Search

Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, β, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, β, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry., (Copyright © 2011 Elsevier B.V. All rights reserved.)

As regulation of CD8 T cell homeostasis is incompletely understood, we investigated the expression profile of the vasoactive intestinal peptide (VIP) receptors, VPAC1 and VPAC2, on CD8 T cells throughout an in vivo immune response. Herein, we show that adoptively transferred CD8 T cells responding to a Listeria monocytogenes infection significantly downregulated, functionally active VPAC1 protein expression during primary and secondary expansion. VPAC1 mRNA expression was restored during contraction and regained naïve levels in primary, but remained low during secondary, memory generation. VIP co-administration with primary infection suppressed CD8 T cell expansion (≈ 50%). VPAC2 was not detected at any time points throughout primary and secondary infections. Collectively, our data demonstrate that functionally active VPAC1 is dynamically downregulated to render expanding CD8 T cells unresponsive to VIP., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO) resistant (SWR/J) and susceptible (C57BL/6) mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ) potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4(+)/CD44(hi) and CD8(+)/CD44(hi) cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.

More than 40 years after the discovery of vasoactive intestinal peptide (VIP), its transcriptome in the immune system has still not been completely elucidated. In an attempt to understand the biological role of this neuropeptide in immunity, we chose CD4 T cells as a cellular system. Agilent Mouse Whole Genome microarrays were hybridized with fluorescently labeled total RNA isolated from resting CD4 T cells cultured +/-10(-7)M VIP for 5h or PMA/ionomycin activated CD4 T cells cultured +/-10(-7)M VIP for 5h. These VIP-regulated transcriptomes were analyzed by Significance Analysis of Microarrays (SAM) and Ingenuity Pathway Analysis (IPA) software to identify relevant signaling pathways modulated by VIP in the absence and presence of T cell activation. In resting CD4 T cells, VIP-modulated 368 genes, ranging from 3.49 to -4.78-fold. In the PMA/ionomycin activated CD4 T cells, 326 gene expression levels were changed by VIP, ranging from 2.94 to -1.66-fold. IPA analysis revealed that VIP exposure alters cellular function through EGFR signaling in resting CD4 T cells, and modulates immediate early genes, Fos and CREM/ICER, in activated CD4 T cells. These gene expression changes are suggested to explain at a molecular level how VIP can regulate T cell homing to the gut and induce regulatory T cell generation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ralpha and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized beta-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized beta-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

T cells express receptors for neuropeptides that mediate immunological activities. Vasoactive intestinal peptide receptor-1 (VPAC1), the prototypical group II G protein coupled receptor, binds two neuropeptides with high-affinity, called vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide. During T cell signaling, VPAC1 mRNA expression levels are significantly downregulated through a Src kinase dependent mechanism, thus altering the sensitivity for these neuropeptides during an immune reaction. Presently, it is unknown whether the mechanism that regulates VPAC1 during T cell signaling involves epigenetic changes. Therefore, we hypothesized that the epigenetic landscape consisting of diacetylation at H3K9/14 and trimethylation at H3K4, two transcriptionally permissive histone modifications, would parallel VPAC1 expression showing high enrichment in untreated T cells, but lower enrichment in alpha-CD3 treated T cells. To this end, quantitative chromatin immunoprecipitation (ChIP) analysis of H3K9/14ac and H3K4me3 was conducted using purified CD4(+) T cells, with CD45R(+) B cells as a negative control. Our data revealed that these histone modifications at the VPAC1 promoter did indeed parallel its mRNA levels between T and B lymphocytes, but did not decrease during T cell signaling. Collectively, these data strongly imply a euchromatin nuclear position for the VPAC1 locus irrespective of the activation status of T cells.

Intranasal mouse hepatitis virus type 1 (MHV-1) infection of mice induces lung pathology similar to that observed in severe acute respiratory syndrome (SARS) patients. However, the severity of MHV-1-induced pulmonary disease varies among mouse strains, and it has been suggested that differences in the host immune response might account for this variation. It has also been suggested that immunopathology may represent an important clinical feature of SARS. Little is known about the host immune response to MHV-1 and how it might contribute to some of the pathological changes detected in infected mice. In this study we show that an intact type I interferon system and the adaptive immune responses are required for controlling MHV-1 replication and preventing morbidity and mortality in resistant C57BL/6J mice after infection. The NK cell response also helps minimize the severity of illness following MHV-1 infection of C57BL/6J mice. In A/J and C3H/HeJ mice, which are highly susceptible to MHV-1-induced disease, we demonstrate that both CD4 and CD8 T cells contribute to morbidity during primary infection, and memory responses can enhance morbidity and mortality during subsequent reexposure to MHV-1. However, morbidity in A/J and C3H/HeJ mice can be minimized by treating them with immune serum prior to MHV-1 infection. Overall, our findings highlight the role of the host immune response in contributing to the pathogenesis of coronavirus-induced respiratory disease.

