bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determining intracellular cytokine expression and viability simultaneously in differentiated mouse CD4+ T-cells through flow cytometry (152.8)
Authors: Weldon Don , Williams Yuko , Hsu Matthew 
Published: January 2023

Abstract
The study of CD4+ T-helper (TH) cell differentiation is an important area of research that will aid in the understanding of inflammation and autoimmunity. CD4 + T cells can give rise to many subtypes depending on type of immune response. This study focuses on the TH1, TH2 and TH17 CD4+ T cell subtypes. Each subtype expresses a signature cytokine that directs the type of immune response needed. In order to analyze cytokine expression in TH cultures by flow cytometry, we employed a fixable viability dye to gate out dead cells that can accumulate in long-term differentiation cultures. In addition, flow cytometric analysis provides cell-specific information not obtained by ELISA analysis. Using standard TH culture protocols we differentiated naïve CD4+ T cells into TH1, TH2, and TH17 subtypes. We first stained the cells with the viability dye and then stained with antibodies against the signature cytokine of interest in less than 4 hours. Our data shows that we can easily obtain viability information while simultaneously evaluating cytokine production within our TH culture system. Using the fixable viability dye, we can exclude false positive cytokine staining and therefore obtain more accurate and reproducible intracellular expression data from cultured CD4+ T cells.
COL2A1 gene expression Is modulated by TSH In bone marrow-derived mesenchymal stem cells (153.12)
Authors: Bagriacik Emin , Yaman Melek , Savas Didem , Uslu Kadriye 
Published: January 2023

Abstract
Effect of interactions between the Thyroid stimulating hormone (TSH) and its receptor in mesenchymal stem cell (MSCs) biology has not been studied. We investigated gene expression by bone marrow-derived mesenchymal stem cells stimulated by TSH. Quantitative polymerase chain reaction and immunohistochemistry were employed to assess gene expression at both RNA and protein levels. We found that TSH increased expression of several genes that were crucial in self-renewal and differentiation processes of MSCs. Particularly SOX2 and COL2A1 mRNA levels increased significantly in comparison to untreated, control MSCs. This data was confirmed by COL2A1-specific antibody staining in immunohistochemistry experiments. TSH-treated MSCs elevated COL2A1 expression at protein level as well. Our findings suggest that TSH may play an important role in self-renewal and differentiation of MSCs and stroma of the bone marrow.
Pax5 control the B lineage specific gene expression program through association with the nuclear matrix (153.14)
Authors: Hong Sang Yong , He Ti , Huang Lin , Xie Wanqin , Yu Zhihong , Su Kaihong , Zhang Zhixin 
Published: January 2023

Abstract
Accumulating studies indicate that the nuclear matrix provides a dynamic structural support for various biological reactions inside of nuclei such as DNA replication, RNA transcription, and RNA splicing. Pax5 is an important regulator for B lineage cell development, which controls the B lineage specific gene expression program involving hundreds of positive and negative target genes. Here, we show that majority of the endogenous Pax5 proteins in human and murine B lineage cells are associated with the nuclear matrix, where they occupy the centers for transcription on the nuclear matrix as indicated by co-localization with the RNA polymerase II or the TATA box binding protein (TBP). Detailed analyses identified that two different domains of Pax5 are required for its association with the nuclear matrix. Interestingly, lysine 67, 87, and 89 residues within the PRD domain of Pax5 are essential for its association with the nuclear matrix. Microarray analysis showed that mutation of these lysine residues compromise Pax5 mediated activation and repression of hundreds target genes. Furthermore, chromatin immunoprecipitation (ChIP) and nuclear matrix foot printing assays indicated that Pax5 is responsible for recruitment of Cd19 loci to the nuclear matrix bound RNA polymerase complex. These results demonstrate that association with the nuclear matrix is essential for Pax5 to control the B lineage specific gene expression program.
FCRL6 distinguishes a subset of biologically impaired mouse pro B cells (153.16)
Authors: Won Woong-Jai , Stephan Robert , Davis Randall 
Published: January 2023

