Ncoa2 Promotes CD8+ T cell-Mediated Antitumor Immunity by Stimulating T-cell Activation via Upregulation of PGC-1α Critical for Mitochondrial Function.

Nuclear receptor coactivator 2 (Ncoa2) is a member of the Ncoa family of coactivators, and we previously showed that Ncoa2 regulates the differentiation of induced regulatory T cells. However, it remains unknown if Ncoa2 plays a role in CD8+ T-cell function. Here, we show that Ncoa2 promotes CD8+ T cell-mediated immune responses against tumors by stimulating T-cell activation via upregulating PGC-1α expression to enhance mitochondrial function. Mice deficient in Ncoa2 in T cells (Ncoa2fl/fl/CD4Cre) displayed defective immune responses against implanted MC38 tumors, which associated with significantly reduced tumor-infiltrating CD8+ T cells and decreased IFNγ production. Consistently, CD8+ T cells from Ncoa2fl/fl/CD4Cre mice failed to reject tumors after adoptive transfer into Rag1-/- mice. Further, in response to TCR stimulation, Ncoa2fl/fl/CD4Cre CD8+ T cells failed to increase mitochondrial mass, showed impaired oxidative phosphorylation, and had lower expression of PGC-1α, a master regulator of mitochondrial biogenesis and function. Mechanically, T-cell activation-induced phosphorylation of CREB triggered the recruitment of Ncoa2 to bind to enhancers, thus, stimulating PGC-1α expression. Forced expression of PGC-1α in Ncoa2fl/fl/CD4Cre CD8+ T cells restored mitochondrial function, T-cell activation, IFNγ production, and antitumor immunity. This work informs the development of Ncoa2-based therapies that modulate CD8+ T cell-mediated antitumor immune responses.

SRC2 controls CD4+ T cell activation via stimulating c-Myc-mediated upregulation of amino acid transporter Slc7a5.

T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C. rodentium) infection. Adoptive transfer of naive CD4+ T cells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/ recipients. Further, CD4+ T cells from SRC2fl/fl/CD4Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4+ T cells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ T cells to induce EAE. Therefore, SRC2 is essential for CD4+ T cell activation and, thus, a potential drug target for controlling CD4+ T cell-mediated autoimmunity.

Decoupling the role of RORγt in the differentiation and effector function of TH17 cells.

RORγt is known to instruct the differentiation of T helper 17 (TH17) cells that mediate the pathogenesis of autoimmune diseases. However, it remains unknown whether RORγt plays a distinct role in the differentiation and effector function of TH17 cells. Here, we show that mutation of RORγt lysine-256, a ubiquitination site, to arginine (K256R) separates the RORγt role in these two functions. Preventing ubiquitination at K256 via arginine substitution does not affect RORγt-dependent thymocyte development, and TH17 differentiation in vitro and in vivo, however, greatly impaired the pathogenesis of TH17 cell-mediated experimental autoimmune encephalomyelitis (EAE). Mechanistically, K256R mutation impairs RORγt to bind to and activate Runx1 expression critical for TH17-mediated EAE. Thus, RORγt regulates the effector function of TH17 cells in addition to TH17 differentiation. This work informs the development of RORγt-based therapies that specifically target the effector function of TH17 cells responsible for autoimmunity.

A Case-Control Study of Follicular Fluid Cytokine Profiles in Women with Diminished Ovarian Reserve.

Ovarian reserve is an important determinant of a woman's reproductive potential, and women with diminished ovarian reserve (DOR) often seek in vitro fertilization (IVF). The underlying etiology of DOR is unknown, but follicular fluid cytokine concentrations likely play a role in follicular development and maturation. The present study seeks to investigate the expression of cytokines in follicular fluid (FF) of women with DOR undergoing IVF and explore correlated functional pathways. One hundred ninety-four women undergoing ovarian stimulation were recruited at the time of oocyte retrieval. Women were classified as having DOR if they met one or more of the following criteria: AMH < 1 ng/ml, FSH > 10 mIU/ml, and/or AFC < 10. Controls included women undergoing IVF for male factor, tubal factor due to tubal ligation, or planned oocyte cryopreservation (non-oncologic). The concentrations of 480 cytokines and related growth factors in follicular fluid were determined using a multiplex immunoassay. Fifty-nine cytokines had significantly different concentrations (53 higher and 6 lower) in the DOR relative to the control group after adjusting for age and body mass index (BMI) (false discovery rate; FDR < 0.1). Using the most informative 44 biomarkers as indicated by a random forest (RF) model, an area under the curve (AUC) of 0.78 was obtained. Thus, follicular microenvironment differs between women with DOR and normal ovarian reserve. The differentially expressed cytokines belong to diverse processes that are primarily involved in follicular maturation and ovulation. These changes may play an important role in treatment outcomes in women with DOR.

A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells.

Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (C x 43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on C x 43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of C x 43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of C x 43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of C x 43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.