Proinflammatory cytokines, such as gamma interferon (IFN-gamma), impact aspects of T-cell responses after infection, including expansion, contraction, and memory formation. Interleukin-18 (IL-18) functions as a proinflammatory cytokine by stimulating the production of IFN-gamma from multiple cell types and accentuating the development of Th1 CD4 T-cell responses. Focused microarray analyses revealed upregulation of IL-18 and IL-18 receptor genes in CD8 T cells during the contraction phase. Based on these findings we investigated if and how signaling through the IL-18 receptor influences the development and kinetics of antigen (Ag)-specific CD8 and CD4 T-cell responses following infection. IL-18Ralpha(-/-) and IL-18(-/-) mice developed frequencies and total numbers of Ag-specific CD8 T cells after Listeria monocytogenes infection that were similar to those of wild-type C57BL/6 mice. The kinetics of expansion, contraction, and memory CD8 T-cell maintenance were also similar. When IL-18Ralpha deficiency was isolated to Ag-specific CD8 T cells, the kinetics of the expansion and contraction phases were also normal. These basic findings were confirmed by examining the response to vaccinia virus infection. In contrast, the expansion of Ag-specific CD4 T cells was slightly curtailed by the absence of IL-18Ralpha; however, contraction and the maintenance of memory were not altered. Importantly, both memory Ag-specific CD8 and CD4 T cells generated in the absence of IL-18Ralpha expanded appropriately after secondary antigen exposure and were protective, indicating that signaling through the IL-18 receptor is not required for normal T-cell response kinetics and survival of immunized mice challenged with a lethal L. monocytogenes infection.

Receptors belonging to the TNF-receptor (TNF-R) superfamily include important costimulatory molecules, many of which specifically affect T cell activation. TNF receptor-associated factors (TRAFs) are recruited to many TNF-R superfamily members and are important modulators of the proximal signaling events that occur at the time of receptor engagement and activation. TRAF5 has been shown to be a positive regulator of a number of these receptors that are involved in T cell costimulation. However, the potential importance of TRAF5 in cellular immune responses to infection or in T cell expansion and memory have not been studied. We report in this study that TRAF5 was required for optimal CD8(+) T cell responses following infection with Listeria monocytogenes expressing OVA (LM-OVA). TRAF5 was necessary for optimal T cell expansion following primary infection with LM-OVA, and its absence resulted in fewer memory CD8(+) T cells following LM-OVA infection, together with higher bacterial loads in the liver. The effect of TRAF5 on CD8(+) T cell expansion was T cell intrinsic and not due to effects of TRAF5 deficiency on APCs. Although their proliferative ability remained intact, CD8(+) T cells from TRAF5(-/-) mice were more sensitive to apoptosis and were unresponsive to the prosurvival effects of the TNF-R superfamily costimulator CD27. Collectively, these studies identify TRAF5 as an important positive signaling element that enhances T cell expansion and pathogen containment by providing a survival advantage to responding Ag-specific CD8(+) T cells during infection.

GA binding protein (GABP) is a ubiquitously expressed Ets family transcription factor that consists of two subunits, GABPalpha and GABPbeta. GABPalpha binds to DNA, and GABPbeta heterodimerizes with GABPalpha and possesses the ability to transactivate target genes. Our previous studies using GABPalpha-deficient mice revealed that GABPalpha is required for the development of both T and B cells. Two splice variants of GABPbeta are generated from the Gabpb1 locus and differ in their carboxy-terminal lengths and sequences. The longer isoform (GABPbeta1L) can homodimerize and thus form alpha(2)beta(2) tetramers depending on the gene context, whereas the shorter isoform (GABPbeta1S) cannot. In this study, we generated mice that are deficient in GABPbeta1L but that retain the expression of GABPbeta1S. Surprisingly, GABPbeta1L-/- mice had normal T- and B-cell development, and mature T and B cells showed normal responses to various stimuli. In contrast, targeting both GABPbeta1L and GABPbeta1S resulted in early embryonic lethality. Because of its incapability of forming homodimers, GABPbeta1S has been suspected to have a dominant negative role in regulating GABP target genes. Our findings argue against such a possibility and rather suggest that GABPbeta1S has a critical role in maintaining the transcriptional activity of the GABPalpha/beta complex.

