bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-FcϵRIα mAb-based therapy can suppress IgE-mediated disease (151.4)
Authors: Khodoun Marat , Krishnamurthy Durga , Strait Richard , Kucuk Yesim , Finkelman Fred
Published: January 2023

IgE contributes to allergy pathogenesis by mediating Ag crosslinking of the high affinity IgE receptor, FcϵRI, on mast cells and basophils, which results in massive secretion of cytokines, vasoactive mediators, and proteolytic enzymes. We evaluated the ability of MAR-1, a hamster anti-mouse FcϵRIα mAb, to suppress murine IgE-mediated disease. MAR-1 and IgE compete for binding to FcϵRI, but MAR-1, unlike IgE, removes mast cell FcϵRI and kills basophils. Although MAR-1 does not remove FcϵRI/IgE complexes from mast cells, receptor turnover allows in vivo treatment with MAR-1 for 22 days to remove nearly all mast cell FcϵRI. MAR-1 itself activates basophils and mast cells and induces anaphylaxis, but anaphylaxis can be prevented by either antihistamine plus corticosteroid pretreatment or serial injection of increasing doses of MAR-1 (rapid desensitization). MAR-1 treatment immediately prevents subsequent mast cell priming with Ag-specific IgE mAb, while treatment for 22 days prevents Ag-induced mast cell activation in mice injected with Ag-specific IgE mAb prior to treatment initiation. A single dose of MAR-1 fully suppresses established moderate food allergy. Although MAR-1 suppression of established severe food allergy is limited by development of anti-hamster IgG Ab; this is prevented and disease fully suppressed by additional treatment with a single dose of anti-CD4 mAb. These observations suggest practical approaches to treat established IgE-mediated allergic disorders.
Expression of mouse and human TIGIT on follicular T helper cells and other lymphocytes (152.10)
Authors: Funatake Castle , Kang William , Araji Soha , Li Shourong , Atteberry Brandi , Huang Qiulong , Just Peggy
Published: January 2023

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a newly identified member of the polio virus receptor family. In humans, its expression has been reported on NK cells, regulatory T cells (Treg), memory CD4 T cells, follicular T helper cells (Tfh), and CD8 T cells, but not on B cells or naïve CD4 T cells. In mice, mRNA for TIGIT has been observed in Tfh but not naïve T cells. We examined the expression of TIGIT on lymphocytes in mice and humans using two novel antibodies, clone GIGD7 and MBSA43, respectively. We found that the expression patterns on unstimulated lymphocytes were dissimilar between mice and humans, whereas expression following activation was more consistent. For the first time, we show that TIGIT is expressed on the surface of mouse Tfh, consistent with studies examining mRNA and similar to previous observations in humans. Finally, the response of memory T cells in vitro was slightly increased when normal human peripheral blood cells were stimulated in the presence of MBSA43 to block TIGIT, consistent with its role as an inhibitory receptor. These two antibodies will facilitate the characterization of TIGIT on Tfh and Treg, as well as human memory T cells and NK cells and will help to elucidate the role of this inhibitory receptor during immune responses.
C6-ceramide facilitates IL-12-mediated T helper-1 cell differentiation through a COX-2/PGE2-dependent pathway (152.17)
Authors: Kue Chin Siang , Jung Mi Young , Kim Tae Sung
Published: January 2023

Ceramides, lipid molecules that are found predominantly in the plasma membrane of a cell, can serve as a second messenger and have been known to perform a number of cellular functions. Our study was designed to investigate the effect of ceramides on T helper cell responses and their underlying mechanisms. We found that a cell-permeable C6-ceramide (C6) together with IL-12 synergistically increased the populations of IFN-γ+ T cells and up-regulated IFN-γ production from CD4+ T cells, whereas C2-ceramide and long chain ceramides (C16 and C24) did not affect. C6 treatment increased the expression of T-bet, a master transcription factor of Th1 responses, in a dose-dependent manner. Moreover, C6 induced expression of cyclooxygenase-2 (COX-2) accompanied by prostaglandin E2 (PGE2) production in CD4+ T cells. The IFN-γ+ T cells populations and IFN-γ production induced by C6 were significantly suppressed by both a COX-2 specific inhibitor (NS398) and an anti-PGE2 neutralizing antibody in a dose-dependent manner. T-bet expression was also decreased by NS398 treatment. Taken together, C6 promotes the production of PGE2 in CD4+ T cells through a COX-2 dependent mechanism, thereby facilitating Th1 cell differentiation. These results suggest that C6 can be utilized in the therapeutics of diseases such as cancer and allergic by enhancing the Th1 activity.
Egr3 promotes the development of inflammatory γδ T cells (152.2)
Authors: Parkinson Rose , Powell Jonathan
Published: January 2023