Analysis of follicular fluid retinoids in women undergoing in vitro fertilization: retinoic acid influences embryo quality and is reduced in women with endometriosis.

Retinol (ROL) and its biologically active metabolite, all-trans retinoic acid (ATRA), are essential for a number of reproductive processes. However, there is a paucity of information regarding their roles in ovarian folliculogenesis, oocyte maturation, and early embryogenesis. The objectives of this study were to quantify and compare peripheral plasma (PP) and follicular fluid (FF) retinoid levels, including ATRA in women undergoing in vitro fertilization (IVF) and to investigate the relationship between retinoid levels and embryo quality. Retinoid levels were evaluated in PP and FF from 79 women undergoing IVF at the time of oocyte retrieval and corresponding embryo quality assessed on a daily basis after retrieval for 3 days until uterine transfer. Analysis compared the retinoid levels with day 3 embryo grades and between endometriosis versus control patients. Results demonstrated distinctive levels of retinoid metabolites and isomers in FF versus PP. There was a significantly larger percentage of high-quality grade I embryos derived from the largest versus smallest follicles. An increase in follicle size also correlated with a >50% increase in FF ROL and ATRA concentrations. Independent of follicle size, FF yielding grade I versus nongrade I embryos showed higher mean levels of ATRA but not ROL. In a nested case-control analysis, control participants had 50% higher mean levels of ATRA in their FF and PP than women with endometriosis. These findings strongly support the proposition that ATRA plays a fundamental role in oocyte development and quality, and that reduced ATRA synthesis may contribute to decreased fecundity of participants with endometriosis.

Ameliorated ConA-induced hepatitis in the absence of PKC-theta.

Severe liver injury that occurs when immune cells mistakenly attack an individual's own liver cells leads to autoimmune hepatitis. In mice, acute hepatitis can be induced by concanavalin A (ConA) treatment, which causes rapid activation of CD1d-positive natural killer (NK) T cells. These activated NKT cells produce large amounts of cytokines, which induce strong inflammation that damages liver tissues. Here we show that PKC-θ(-/-) mice were resistant to ConA-induced hepatitis due to essential function of PKC-θ in NKT cell development and activation. A dosage of ConA (25 mg/kg) that was lethal to wild-type (WT) mice failed to induce death resulting from liver injury in PKC-θ(-/-) mice. Correspondingly, ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in PKC-θ(-/-) mice. Peripheral NKT cells had developmental defects at early stages in the thymus in PKC-θ(-/-) mice, and as a result their frequency and number were greatly reduced. Furthermore, PKC-θ(-/-) bone marrow adoptively transferred to WT mice displayed similar defects in NKT cell development, suggesting an intrinsic requirement for PKC-θ in NKT cell development. In addition, upon stimulation with NKT cell-specific lipid ligand, peripheral PKC-θ(-/-) NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells also requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cell to induce hepatitis, and thus, is a potential drug target for prevention of autoimmune hepatitis.

Developing and activated T cell survival depends on differential signaling pathways to regulate anti-apoptotic Bcl-x(L).

Survival of T cells in both the central and peripheral immune system determines its ultimate function in the regulation of immune responses. In the thymus, developing T cells undergo positive and negative selection to generate a T cell repertoire that responds to foreign, but not self, antigens. During T cell development, the T cell receptor α chain is rearranged. However, the first round of rearrangement may fail, which triggers another round of α chain rearrangement until either successful positive selection or cell death occurs. Thus, the lifespan of double positive (CD4(+)CD8(+); DP) thymocytes determines how many rounds of α chain rearrangement can be carried out and influences the likelihood of completing positive selection. The anti-apoptotic protein Bcl-x(L) is the ultimate effector regulating the survival of CD4(+)CD8(+) thymocytes subject to the selection process, and the deletion of Bcl-x(L) leads to premature apoptosis of thymocytes prior to the completion of the developmental process. In addition to its critical function in the thymus, Bcl-x(L) also regulates the survival of peripheral T cells. Upon engagement with antigens, T cells are activated and differentiated into effectors. Activated T cells upregulate Bcl-x(L) to enhance their own survival. Bcl-x(L)-mediated survival is required for the generation of effectors that carry out the actual immune responses. In the absence of Bcl-x(L), mature T cells undergo apoptosis prior to the completion of the differentiation process to become effector cells. Therefore, Bcl-x(L) ensures the survival of both developing and peripheral T cells, which is essential for a functional immune system.

Current status of the approach to assisted reproduction.

Assisted reproductive technologies are important tools in the clinical armamentarium used to treat both female and male infertility disorders. Pre-implantation genetic diagnosis offers couples at risk of having children with inheritable disorders the ability to analyze the genetic make-up of embryos before transfer. For patients undergoing treatment of cancer with chemotherapy or radiation therapy, these technologies offer the potential for the preservation of future fertility. As technology evolves, it is likely the clinical applications of assisted reproduction will continue to develop and expand in the future to enhance fertility.

Differential requirement of PKC-theta in the development and function of natural regulatory T cells.