Expression of IL-7Ralpha (CD127) has been suggested as a major determinant in the survival of memory T cell precursors. We investigated whether constitutive expression of IL-7Ralpha on T cells increased expansion and/or decreased contraction of endogenous Ag-specific CD4 and CD8 T cells following infection with Listeria monocytogenes. The results indicate that constitutive expression of IL-7Ralpha alone was not enough to impart an expansion or survival advantage to CD8 T cells responding to infection, and did not increase memory CD8 T cell numbers over those observed in wild-type controls. Constitutive expression of IL-7Ralpha did allow for slightly prolonged expansion of Ag-specific CD4 T cells; however, it did not alter the contraction phase or protect against the waning of memory T cell numbers at later times after infection. Memory CD4 and CD8 T cells generated in IL-7Ralpha transgenic mice expanded similarly to wild-type T cells after secondary infection, and immunized IL-7Ralpha transgenic mice were fully protected against lethal bacterial challenge demonstrating that constitutive expression of IL-7Ralpha does not impair, or markedly improve memory/secondary effector T cell function. These results indicate that expression of IL-7Ralpha alone does not support increased survival of effector Ag-specific CD4 or CD8 T cells into the memory phase following bacterial infection.

Adoptive-transfer experiments with relatively large input numbers ( approximately 10(6)) of T cell receptor-transgenic (TCR-tg) T cells are widely used to model endogenous T cell responses to infection or immunization. We show that input numbers of naive TCR-tg T cells sufficient to squelch the endogenous response to the same epitope substantially alter the kinetics, proliferative expansion, phenotype, and efficiency of memory generation by the TCR-tg T cells in response to infection. Thus, responses from nonphysiologic input numbers of TCR-tg T cells fail to accurately mimic the endogenous T cell response. Importantly, seeding as few as approximately 10-50 TCR-tg T cells, which constitute a fraction of the endogenous repertoire, allowed vigorous proliferation and analysis of TCR-tg cells after infection in a scenario representing normal physiology for any individual TCR. These data strongly suggest that modeling the endogenous T cell response with TCR-tg cells will require every effort to approximate the endogenous precursor frequency.

Several lines of evidence from different model systems suggest that gamma interferon (IFN-gamma) is an important regulator of T-cell contraction after antigen (Ag)-driven expansion. To specifically investigate the role of IFN-gamma in regulating the contraction of Ag-specific CD4 T cells, we infected IFN-gamma-/- and IFN-gammaR1-/- mice with attenuated Listeria monocytogenes and monitored the numbers of Ag-specific CD4 T cells during the expansion, contraction, and memory phases of the immune response to infection. In the absence of IFN-gamma or the ligand-binding portion of its receptor, Ag-specific CD4 T cells exhibited normal expansion in numbers, but in both strains of deficient mice there was very little decrease in the number of Ag-specific CD4 T cells even at time points later than day 90 after infection. This significant delay in contraction was not due to prolonged infection, since mice treated with antibiotics to conclusively eliminate infection exhibited the same defect in contraction. In addition to altering the number of Ag-specific CD4 T cells, the absence of IFN-gamma signaling also changed the phenotype of cells generated after infection. IFN-gammaR1-/- Ag-specific CD4 T cells reacquired expression of CD127 more quickly than wild-type cells, and more IFN-gammaR1-/- CD4 T cells were capable of producing both IFN-gamma and interleukin 2 following Ag stimulation. From these data we conclude that IFN-gamma regulates the contraction, phenotype, and function of Ag-specific CD4 T cells generated after infection.

Although inflammatory cytokines induced by infection or vaccination with adjuvants have long been known to stimulate optimal antigen-presenting cell function, recent evidence indicates that some inflammatory cytokines also act directly on the responding T cells to control their response to infection. Here, we review the evidence that specific inflammatory cytokines act to control the magnitude of expansion, the degree of contraction, and the rate of memory cell development. These data may suggest new strategies for manipulating vaccine efficacy in the quest to protect against pathogenic microbes.

The precise mechanisms that govern the commitment of CD4 T cells to become Th1 or Th2 cells in vivo are incompletely understood. Recent experiments demonstrate colocalization of the IFN-gammaR chains with the TCR during activation of naive CD4 T cells, suggesting that association of these molecules may be involved in determining lineage commitment. To test the role of IFN-gamma and its receptor in the generation of Th1 Ag-specific CD4 T cells, we analyzed mice after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus. In the absence of IFN-gamma, Ag-specific CD4 T cells were generated in response to both these infections. In addition, IFN-gamma-producing (Th1) Ag-specific CD4 T cells were generated in mice lacking the ligand-binding chain of the IFN-gammaR (IFN-gammaR1-/-) or the signaling chain (IFN-gammaR2-/-). There was no increase in the number of IL-4-producing Ag-specific CD4 T cells, nor was there a decrease in the expression of T-bet in the absence of functional IFN-gamma signaling, indicating that the cells were committed Th1 cells. Thus, both chains of the IFN-gammaR are dispensable for the generation of Th1 Ag-specific CD4 T cells after infection in vivo.