Though Egr3 is known to inhibit a Th1 response, its role in Th17 development is unclear. We show that CD4+ T cells from mice overexpressing Egr3 (Egr3 TG) produce more IL-17 than wildtype controls. This increase is not cell intrinsic, as naïve CD4+ T cells from Egr3 TG mice do not produce IL-17 upon activation and IL-6 and IL-23 mRNA are elevated in lymphoid organs. Egr3 TG mice also have increased numbers of CD4- CD8- T cells, most of which express the γδ TCR. These γδ T cells strongly express IL-17 and IL-6, suggesting that they are capable of directing CD4 T cells toward a Th17 phenotype. We find a similar increase in the number of γδ T cells in the mucosal epithelia of Egr3 TG mice. Consistent with our previous results, Egr3 TG mice exhibit more lung inflammation, IL-17 production, weight loss, and mortality in a bleomycin-induced pulmonary fibrosis model. We also tested the susceptibility of these mice in a DSS-induced colitis model, where the role of IL-17 is less clear and γδ T cells may serve a protective role. In this model, Egr3 TG mice have decreased TNFα production, decreased rectal bleeding and increased overall survival. Our data suggest that Egr3 induces of one or more subsets of γδ T cells, which are inflammatory in select mucosal settings. Future studies will clarify the precise mechanism of enhancement or protection in models of mucosal inflammatory disease.
Curcumin attenuates decline of CD69 expression on CD4+ T cells and induces generation of CD25hiFoxp3+ T cells through TGF-β1 production (152.22)
Authors: Kim Gi Rak , Jang Mi Seon , Son Young Min , Han Seung Hyun , Yun Cheol-Heui
Published: January 2023

Curcumin is a promising candidate as a natural medicinal agent to treat chronic inflammatory diseases. However, little is known about curcumin-mediated CD4+ T cell regulation, which contributes to the therapeutic effects of curcumin. Here, we report that curcumin inhibits T-cell receptor (TCR)-triggered immune responses of CD4+ T cells through suppression of proliferation, cytokine production and differentiation of CD4+ T cells. Intriguingly, spontaneous decline of CD69 expression was attenuated by curcumin treatment, which continued until 6 days of culture. We showed that curcumin-mediated CD69 up-regulation was dependent on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Concomitantly, the expressions of C-C motif chemokine receptor 7 (CCR7), L-selectin and transforming growth factor-β1 (TGF-β1) were also increased. TGF-β1 production was moderate, but was high enough to lead to the late generation of CD25hiFoxp3+CD4+ T cells and to maintain prolonged suppression of CD4+ T cell activation. These findings suggest that the immunoregulatory role of curcumin is effective for CD4+ T cells.
Suppressor of cytokine signaling-1 contributes to peripheral regulatory T cell stability in an inflammatory environment. (152.24)
Authors: Collins Erin , Jager Lindsey , Dabelic Rea , Lau Ken , Haider Mohammed , Johnson Howard , Larkin III Joseph
Published: January 2023

Suppressor of cytokine signaling-1 deficient mice (SOCS1-/-) die of a T cell mediated inflammatory, autoimmune disease by 3 weeks of age. The inflammation mediated by excessive interferon γ signaling and leukocyte infiltration results in the destruction of many vital organs. Significantly, numerous mouse models of inflammatory autoimmune disease have been associated with a deficiency in Foxp3+ regulatory T cells. Indeed, SOCS1-/- mice possessed a reduction in peripheral Tregs, despite enhanced thymic development. The adoptive transfer SOCS1+/+ Tregs or CD4+ T lymphocytes mediated an increased, yet limited, survival in the SOCS1-/- mice. However, the adoptive transfer of CD4+ T lymphocytes in conjunction with SOCS1-KIR, a mimetic peptide sufficient to partially restore SOCS1 function, resulted in 30% of the mice living beyond 5 weeks. Moreover, the combined treatment mediated a decrease in leukocyte infiltration into vital organs, and a decrease in effector CD4+ cells. Additionally, the decrease in inflammation was associated with an increase in the peripheral Foxp3+ regulatory T cell population. Collectively, these results suggest that the SOCS1-KIR/CD4+ T cell combined treatment synergistically promoted long-term survival of the SOCS1-/- mice by restoring peripheral Tregs. Furthermore, these data propose that SOCS1 contributes to peripheral Treg stability under inflammatory conditions.
Identification of the E3 deubiquitinase USP21 as a positive regulator of GATA3 (152.25)
Authors: Zhang Jing , Chen Chen , Gao Yayi , Lin Fang , Tsun Andy , Li Bin
Published: January 2023