Abstract
Human Fc receptor-like 6 (FCRL6) is discretely expressed by cytotoxic lymphocytes, has ITIM-based signaling potential, and interacts with MHC II. To investigate the biology of its counterpart in mice, a panel of FCRL6-specific monoclonal antibodies was generated. In contrast to its expression in humans, FCRL6 could not be detected on mouse NK or CD8+ T cells. Instead, the receptor marked a subpopulation of pro B cells in adult bone marrow. FCRL6+ cells were absent in Hardy Fr. A, but became evident in Fr. B, peaked in Fr. C, and diminished in Fr. C’. The developmentally regulated expression of FCRL6 was also indicated by the increased frequency of these pro B cells in RAG2-/- and λ5-/- mice. FCRL6+ pro B cells were slightly smaller by light scatter and expressed relatively lower levels of IL-7Rα compared to FCRL6- cells. In keeping with this phenotypic difference, FCRL6+ pro B cells responded less favorably to IL-7 stimulation. They also had relatively lower proliferative and cycling capacity. Exposure of purified FCRL6- pro B cells to cytokines important for early B lineage differentiation revealed that FCRL6 surface expression could be induced by IL-7 alone, but was augmented by the addition of SCF. Collectively, these results demonstrate that FCRL6 defines a unique subset of mouse pro B cells that competes poorly relative to cells lacking its expression and suggest that FCRL6 may distinguish a subset of biologically impaired pro B cells.
Novel targets of E47 in regulating hematopoietic stem cell proliferation (153.2)
Authors: Santos Patricia , Denzin Lisa , Borghesi Lisa 
Published: January 2023

Abstract
Hematopoietic stem cells (HSCs) continuously replenish all blood lineages thus maintaining a functional immune system throughout adult life. Our lab is interested in defining targets of E47 that control HSC proliferation. Here, we show that E47 and p21 collaborate to regulate HSC proliferation, and identify a novel connection between E47 and tumor associated factor Paf. In vivo assays reveal an increase from 67% BrdU+ LSKs in each WT and animals heterozygous for either E47 or p21 to 75% in E47+/-p21+/- mice. More importantly, approximately 15% of LT-HSCs (CD150+flk2-) from compound hets have incorporated BrDU compared to ~10% LT-HSCs from WT or single HET controls. ST-HSCs from E47+/-p21+/- mice also show increased proliferation compared to controls. Consistent with the increase in proliferation, uncommitted progenitors from E47+/-p21+/- mice are more sensitive to 5-FU treatment. Mice deficient in Paf have a similar phenotype as E47 KO mice. We show that there is a 2-fold increase in Paf mRNA levels in E47 KO flk2- and flk2+ LSKs as compared to E47 WT mice. In addition, LT- HSCs from E47 KO mice show a 1.5-fold increase in Paf mRNA levels relative to WT. While Paf protein levels are comparable in flk2- and flk2+ LSKs, proliferating cells from both subsets obtained from E47 KO mice have a ~30% reduction in protein levels relative to WT. Together, these results directly link E47 and p21 in the regulation of HSC proliferation and also suggests Paf as a novel E47 target.
C/EBPβ mediates the differentiation of peritoneal, but not splenic or liver, tissue macrophages (153.28)
Authors: Cain Derek , Womble Mandy , Kelsoe Garnett 
Published: January 2023

Abstract
Macrophages are dispersed throughout the body but exhibit tissue-specific phenotypes and functions. The extracellular and transcriptional pathways that regulate the generation of tissue-specific macrophages are largely unknown. In mice, the peritoneal cavity contains two distinct populations of macrophages, small (CD11b+F4/80lowCD93-) and large (CD11b+F4/80hiCD93+) peritoneal macrophages (SPM and LPM, respectively). Here, we show that mice deficient for the transcription factor C/EBPβ have increased numbers of SPM compared to control littermates, but lack LPM. In C/EBPβ-sufficient and deficient mice, however, splenic (MOMA-1+ and F4/80+) and liver (F4/80+) macrophage compartments are equivalent. When adoptively transferred into C/EBPβ-/- mice, wild-type SPM give rise to LPM, revealing a differentiative pathway from SPM to LPM that requires C/EBPβ. A role for C/EBPβ in the differentiation of certain macrophages is also evident in vitro, as bone marrow cells from C/EBPβ-sufficient and -deficient mice produce macrophages equivalently in cultures containing M-CSF, but C/EBPβ-/- bone marrow cells fail to generate normal macrophages in GM-CSF cultures. These findings demonstrate that tissue-specific macrophages arise through distinct transcriptional pathways, and reveal C/EBPβ as a master regulator of differentiation for a restricted subset of tissue macrophages.
Cancer cells induce a self-amplifying loop of splenic myelopoiesis in the mouse (153.30)
Authors: Ren Guangwen , Zhao Xin , Zheng Betty , Yuan Zeng-rong , Tischfield Jay , Shi Yufang , Shao Changshun 
Published: January 2023

