes of nonobese diabetic <--> C57BL/6 mouse embryo aggregation chimeras.

To determine whether the genetic background of the insulin-producing beta cells of the pancreas contributes to autoimmune diabetes susceptibility, we have used a model of the disease based on transferring spleen cells from nonobese diabetic (NOD) <--> C57BL/6 (B6) embryo aggregation (EA) chimeras into B6 and NOD irradiated mice. Insulitis and diabetes could be induced into both B6 and NOD hosts, albeit with low incidence. Cyclophosphamide (CY) treatment, known to accelerate diabetes in prediabetic NOD mice, was found to increase diabetes incidence up to 50-60% in both B6 and NOD mice reconstituted with chimeric splenocytes, while diabetes did not occur in CY-treated B6 mice reconstituted with B6 splenocytes. We conclude that the genetic make-up of the target organ does not affect the final stage of the pathogenesis of insulin-dependent diabetes mellitus.

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region.

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

Induction of diabetes in NOD<-->C57BL/6 embryo aggregation chimeras by cyclophosphamide through preferential depletion of C57BL/6 lymphocytes.

The majority of embryo aggregation (EA) mouse chimeras between non-obese diabetic (NOD) mice and C57BL/6 (B6) mice show clear signs of insulitis frequently accompanied by beta-cell destruction. Less than 5% of these chimeras, however, spontaneously progress to autoimmune diabetes, an incidence far lower than observed in NOD mice. The resistance in chimeras can be accounted for by the target organ chimerism and/or the immune system chimerism. To investigate the mechanism(s) controlling diabetes resistance in these mice, we studied a total of 92 NOD<-->B6 EA chimeras that showed overt lymphoid chimerism and treated 34 chimeras with cyclophosphamide (CY), a compound known to precipitate an acute form of insulin-dependent diabetes mellitus (IDDM) in pre-diabetic NOD mice, by interfering with regulatory mechanisms. We found that CY-treated EA chimeras displayed an increase in the NOD:B6 lymphocyte ratio and 32% of them developed diabetes that could be adoptively transferred to irradiated NOD or NOD-rag-2-/- mice. These findings suggest that lymphocyte chimerism rather than beta-cell chimerism accounts for diabetes resistance in NOD<-->B6 EA chimeras and that the susceptibility to CY-induced diabetes may be related to the proportion of NOD versus B6 lymphoid cells.

Establishment and characterization of RAG-2 deficient non-obese diabetic mice.

The authors have established a new immunodeficient mouse strain on the genetic background of the diabetes prone non-obese diabetic (NOD) mouse. A deletion mutant of the RAG-2 gene was back crossed 10 generations onto the NOD/Bom strain background. The homozygous NODrag-2-/- mice lack functionally mature B and T lymphocytes and do not develop insulitis or diabetes throughout life. In contrast, heterozygous NODrag-2+/- develop both insulitis and diabetes with an incidence similar to the wild type NOD mice. In transfer experiments, spleen cells from diabetic NOD donors were found to transfer disease to NODrag-2-/- recipients similar to what has been previously observed in transfer to irradiated NOD recipients or to immunodeficient NOD-scid/scid mice. While resembling the recently established NOD-scid/scid mice in many respects, the NODrag-2-/- mice represents an advantageous model for reconstitution of the pathogenesis of murine IDDM as it does not produce any endogenous, mature T or B lymphocytes.