TCR signaling induces GATA3 expression in FOXP3+ Treg, but the underlying mechanism for GATA3 accumulation and its role in Treg function remains unclear. Here we identify the E3 deubiquitinase USP21 as a positive regulator of GATA3 in FOXP3+ T cells. By screening for regulators which may affect the function of key transcription factors essential for T cell lineage differentiation and function, we found that USP21 dramatically increases the cellular level of GATA3. Reciprocal co-immunoprecipitation studies indicated that USP21 can interact with GATA3 in vivo. Overexpression of wild type USP21, but not the enzymatically inactive mutant C221A, specifically promotes the up-regulation of GATA3 protein expression, suggesting that the deubiquitinase activity of USP21 is essential for the upregulation of GATA3. Moreover, we found that T cell activation via TCR and CD28 costimulation may upregulate the level of USP21 and GATA3 in FOXP3 expressing T cells. shRNA mediated knockdown of USP21 can prevent GATA3 accumulation after T cell activation, suggesting that the mechanism by which USP21 stablizes GATA3 may occur through preventing GATA3 degradation. Functionally, overexpression of wild-type USP21 promotes GATA3 mediated upregulation of IL-4. Our studies have identified a unique pathway to upregulate GATA3 activity at posttranslational level by reversing ubiqutination-mediated protein degradation.
Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells. (152.3)
Authors: Beurel Eleonore , Yeh Wen-I , Michalek Suzanne , Harrington Laurie , Jope Richard
Published: January 2023

CD4+ T cells are critical for host defense but also drive immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4+ T cells was recently revised by the identification of the Th17 lineage that produces IL-17 and are critical in autoimmune disease pathogenesis. Mechanisms controlling the differentiation of Th17 cells have been described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4+ T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4+ T cells to Th17 cells increased by 10-fold GSK3β protein levels in Th17 cells, while GSK3β was unaltered in Treg cells. Diminishing GSK3 activity pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung following Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis (EAE) mouse MS model. These findings identify GSK3 as a critical mediator of Th17 cell production, and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.
Identification and characterization of a novel and functionally distinct human memory CD4+CD25INTFOXP3- population (152.30)
Authors: Triplett Todd , Curti Brendan , Weinberg Andrew
Published: January 2023

Surface expression of the IL-2 receptor α-chain (CD25) has been used as a marker to discriminate between CD4+CD25HiFOXP3+ Tregs and resting CD4+CD25-FOXP3- non-Tregs. However, using a new monoclonal antibody we have revealed a novel human CD4+CD25INTFOXP3- T-cell population that expresses intermediate levels of CD25 and constitutes ~ 40% of resting human CD4+ T cells, the majority of which are memory cells. Further analysis revealed that the CD25NEG and CD25INT memory populations are stable populations composed of distinct memory subsets. The CD25NEG population contains the vast majority of late differentiated EOMES+CD28-GranzymeB+ memory cells that are expanded in Lupus patients and respond to antigens associated with chronic immune responses (CMV). In contrast, the CD25INT population contains memory cells that primarily respond to antigens associated with recall antigen responses (FLU) and produce a wider array of cytokines (IL-17, IL-10, IL-5, IL-9). We also found that compared to the CD25NEG and Treg memory populations, the CD25INT population is differentially affected by rhIL-2 in vitro and in vivo when cancer patients are treated with IL-2-specific immunotherapy. Collectively, these results show that in humans a larger proportion of CD4+ T cells express the high affinity IL-2 receptor than originally thought and that expression of CD25 may be used as a surface marker to discriminate functionally distinct memory populations that are uniquely affected by IL-2.
Denditic cells and regulatory T cells: development, crosstalk and consequences (152.31)
Authors: Darrasse-Jeze Guillaume
Published: January 2023