Abstract
Individuals with cancer often show abnormal hematopoiesis that contributes to the formation of tumor-associated immune cells and tumor microenvironment. We here showed that in lymphoma-bearing mice, hematopoietic stem/progenitor cells (HSPCs) underwent a significant expansion in the spleen, and preferentially differentiated into myeloid lineage, predominantly the myeloid-derived suppressor cells (MDSCs). The myelopoiesis-skewing differentiation program depended on tumor environment, because HSPCs from tumor-free (healthy) donors also underwent myelopoiesis-skewing differentiation in tumor-bearing hosts, and HSPCs from tumor-bearing donors can give rise to a less skewed differentiation spectrum of cell lineages in tumor-free hosts. Furthermore, we tested whether MDSCs (or other myeloid cells) and HSPCs formed a positive feedback loop in driving myelopoiesis. We found that donor MDSCs were able to facilitate the expansion of HSPC population in the spleen in tumor-bearing hosts. The donor MDSCs, however, quickly lost their original phenotype in the hosts and a proportion of them became macrophage-like cells (F4/80+) in the spleen. Depletion of macrophage-like cells, but not MDSCs, strikingly attenuated the generation of HSPCs in tumor-bearing hosts, suggesting that MDSCs may only indirectly contribute to splenic hematopoiesis. Our results provide a paradigm that depicts the cancer-associated abnormalities in hematopoiesis, an important aspect of cancer progression.
microRNA miR-150 regulates thymic NKT cell development and function (153.38)
Authors: ZHou Li , Zheng Quan-Hui , Mi Qing-Sheng 
Published: January 2023

Abstract
CD1d-restricted invariant natural killer T (iNKT) cells play an important role in the regulation of diverse immune responses. MicroRNAs (miRNAs), a class of 21-25 nt single-stranded non-coding small RNAs, are increasingly being recognized as important regulators of gene expression through the inhibition of effective mRNA translation. We recently reported that miRNAs expressed in iNKT cell lineage are potent regulators of iNKT cell development, maturation, and function. However, the role of specific miRNAs in the iNKT cells has not been addressed so far. Using miRNA arrays, we found that the expression of miR-150 was significantly upregulated in mature iNKT cells compared to immature iNKT cells in the thymus. Interestingly, mice with miR-150 deficiency have a defect in iNKT cell development in the thymus and iNKT cell maturation was blocked at developmental stage 1 and 2, while the peripheral iNKT cells maturated normally. Furthermore, miR-150-deficient peripheral iNKT cells produce more cytokine interferon-gamma compared to that from wild type after PMA/Ionomycin stimulation. Thus, our results suggest that miR-150 controls iNKT cell thymic maturation and regulates iNKT cell function.
Neonatal naive CD4+ T cells have increased calcium flux and higher expression of miR-181a relative to adult naive CD4+ T cells (153.39)
Authors: Palin Amy , Venkatasubrahmanyam Shivkumar , Butte Atul , Lewis David 
Published: January 2023

Abstract
Neonates are highly susceptible to severe infection by a variety of intracellular pathogens, including M. tuberculosis and T. gondii. A large body of evidence suggests that a limitation in T helper 1 (Th1) responses accounts for much of this susceptibility. Our lab has previously defined a number of limitations in neonatal naive CD4+ T cells obtained from umbilical cord blood that restrict Th1 generation in vitro. These include decreased expression by naive CD4+ T cells of CD40 ligand and IFN-γ. Unexpectedly, we have also observed increased calcium flux in neonatal cells in response to CD3 mAb cross-linking. Because of the diverse molecular differences between neonatal and adult naive CD4+ T cells, we investigated differences in microRNA expression. miR-181a, which has been shown to regulate calcium flux positively in murine T cells, was expressed at higher levels in neonatal naive CD4+ T cells, consistent with increased calcium flux. Its predicted mRNA targets are expressed at lower levels in neonatal naive CD4+ T cells by gene set enrichment analysis (GSEA). Others have shown that over-expression of miR-181a results in increased phosphorylation of Erk1/2 after CD3 mAb crosslinking, and we are examining the activity of the Erk pathway in neonatal and adult naive CD4+ T cells. Also in progress are studies using a stable, lentiviral system to determine the effects of miR-181a over-expression and knockdown in human CD4 T cells focusing on validated murine mRNA targets.
Hematopoietic stem cells undergo an IFNγ-dependent, MyD88-independent transition from dormancy to activity during acute bacterial infection (153.8)
Authors: MacNamara Katherine , McLaughling Maura , Martin Olga , Winslow Gary 
Published: January 2023

