Reproductive Ageing: Inflammation, immune cells, and cellular senescence in the aging ovary.

In brief: Recent reports suggest a relationship between ovarian inflammation and functional declines, although it remains unresolved if ovarian inflammation is the cause or consequence of ovarian aging. In this review, we compile the available literature in this area and point to several current knowledge gaps that should be addressed through future studies. Abstract: Ovarian aging results in reduced fertility, disrupted endocrine signaling, and an increased burden of chronic diseases. The factors contributing to the natural decline of ovarian follicles throughout reproductive life are not fully understood. Nevertheless, local inflammation may play an important role in driving ovarian aging. Inflammation progressively rises in aged ovaries during the reproductive window, potentially affecting fertility. In addition to inflammatory markers, recent studies show an accumulation of specific immune cell populations in aging ovaries, particularly lymphocytes. Other hallmarks of the aging ovary include the formation and accumulation of multinucleated giant cells, increased collagen deposition, and increased markers of cellular senescence. Collectively, these changes significantly impact the quantity and quality of ovarian follicles and oocytes. This review explores recent literature on the alterations associated with inflammation, fibrosis, cell senescence, and the accumulation of immune cells in the aging ovary.

A single-cell atlas of the aging mouse ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Female humans experience a sharp decline in fertility around 35 years of age, which corresponds to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging mouse ovary to identify early drivers of ovarian decline. To fill this gap we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress-response, immunogenic and fibrotic signaling pathway inductions with aging. This report provides critical insights into mechanisms responsible for ovarian aging phenotypes. The data can be explored interactively via a Shiny-based web application.

A single-cell atlas of the aging murine ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling, and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Around 35 years old, women experience a sharp decline in fertility, corresponding to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging ovary to identify early drivers of ovarian decline. To fill this gap, we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress response, immunogenic, and fibrotic signaling pathway inductions with aging. This report raises provides critical insights into mechanisms responsible for ovarian aging phenotypes.

Development of αß T Cells with Innate Functions.

Although we mostly think of αß T cells as components of the adaptive immune system, a number of them differentiate into alternative lineages. These lineages express TCRs with limited diversity, and functionally bridge the gap between innate and adaptive immunity. They tend to be tissue resident, and mount potent cytokine responses very rapidly after activation, and their development and functional maturation are strongly influenced by the microbiome. Here, we compare the development pathways and interactions with the microbiome of natural killer T (NKT) cells and mucosal-associated invariant T (MAIT cells), the two best studied "innate-like" αß T cell populations.

Multifaceted effects of soluble human CD6 in experimental cancer models.

BACKGROUND: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. METHODS: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEµTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. RESULTS: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. CONCLUSIONS: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.

Development of Type 2 Innate Lymphoid Cells Is Selectively Inhibited by Sustained E Protein Activity.

Innate lymphoid cells (ILCs) are tissue-resident lymphoid cells that reside mostly at barrier surfaces and participate in the initial response against pathogens. They are classified into different types based on effector programs that are based on cytokine production and transcription factor expression. They all derive from the common lymphoid precursor, but the molecular mechanisms regulating ILC subset development is not well understood. Experiments using Id2 knockout mice have previously shown that E protein activity inhibition is an absolute requirement for the development of all ILC subsets. In this study, we use a genetic approach to demonstrate that small increases in E protein activity during ILC development selectively inhibit type 2 ILC development. Type 1 ILCs are mostly unperturbed, and type 3 ILC show only a minor inhibition. This effect is first evident at the ILC2 progenitor stage and is ILC intrinsic. Therefore, our results demonstrate that modulation of E protein activity can bias cell fate decisions in developing ILCs.

Suppression of ILC2 differentiation from committed T cell precursors by E protein transcription factors.

Current models propose that group 2 innate lymphoid cells (ILC2s) are generated in the bone marrow. Here, we demonstrate that subsets of these cells can differentiate from multipotent progenitors and committed T cell precursors in the thymus, both in vivo and in vitro. These thymic ILC2s exit the thymus, circulate in the blood, and home to peripheral tissues. Ablation of E protein transcription factors greatly promotes the ILC fate while impairing B and T cell development. Consistently, a transcriptional network centered on the ZBTB16 transcription factor and IL-4 signaling pathway is highly up-regulated due to E protein deficiency. Our results show that ILC2 can still arise from what are normally considered to be committed T cell precursors, and that this alternative cell fate is restrained by high levels of E protein activity in these cells. Thymus-derived lung ILC2s of E protein-deficient mice show different transcriptomes, proliferative properties, and cytokine responses from wild-type counterparts, suggesting potentially distinct functions.

Retroviral Transduction of T Cells and T Cell Precursors.

Transduction of lymphoid progenitors with retroviral or lentiviral vectors is a powerful experimental strategy to tease out the role of a gene or pathway in T cell development via gain-of-function or loss-of-function strategies. Here we discuss different approaches to use this powerful technology, and present some protocols that we use to transduce murine HSCs, thymocytes, and lymphoid cell lines with these viral vectors.

Transgenic expression of soluble human CD5 enhances experimentally-induced autoimmune and anti-tumoral immune responses.

CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L) of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EµTg), expressing a circulating soluble form of human CD5 (shCD5) as a decoy to impair membrane-bound CD5 function. These shCD5EµTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE), as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma). This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+), and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EµTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens.

Increased level of E protein activity during invariant NKT development promotes differentiation of invariant NKT2 and invariant NKT17 subsets.

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.

Control of early stages in invariant natural killer T-cell development.

Natural killer T (NKT) cells develop in the thymus from the same precursors as conventional CD4(+) and CD8(+) αß T cells, CD4(+) CD8(+) double-positive cells. In contrast to conventional αßT cells, which are selected by MHC-peptide complexes presented by thymic epithelial cells, invariant NKT cells are selected by lipid antigens presented by the non-polymorphic, MHC I-like molecule CD1d, present on the surface of other double-positive thymocytes, and require additional signals from the signalling lymphocytic-activation molecule (SLAM) family of receptors. In this review, we provide a discussion of recent findings that have modified our understanding of the NKT cell developmental programme, with an emphasis on events that affect the early stages of this process. This includes factors that control double-positive thymocyte lifespan, and therefore the ability to generate the canonical Vα rearrangements that characterize this lineage, as well as the signal transduction pathways engaged downstream of the T-cell receptor and SLAM molecules.

The Ras/MAPK pathway is required for generation of iNKT cells.

iNKT cells derive from CD4(+)CD8(+) DP thymocytes, and are selected by thymocyte-thymocyte interactions through signals from their invariant Vα14-Jα18 TCR and from the costimulatory molecules SLAMF1 and SLAMF6. Genetic studies have demonstrated the contribution of different signaling pathways to this process. Surprisingly, current models imply that the Ras/MAPK pathway, one of the critical mediators of conventional αß T cell positive selection, is not necessary for iNKT cell development. Using mice defective at different levels of this pathway our results refute this paradigm, and demonstrate that Ras, and its downstream effectors Egr-1 and Egr-2 are required for positive selection of iNKT cells. Interestingly our results also show that there are differences in the contributions of several of these molecules to the development of iNKT and conventional αß T cells.

Regulation of GATA-3 expression during CD4 lineage differentiation.

GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.

The transcription factor c-Myb primes CD4+CD8+ immature thymocytes for selection into the iNKT lineage.

Type I invariant NKT cells (iNKT cells) are a subset of alphabeta T cells characterized by the expression of an invariant alpha-chain variable region 14-alpha-chain joining region 18 (V(alpha)14J(alpha)18) T cell antigen receptor (TCR) alpha-chain. The iNKT cells derive from CD4(+)CD8(+) double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow V(alpha)14-J(alpha)18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM-adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4.

During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.

Egr2 is required for Bcl-2 induction during positive selection.

The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2(-/-) thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.

Phosphatidylinositol 3-kinase improves the efficiency of positive selection.

We have generated transgenic mice expressing the amino-terminal fragment of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110ABD) in thymocytes. Expression of p110ABD results in constitutive activation of PI3K and in significant increases in the numbers of mature, single-positive thymocytes. We previously reported that the increase in mature cells was in part due to a defect in thymic emigration. In this study we identify another component to this phenotype. Expression of p110ABD results in an enhancement of positive selection, without alterations in thymocyte lifespan or negative selection. Since PI3K can affect activation of Btk, which in turn potentiates calcium fluxes, during B cell development, our results suggest that PI3K could play a role in the regulation of Itk kinases in T cells, and that both cell types share a common signaling network to modulate calcium responses downstream of their antigen receptor.

Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity.

BACKGROUND: Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. RESULTS: ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. CONCLUSION: EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.

Phosphatidylinositol 3-kinase regulates thymic exit.

To understand the role of PI3K during T cell development, we generated transgenic mice expressing the N terminus of the PI3K catalytic subunit (p110(ABD); ABD, adaptor binding domain) in thymocytes. Expression of p110(ABD) activates endogenous p110 and results in the accumulation of mature single-positive CD3(high)heat-stable Ag(low) thymocytes. This is mostly due to a defect in emigration of those cells, as shown by the delayed appearance of peripheral T cells in neonatal transgenic mice and by competitive adoptive transfer experiments. Although the mechanisms underlying these effects of PI3K are not yet clear, our results show an important role for PI3K activity in the regulation of mature thymocyte exit to the periphery.

Analysis of T-cell development by using short interfering RNA to knock down protein expression.

We have applied RNA interference (RNAi) technology to the analysis of genes involved in T-cell development, combining a reaggregate fetal thymic organ culture (rFTOC) system with retroviral delivery of short interfering RNA (siRNA) hairpins. The process involves the isolation of murine fetal liver or fetal thymocytes, infection with retroviral particles carrying the construct of interest, followed by reaggregation of the transduced precursors with fetal thymic stroma into lobes. Subsequently, individual lobes are harvested and analyzed for development at various time points. These reaggregate cultures recapitulate most features of T-cell development in vivo, including pre-TCR selection and expansion, positive selection of CD4 and CD8 T cells, and negative selection. In our hands, the combination of retroviral delivery of RNAi and rFTOCs is a quick alternative to conventional knockouts for the analysis of gene function during T-cell development. This chapter describes the methods we have developed to knock down gene expression in T-cell precursors, using retroviral delivery of siRNA hairpins.