Dendritic cells (DCs) play a major role of immune sentinels of our body. These cells capture, process and presents both foreign and self antigens to lymphocytes that activate immune responses or maintain immune tolerance. Despite a growing interest, their behavior remains mysterious. We have characterized in vivo at the cellular and molecular level, their development, their homeostasis and their intercellular communication with natural regulatory T cells (Tregs). We had previously found that Tregs were essential for maintaining normal numbers of dendritic cells (DCs): when Tregs are depleted, there is a compensatory Flt3-dependent increase in DCs. We now show that Treg homeostasis is maintained in vivo by the existence of a feedback regulatory loop between DCs and Tregs. We find that loss of DCs leads to a loss of Tregs, and that the remaining Tregs exhibit decreased Foxp3 expression. The DC-dependent loss in Tregs leads to an increase in the number of T cells producing inflammatory cytokines, such as interferon gamma and interleukin 17. Conversely, increasing the number of DCs leads to increased Treg division and accumulation by a mechanism that requires major histocompatibility complex II expression on DCs. The increase in Tregs induced by DC expansion is sufficient to prevent type 1 autoimmune diabetes and IBD, which suggests that interference with this feedback loop will create new opportunities for immune-based therapies.
Modeling the mechanisms of action underlying the plasticity of the CD4+ T cell differentiation process (152.34)
Authors: Carbo-Barrios Adria , Hontecillas Raquel , Climent Montse , Hoops Stefan , Lu Pinyi , Wendelsdorf Katherine , Eubank Stephen , Marathe Madhav , Bassaganya-Riera Josep
Published: January 2023

Mathematical and computational modeling facilitates concurrent multiparametric analyses of dynamic biological processes. Herein we describe a network model illustrating intracellular pathways controlling a naïve T cell differentiation into Th1, Th2, Th17 or iTreg phenotypes. The model is comprised of 37 differential equations representing 40 reactions and 81 species. The COmplex PAthway SImulator software has been used for model calibration and it shows that our model adequately computes the differentiation of CD4 T cells into the four phenotypes. Moreover, our network includes the nuclear transcription factor peroxisome proliferator activated receptor γ (PPARγ) that modulates the Th17/iTreg plasticity and is highly expressed during IL-4-induced Th2 differentiation. Local model sensitivities demonstrate that PPARγ activation increases the production of FOXP3 when the system is driven into Th17 differentiation. The prediction of this model was validated in vitro using primary mouse CD4+ T cells differentiated into Th17 and treated with 0, 0.25, 0.5, 1, 2, 4, 10 and 40 μM rosiglitazone, a PPARγ agonist. Our differentiation studies confirmed the prediction of our model that increasing concentrations of rosiglitazone in Th1-induced CD4 T cells decrease INFγ and Tbet levels, and increase GATA3 expression in Th2-induced T cells. These data support the prediction of our model regarding the modulation of intracellular networks controlling fate and function of CD4+ T cells by PPARγ.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
A recombinant adenovirus for targeted gene therapy of pulmonary inflammation (155.23)
Authors: Alberti Michael , Deshane Jessy , Pereboeva Larisa , Tsuruta Yuko , Abraham Ed , Chaplin David , Gerson Stanton , Curiel David , Roth Justin
Published: January 2023

Inflammation is a pathological component of many debilitating lung diseases. Thus, therapeutics that suppress the inflammatory response should also minimize inflammation-induced airway destruction. In this regard, Adenovirus type 5 (Ad5) vectors have a long history as therapeutic agents, due to their in vivo stability and amenability to genetic manipulation. Since circulating leukocytes infiltrate and mediate damage to the sites of pathology, we proposed to modulate Ad5 tropism to the dense leukocyte pool in the lung microvasculature for localized therapeutic intervention of inflammation. However, leukocytes do not express the coxsackie and adenovirus receptor (CAR) and are thus resistant to Ad5 infection. Therefore, we utilized phage display to identify a myeloid-binding peptide (MBP) and then genetically incorporated this sequence into an Ad5 fiber lacking the native CAR-binding domain (knob). Non-replicative Ad reporter vectors containing these MBP-fibers (Ad.MBP) maintained myeloid-binding specificity, and upon intravenous delivery the altered tropism enhanced lung gene transfer by five orders of magnitude over that obtained with unmodified Ad5. Disparity between cell types bound and those transduced suggest Ad.MBP is first sequestered by circulating leukocytes and subsequently “handed-off” to transduce pulmonary endothelium. These studies highlight the importance of pulmonary leukocytes as a target for therapeutic delivery in inflammatory lung disease.
Reversing the suppressive effects of regulatory T cells by targeting toll-like receptors and OX40 in human follicular lymphoma (156.11)
Authors: Voo Kui , Foglietta Myriam , Harlin Megan , Bover Laura , Kwak Larry , Liu Yong-Jun , Neelapu Sattva
Published: January 2023