Abstract
How hematopoietic stem cells (HSCs) respond to and recover from immunologic stress is not well-understood. We demonstrate that infection with the intracellular bacteria, Ehrlichia muris, induces a transient expansion of bone marrow Lin-negative Sca-1+ cKit+ (LSK) progenitor cells. As inflammation can modulate the expression of surface markers used to phenotype stem and progenitor cells, we addressed whether the phenotypic changes correlated with functional changes in the progenitor cell population. The expansion of LSK cells was associated with a loss of dormant, long-term repopulating HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation. The infection-induced changes required IFNγ signaling, and were accompanied by an expansion of more differentiated multipotent progenitor cells. IFNγ was required for the infection-induced loss of functionally-defined dormant HSCs in label retaining-cell assays. In competitive reconstitution experiments, LSK cells purified from infected IFNγR-deficient mice preferentially engrafted, relative to LSK cells purified from infected wild type mice. Furthermore, the infection-induced changes in progenitor cells were independent of intrinsic MyD88-signaling. We propose that IFNγ elicited during acute infection, provides a signal to dormant HSCs to undergo a rapid transition from dormancy to activity, ostensibly, to provide the host with additional or better-armed innate cells for host defense.
Viral and bacterial antigens as well as agonists for TLR4 and TLR9 can trigger granzyme B secretion by human B cells in the presence of interleukin 21 (154.21)
Authors: Kaltenmeier Christof , Brüggeman Sabine , Beyer Thamara , Sontheimer Kai , Hagn Magdalena , Simmet Thomas , Jahrsdörfer Bernd 
Published: January 2023

Abstract
B cells play a key role for adaptive immune responses by secreting antigen-specific immunoglobulins, for example in the course of infections. Recently, we found that human B cells are able to express granzyme B (GzmB) in response to IL-21. Here we demonstrate that viruses including tick-borne encephalitis, polio, hepatitis B, measles and human immunodeficiency virus (HIV) as well as Bacille Calmette-Guérin can enhance the GzmB response by B cells from healthy individuals. In contrast, B cells from patients with active HIV infection showed a lower capacity to produce GzmB in response to HIV, suggesting a novel pathogenetic mechanism in HIV patients involving B cells. Furthermore, we reveal that next to B cell receptor (BCR) stimulation ligation of toll-like receptors including TLR4 and TLR9 can be involved in B cell GzmB enhancement. After 2 days incubation, up to 77% of freshly isolated B cells directly respond with GzmB expression to stimulation with IL-21, BCR stimulation and TLR ligation. Of note, GzmB is secreted by B cells in an enzymatically active state, reaching levels comparable to those secreted by cytotoxic cells and its induction requires activation of similar signaling pathways as in CTL and NK cells. We suggest GzmB-secreting B cells may play a role in early immune responses, and may contribute to elevated serum GzmB levels found in viral diseases and sepsis. Further studies have to elucidate whether B cells exhibit cytotoxicity towards infected cells.
Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade (154.37)
Authors: Li Wen , Green William 
Published: January 2023

Abstract
LP-BM5 retrovirus induces a disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are critical to MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously un-recognized LP-BM5 retroviral alternative (+1 NT) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response is critical to MAIDS pathogenesis. However, uninfected B6 mice can generate CTL responses to LP-BM5 retrovirus-associated epitope(s) especially prevalent on LP-BM5-induced tumors. Here, we utilized this LP-BM5 retrovirus-induced disease model to test whether modulation of normal immune down-regulatory mechanisms can modulate retroviral pathogenesis. Following in vivo depletion of CD4 T regulatory (Treg) cells, and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis, but not viral load, was substantially decreased, with the combined treatment of CD4 Treg depletion and PD-1 blockade working in a synergistic fashion to eliminate induction of MAIDS.
Viral infection duration mediates locus specific de novo DNA methylation in antigen-specific CD8 T cells (154.41)
Authors: Youngblood Benjamin , Oestreich Kenneth , Ha Sang-Jun , Duraiswamy Jaikumar , Akondy Rama , Wei Zhengyu , Austin James , Riley James , Boss Jeremy , Ahmed Rafi 
Published: January 2023