CD4+CD25+ natural Treg cells, which are developed in the thymus, migrate to the periphery to actively maintain self-tolerance. Similar to conventional T cells, TCR signals are critical for the development and activation of Treg cell inhibitory function. While PKC-theta-mediated TCR signals are required for the activation of peripheral naïve T cells, they are dispensable for their thymic development. Here, we show that mice deficient in PKC-theta had a greatly reduced number of CD4+Foxp3+ Treg cells, which was independent of PKC-theta-regulated survival, as transgenic Bcl-x(L) could not restore the Treg cell population in PKC-theta(-/-) mice. Active and WT PKC-theta markedly stimulated, whereas inactive PKC-theta and dominant negative NFAT inhibited Foxp3 promoter activity. In addition, mice-deficient in calcineurin Abeta had a decreased Treg cell population, similar to that observed in PKC-theta deficient mice. It is likely that PKC-theta promoted the development of Treg cells by enhancing Foxp3 expression via activation of the calcineurin/NFAT pathway. Finally, Treg cells deficient in PKC-theta were as potent as WT Treg cells in inhibiting T cell activation, indicating that PKC-theta was not required for Treg cell-mediated inhibitory function. Our data highlight the contrasting roles PKC-theta plays in conventional T cell and natural Treg cell function.

Stabilized beta-catenin potentiates Fas-mediated T cell apoptosis.

In response to Ag stimulation, Ag-specific T cells proliferate and accumulate in the peripheral lymphoid tissues. To avoid excessive T cell accumulation, the immune system has developed mechanisms to delete clonally expanded T cells. Fas/FasL-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) T cells. Using transgenic mice expressing a stabilized beta-catenin (beta-cat(Tg)), we show here that beta-catenin was able to enhance apoptosis of activated T cells by up-regulating Fas. In response to staphylococcal enterotoxin B stimulation, beta-cat(Tg) mice exhibited accelerated deletion of CD4(+)Vbeta8(+) T cells compared with wild type mice. Surface Fas levels were significantly higher on activated T cells obtained from beta-cat(Tg) mice than that from wild type mice. Additionally, T cells from beta-cat(Tg) mice were more sensitive to apoptosis induced by crosslinking Fas, activation-induced cell death, and to apoptosis induced by cytokine withdrawal. Lastly, beta-catenin bound to and stimulated the Fas promoter. Therefore, our data demonstrated that the beta-catenin pathway was able to promote the apoptosis of activated T cells in part via up-regulation of Fas.

Mechanisms regulating the development and function of natural regulatory T cells.

The key of the immune system is to protect the host from foreign threat posed by pathogens and from the internal threat posed by self-attacking lymphocytes. The ability to discriminate self versus non-self ensures that only "non-self" pathogens, but not the self antigens, are attacked. Such tolerance to "self" arises from the central tolerance mechanisms that include the deletion of thymocytes with high reactivity to self antigens and also the induction of unresponsiveness of autoreactive T cells in the periphery. Natural regulatory T cells (nTregs) directly inhibit effector T cells, and keep their proliferation in control. Apart from preventing autoimmune reactions, Tregs also contribute to peripheral immune homeostasis as evidenced by the excessive lymphocyte accumulation in peripheral lymphoid organs and intestinal inflammation in the absence of nTregs. Here we discuss the molecular aspects of the development and suppressive function of naturally occurring Tregs. Accumulating evidence shows the importance of these Tregs in autoimmunity, tumor immunity, organ transplantation, allergy, and microbial immunity.

Requirement of calcineurin a beta for the survival of naive T cells.

Calcineurin (Cn) is a Ca2+/calmodulin-dependent phosphatase that dephosphorylates and activates NFAT, a transcription factor essential for T cell activation. T lymphocytes predominantly express the calcineurin Abeta (CnAbeta) isoform, and the deletion of the CnAbeta gene results in defective T cell proliferation and IL-2 production in response to TCR stimulation. In this study, we show that CnAbeta enhances the spontaneous survival of naive T cells by maintaining high levels of Bcl-2, a critical homeostatic survival factor for naive T cells. T cells obtained from CnAbeta-/- mice displayed accelerated spontaneous apoptosis. The observed apoptosis of the CnAbeta-/- T cells was prevented by IL-7 and IL-15, two cytokines critical for the homeostatic survival of naive T cells. Furthermore, CD4+ or CD8+ single positive CnAbeta-/- thymocytes also underwent accelerated apoptosis. However, no obvious difference in the apoptosis of CD4+CD8+ double positive thymocytes was observed between CnAbeta-/- and wild-type mice, suggesting a specific function of CnAbeta in the survival of single positive T cells. Bcl-2 levels were found to be significantly lower in CnAbeta-/- T cells. Transgenic expression of Bcl-xL restored the survival of the CnAbeta-/- T cells. Thus, in addition to its role in mediating TCR signals essential for T cell activation, CnAbeta is also required for the homeostatic survival of naive T cells.