Active immunotherapy is a promising approach for the treatment of follicular lymphoma (FL), an incurable B-cell lymphoma that accounts for 22% of all non-Hodgkin’s lymphomas worldwide. However, the clinical response rates following therapeutic cancer vaccination have been low in FL. We and others have shown that CD4+Foxp3+ regulatory T cells (Tregs) are increased in the peripheral blood and at the tumor site and are induced by tumor cells in FL. More importantly, Tregs potently suppressed the proliferation and cytokine production by FL tumor-specific effectors T cells. We previously reported that OX40 signaling shuts down the generation of IL-10-producing Tr1 cells. In this study, we showed that OX40 signaling blocks the suppressive function of Tregs and promotes tumor-specific effector T-cell proliferation in FL. We also showed that TLR ligands Pam3CSK4, flagellin, and CpG-B potently blocked the suppressive function of Tregs and restored the function of FL-specific effector T cells. However, agonists of TLR2/6, TLR3, TLR4, TLR7/8, and TLR8 were ineffective. These results suggest the potential usefulness of these agents as adjuvants to enhance the efficacy of therapeutic vaccines and other immunotherapeutic strategies in FL patients. Furthermore, they support the development of combinatorial strategies to promote effector T cell function and survival and block Treg function in FL and other malignancies.
Inflammation, apoptosis, and necrosis induced by neoadjuvant Fas ligand gene therapy improves survival of dogs with spontaneous bone cancer (156.18)
Authors: Modiano Jaime , Bellgrau Donald , Cutter Gary , Lana Susan , Ehrhart Nicole , Ehrhart Eugene , Wilke Vicki , Charles J. , Scott Milcah , Pozniak John , Schaack Jerome , Duke Richard
Published: January 2023

The use of Fas ligand (FasL) gene therapy for cancer has shown promise in laboratory animals. The mechanism by which this treatment works appears to be through induction of tumor inflammation and anti-tumor immunity. However, the efficacy of FasL to treat spontaneous tumors is unknown. To investigate this, we used the intratumoral FasL gene delivered in an adenovirus vector as neoadjuvant to standard of care in 56 dogs with naturally occurring appendicular osteosarcoma. Tumors from dogs treated with FasL had greater inflammation, necrosis, apoptosis, and fibrosis at day-10 (amputation) than tumors from controls dogs that did not receive FasL. These parameters also were increased when compared to pre-treatment biopsy samples (day-0). Survival improvement was apparent in dogs with greater inflammation or lymphocyte infiltration scores and in the top 50th percentile of apoptosis, whereas it was no different than standard of care alone in dogs with lower inflammation scores and the bottom 50th percentile of apoptosis. Reduced Fas expression in tumor cells was the only variable associated with prognostically advantageous inflammation; this relationship was not seen in dogs that did not receive FasL therapy. Together, the data suggest that FasL gene therapy can improve survival in naturally occurring tumors of large animals, and may be most effective when tumors fail to express Fas receptor, thus allowing FasL to exert its pro-inflammatory effects on the tumor microenvironment.
Functional and structural characterization of potent Th1 biasing 6′-derivatised α-GalCer iNKT cell agonists, and their superior role in tumor protection. (156.4)
Authors: Aspeslagh Sandrine , Li Yali , Yu Esther , Decruy Tine , Van Beneden Katrien , Girardi Enrico , Pauwels Nora , Trappeniers Matthias , Venken Koen , Drennan Michael , Leybaert Luc , Wang Jing , Van Calenbergh Serge , Zajonc Dirk , Elewaut Dirk
Published: January 2023

Invariant natural killer T cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. In this study, we report the first crystal structures of a novel class of strong Th1 biasing structural analogues of α-galactosylceramide by addition of aromatic structures on the 6-OH position of galactose. They are characterized by marked Th1 polarized cytokine patterns that are highly conserved between mice and men, and marked tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1 biasing glycolipid, α-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.