Abstract
Progressive loss of antiviral functions in antigen-specific CD8 T cell during chronic infection is caused by continuous upregulation and signaling by immunoinhibitory receptors including Programmed Death 1(PD-1). Using human and murine model systems of acute and chronic viral infections, we found that differentiation of naïve CD8 T cells into effector cells during acute infection was accompanied by a transient loss of DNA methylation of the PD-1 locus. Viral clearance and the subsequent effector to memory differentiation resulted in memory-specific DNA remethylation of the PD-1 locus. Conversely, chronic infection induced complete and sustained DNA demethylation of the PD-1 regulatory region in exhausted CD8 T cells. Surprisingly, reduction of virus levels and PD-1 expression in chronically infected animals did not result in remethylation and coincided with decreased expression of a specific de novo methyltransferase (Dnmt3a) isoform. The unmethylated PD-1 locus allowed for rapid PD-1 re-expression, potentially prematurely terminating antiviral functions. These studies provide a foundation for efforts towards reprogramming of PD-1 expression to rejuvenate exhausted T cells.
Murine CD8α+ DCs and human BDCA3+ DCs produce large amounts of IFN-λ in response to poly IC and DNA viruses (154.6)
Authors: Lauterbach Henning , Bathke Barbara , Gilles Stefanie , Traidl-Hoffmann Claudia , Luber Christian , Fejer György , Freudenberg Marina , Davey Gayle , Vremec David , Kallies Axel , Wu Li , Shortman Ken , Chaplin Paul , Suter Mark , O‘Keeffe Meredith , Hochrein Hubertus 
Published: January 2023

Abstract
Dendritic cells (DCs) can be segregated into various subsets based on phenotypic and functional differences. Whereas plasmacytoid DCs are known for their type I interferon (IFN) producing capacity, conventional (c) DCs are better known for their roles in T cell homeostasis and priming. Among cDCs the CD8α+ subset is especially efficient in producing IL-12p70 and the induction of immunity against various pathogens and cancer. Here, we reveal a new hallmark function of murine CD8α+ cDCs and their human BDCA3+ counterparts, namely the production of large amounts of IFN-lambda (IFN-λ, also termed IL-28/29) upon stimulation with poly IC. IFN-lambdas are potent immunomodulatory and antiviral cytokines. We demonstrate that the production of IFN-λ upon poly IC injection in vivo depends on hematopoietic cells and the presence of toll-like receptor (TLR)3, interferon regulatory factor (IRF)3, IRF7, IFN-IR, Fms-related tyrosine kinase 3 ligand (FL) and IRF8 but not on myeloid differentiation factor 88 (MyD88), Rig like helicases or lymphocytes. Furthermore, we show that both CD8α+ cDCs and plasmacytoid DCs produce large amounts of IFN-λ in response to HSV-1 or parapoxvirus. Thus, IFN-λ production in response to poly IC is a novel hallmark function of mouse CD8α+ cDCs and their human equivalents.
New vaccination delivery regimen drives enhanced, vaccine-specific immune responses. (155.13)
Authors: Queiroz Rafaella , Allen Kelsey , McEwen Lisa , Ring Jennifer , Harn Donald 
Published: January 2023

Abstract
There is considerable research to define novel and safe adjuvants for use in vaccine formulations. Here, we focused on a gel-slurry delivery method to drive pro-inflammatory vaccine-specific responses using CpGs as adjuvant. The gel slurry hardens at body temperature, forming a gel matrix depot that releases CpGs and antigen, attracting antigen presenting cells. We vaccinated mice with recombinant Hepatitis B antigen using 8 different adjuvant schemes comparing induction of cytokines and antibody subtypes. We compared delivery using two different gel-slurrys with Alhydrogel (Alum) and Complete Freunds adjuvant (CFA). Each was mixed +/- CpGs. Mice vaccinated with gel slurry plus CpGs had significantly higher vaccine-specific antibody responses than mice vaccinated with antigen and CFA. Recall assays showed cells from mice vaccinated with Alhydrogel or CFA upregulated IL-10 at 72h compared to cells from gel-slurry + CpG vaccinated mice. Similar results were seen for IL-4. CpG use reduced levels of IL-5 to background in all groups compared to elevated levels in CFA. No differences in levels of IFNg and TNF. We are currently assaying for Ag-specific antibody isotype titers. The use of gel slurry plus CpGs to deliver vaccine antigens should have great utility for a number of vaccine formulations. Ultimately, this type of vaccine delivery system may facilitate development of therapeutic vaccines as well as prophylactic.