Efficient boosting of memory T-cell numbers to protective levels generally requires a relatively long interval between immunizations. Decreasing this interval could be crucial in biodefense and cancer immunotherapy, in which rapid protective responses are essential. Here, we show that vaccination with peptide-coated dendritic cells (DCs) generated CD8+ T cells with the phenotype and function of memory cells within 4-6 d. These early memory CD8+ T cells underwent vigorous secondary expansion in response to a variety of booster immunizations, leading to elevated numbers of effector and memory T cells and enhanced protective immunity. Coinjection of CpG oligodeoxynucleotides, potent inducers of inflammation that did not alter the duration of DC antigen display, prevented the rapid generation of memory T cells in wild-type mice but not in mice lacking the interferon (IFN)-gamma receptor. These data show that DC vaccination stimulates a pathway of accelerated generation of memory T cells, and suggest that events of inflammation, including the action of IFN-gamma on the responding T cells, control the rate of development of memory CD8+ T cells.

IFN-gamma plays a critical role in the CD8(+) T cell response to infection, but when and if this cytokine directly signals CD8(+) T cells during an immune response is unknown. We show that naive Ag-specific CD8(+) T cells receive IFN-gamma signals within 12 h after in vivo infection with Listeria monocytogenes and then become unresponsive to IFN-gamma throughout the ensuing Ag-driven expansion phase. Ag-specific CD8(+) T cells regain partial IFN-gamma responsiveness throughout the contraction phase, whereas the memory pool exhibits uniform, but reduced, responsiveness that is also modulated during the secondary response. The responsiveness of Ag-specific CD8(+) T cells to IFN-gamma correlated with modulation in the expression of IFN-gammaR2, but not with IFN-gammaR1 or suppressor of cytokine signaling-1. This dynamic regulation suggests that early IFN-gamma signals participate in regulation of the primary CD8(+) T cell response program, but that evading or minimizing IFN-gamma signals during expansion and the memory phase may contribute to appropriate regulation of the CD8(+) T cell response.

Demyelination following infection of mice with the neurotropic coronavirus mouse hepatitis virus strain JHM (MHV) is immune-mediated. It has been demonstrated that MHV-specific CD4 and CD8 T cells are capable of causing demyelination independent of the other T cell subset. Recent work has also demonstrated that activated bystander CD8 T cells mediate significant demyelination. The ability of bystander CD4 T cells to mediate demyelination was investigated using CD4 T cell transgenic mice. The results indicated that bystander CD4 T cells were unable to cause demyelination in MHV-infected mice, despite being recruited into the central nervous system (CNS) and irrespective of activation status. These results suggest that CD4 T cells must recognize antigen in the CNS in order to cause demyelination.

Multiple sclerosis, a chronic inflammatory disease of the CNS, is characterized by immune-mediated demyelination. Many patients have a remitting-relapsing course of disease with exacerbations often following unrelated microbial illnesses. The relationship between the two events remains obscure. One possibility is that T cells specific for the inciting microbial pathogen are able to effect demyelination at a site of ongoing inflammation within the CNS. This possibility was examined in mice infected with mouse hepatitis virus, a well-described model of virus-induced demyelination. Using transgenic TCR/recombination activation gene 2(-/-) mice with only non-mouse hepatitis virus-specific T cells, we show that CD8 T cells are able to cause demyelination in the absence of cognate Ag in the CNS, but only if specifically activated. These findings demonstrate a novel mechanism for immune-mediated neuropathology and show that activated CD8 T cells may serve as important mediators of bystander demyelination during times of infection, including in patients with multiple sclerosis.

The neurotropic JHM strain of mouse hepatitis virus (MHV) causes acute encephalitis and chronic demyelinating encephalomyelitis in rodents. Previous results indicated that CD8 T cells infiltrating the central nervous system (CNS) were largely antigen specific in both diseases. Herein we show that by 7 days postinoculation, nearly 30% of the CD4 T cells in the acutely infected CNS were MHV specific by using intracellular gamma interferon (IFN-gamma) staining assays. In mice with chronic demyelination, 10 to 15% of the CD4 T cells secreted IFN-gamma in response to MHV-specific peptides. Thus, these results show that infection of the CNS is characterized by a large influx of CD4 T cells specific for MHV and that these cells remain functional, as measured by cytokine secretion, in mice with chronic demyelination.